U.S. patent application number 12/077543 was filed with the patent office on 2009-01-29 for microelectromechanical devices useful for manipulating cells or embryos, kits thereof, methods of making same, and methods of use thereof.
Invention is credited to Monica Palacios-Boyce.
Application Number | 20090029471 12/077543 |
Document ID | / |
Family ID | 27384057 |
Filed Date | 2009-01-29 |
United States Patent
Application |
20090029471 |
Kind Code |
A1 |
Palacios-Boyce; Monica |
January 29, 2009 |
Microelectromechanical devices useful for manipulating cells or
embryos, kits thereof, methods of making same, and methods of use
thereof
Abstract
The present invention relates generally to
microelectromechanical systems (MEMS) devices for the manipulation
of cells or groups of cells, such as oocytes, embryos, and sperm.
In particular, the present invention relates to Cell Labeling MEMS
devices (2F), Microinjection MEMS devices, IntraCytoplasmic Sperm
Injection ("ICSI") MEMS devices, Zona Coring MEMS devices,
Enucleation MEMS devices, Enucleation/Nuclear Transfer MEMS
devices, and Cytoplasmic Transfer MEMS devices. The present
invention also relates to kits containing the MEMS devices of the
present invention.
Inventors: |
Palacios-Boyce; Monica;
(Wales, MA) |
Correspondence
Address: |
MATHEWS, SHEPHERD, McKAY & BRUNEAU, P.A.
29 Thanet Road, Suite 201
Princeton
NJ
08540
US
|
Family ID: |
27384057 |
Appl. No.: |
12/077543 |
Filed: |
March 20, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10031259 |
Jun 3, 2002 |
7390648 |
|
|
PCT/US00/11040 |
Apr 24, 2000 |
|
|
|
12077543 |
|
|
|
|
60130802 |
Apr 23, 1999 |
|
|
|
60147802 |
Aug 9, 1999 |
|
|
|
60149269 |
Aug 17, 1999 |
|
|
|
Current U.S.
Class: |
435/455 ;
257/E21.002; 435/285.1; 438/106 |
Current CPC
Class: |
B01L 3/5027 20130101;
C12M 21/06 20130101; C12M 35/04 20130101 |
Class at
Publication: |
435/455 ;
435/285.1; 438/106; 257/E21.002 |
International
Class: |
C12N 15/00 20060101
C12N015/00; C23F 1/00 20060101 C23F001/00; C12M 1/00 20060101
C12M001/00; H01L 21/02 20060101 H01L021/02 |
Claims
1-47. (canceled)
48. A microinjection MEMS device for injecting a fluid, a
suspension or a material into a cell or group of cells comprising:
(a) a first substrate comprising at least one well for holding the
cell or group of cells and wherein the well comprises at least one
hollow protuberance for penetrating the cell or group of cells and
wherein the well is in fluid communication with a fluid transfer
channel wherein the fluid transfer channels permits the fluid to
enter the hollow protuberance and to then enter the cell; and (b) a
second substrate comprising an input manifold in fluid
communication with the fluid transfer channel wherein the input
manifold allows for the input of the fluid, suspension or material
into the hollow protuberances.
49. The microinjection MEMS device of claim 48 further comprising a
pumping means.
50. The microinjection MEMS device of claim 48 wherein the well is
cube-shaped.
51. The microinjection MEMS device of claim 50 wherein the
cube-shaped well is from about 50 .mu.m to about 200 .mu.m in
length per side.
52. The microinjection MEMS device of claim 48 wherein the well is
conical-shaped.
53. The microinjection MEMS device of claim 48 wherein the hollow
protuberance is a needle.
54. The microinjection MEMS device of claim 48 wherein the hollow
protuberance is from about 0.01 .mu.m to about 100 m in
diameter.
55. The microinjection MEMS device of claim 48 further comprising a
coating on the surfaces of the device exposed to the cells.
56. The microinjection MEMS device of claim 55 wherein the coating
is a polypeptide, peptide or protein.
57. The microinjection MEMS device of claim 56 wherein the
polypeptide is polylysine.
58. The microinjection MEMS device of claim 48 wherein the hollow
protuberance acts as an emitter to facilitate piercing the cell or
group of cells.
59. A method of making a microinjection MEMS device comprising the
steps of: (a) etching a plurality of parallel channels on a first
side of a plurality of silicon wafers in which the wafers each have
a second unetched side; (b) silicon fusion bonding the unetched
side of a plurality of silicon wafers of step (a) to the etched
side of a plurality of silicon wafers of step (a) such that the
etched channels are in parallel to form a mega-laminate wherein the
mega-laminate has a plurality of channels; (c) cutting the
mega-laminate at an angle perpendicular to the long axis of the
etched channels thereby forming a slice of the mega-laminate having
a top surface and a bottom surface wherein each surface exposes an
end of the channel; (d) silicon fusion bonding the bottom surface
of the slice of the mega-laminate to the etched side of a
channel-etched base-plate wafer; (e) depositing a first mask on the
top surface of the slice of the mega-laminate such that a region
surrounding each channel end is free of mask; (f) etching the mask
to form a plurality of wells; (g) depositing a second mask on the
mega-laminate top surface such that a boarder forms around each
channel end such that material around the channel is not etched;
and (h) etching the second mask thereby forming a plurality of
hollow protuberances.
60. The method of making a microinjection MEMS device of claim 59
further comprising applying a coating to the mega-laminate top
surface after step (h).
61. The method of making a microinjection MEMS device of claim 60
wherein the coating is a polypeptide, peptide or protein.
62. The method of making a microinjection MEMS device of claim 61
wherein the polypeptide is polylysine.
63. A microinjection MEMS device kit for injecting a fluid, a
suspension or a material into a cell or group of cells comprising:
(a) a centrifugal platter for applying a centripetal force to a
cell or group of cells contained within a MEMS device wherein the
centrifugal platter comprises a circular disk, a plurality of ports
for holding the MEMS devices and a securing means to secure the
platter to a spinner or driving means; and (b) at least one
microinjection MEMS device of claim 48.
64. The microinjection MEMS device kit of claim 63 wherein the
microinjection MEMS device is permanently affixed to the
centrifugal platter.
65. The microinjection MEMS device kit of claim 64 wherein (a) the
centrifugal platter comprises a plurality of grooves arranged in a
concentric pattern and wherein each groove has an inner and outer
edge; (b) at least one microinjection MEMS device is bonded to the
outer edge of a groove in an orientation such that the axis of each
well of the microinjection MEMS device is horizontal to the plane
of the centrifugal platter; and (c) the inner edge of the grooves
forming divided compartments comprising a single well which
restrict the movement of materials from one compartment containing
a containing a single well to another compartment.
66. A method of using a microinjection MEMS device kit of claim 63
comprising the steps of: (a) filling the input manifold of at least
one microinjection MEMS device resident on a centrifugal platter
with a fluid; (b) loading the fluid-filled wells of step (b) with
at least one oocyte or embryo; (c) placing the microinjection
MEMS/centrifugal platter into a centrifuge; and (d) applying a
centripetal force on the microinjection MEMS device kit by rotating
the kit using a spinner or driving means.
67. A method of using a microinjection MEMS device kit of claim 63
comprising the steps of: (a) filling the grooves of the centrifugal
platter with a fluid; (b) loading the grooves of the centrifugal
platter with at least one oocyte or embryo; and (c) applying a
centripetal force to the kit whereby the oocyte or embryo makes
contact with the hollow protuberance of the microinjection MEMS
device and the hollow protuberance penetrates the surface of the
oocyte or embryo.
68. A method of using a microinjection MEMS device kit of claim 63
wherein, upon rotation of centrifugal platter, a volume of fluid is
caused to enter the oocyte or embryo in the cell well through the
hollow protuberance.
69. A microinjection MEMS array for injection of a fluid, a
suspension or a material into a cell or group of cells comprising:
(a) a first substrate comprising at least one well for accepting a
cell or group of cells and wherein the well comprises a hollow
protuberance for penetrating the cell or group of cells; (b) a
second substrate comprising a fluid handling means in fluid
communication with said hollow protuberance; and (c) a central
loading manifold for loading a fluid into the array.
70. A microinjection MEMS array of claim 69 wherein the fluid
handling means is a dynamic hydropressure column.
71. A microinjection MEMS array of claim 69 wherein the device is
embedded in a centrifugal platter.
72. A method of using the microinjection MEMS array of claim 69
comprising: (a) applying an inertial force to the device using a
centripetal (angular) acceleration means brought about by rotation
of the centrifugal platter.
73. A microinjection MEMS array for injecting a fluid, a suspension
or a material into a cell or group of cells comprising: (a) a
central loading manifold for loading the fluid, suspension or
material into the array; (b) a plurality of wells for receiving
cells; (c) a hollow protuberance within each well for penetrating
the cell and injecting the fluid, suspension or material; and (d) a
plurality of dynamic hydropressure columns in fluid communication
with the central loading manifold and with the hollow protuberances
wherein the dynamic hydropressure columns provide pressure for
forcing the fluid, suspension or material through the hollow
protuberance and into the cell.
74. The microinjection MEMS array of claim 73 further comprising at
least one valve in the dynamic hydropressure column for modulating
fluid flow.
75. A microinjection MEMS array of claim 74 wherein each valve is
in operable communication with a controller to control the fluid,
suspension or material flowing into the cell.
76. A microinjection MEMS device of claim 75 wherein the operable
communication is mediated by electronic circuits.
77. A microinjection MEMS device of claim 75 wherein the operable
communication is mediated by Electro-optical circuits.
78. A method of using a microinjection MEMS device of claim 73
comprising: (a) loading at least one cell or group of cells into an
injection domain of the microinjection MEMS device; and (b)
applying a centripetal force to the microinjection MEMS device
thereby causing penetration of the cell or group of cells by the
hollow protuberance of the microinjection MEMS device; and
deposition of a substance in the cell or group of cells from the
hollow protuberance.
79. A method of using a microinjection MEMS device of claim 73
comprising: (a) loading at least one cell or group of cells into
the well of the microinjection MEMS array; and (b) rotating the
microinjection MEMS device whereby the cell or group of cells is
thrust upon the hollow protuberance resident within well and
simultaneously providing for the passive movement of fluid through
dynamic hydropressure columns provides pressure to push fluid into
cell. (c)
80. The method of using the microinjection MEMS of claim 79 further
comprising a valve providing for variable fluid flow from the
dynamic hydropressure column into the cell when activated by a
controller by way of a circuit.
81-134. (canceled)
Description
[0001] The present application is a continuation-in-part
application of U.S. Provisional Application No. 60/130,802 filed
Apr. 23, 1999; U.S. Provisional Application No. 60/147,802 filed
Aug. 9, 1999, and U.S. Provisional Application No. 60/149,269 filed
Aug. 17, 1999. The disclosures of the above-identified applications
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to
microelectromechanical systems (MEMS) devices for the manipulation
of cells or groups of cells, such as oocytes, embryos, and sperm.
In particular, the present invention relates to Cell Labeling MEMS
devices, Labelable Zona Anchor MEMS devices, Microinjection MEMS
devices, IntraCytoplasmic Sperm Injection ("ICSI") MEMS devices,
Zona Coring MEMS devices, Enucleation MEMS devices,
Enucleation/Nuclear Transfer MEMS devices, and cytoplasmic transfer
MEMS devices. The present invention also relates to kits containing
the MEMS devices of the present invention.
[0003] The present invention also relates to devices and articles
of manufacture for manipulating and using MEMS devices of the
invention. More particularly, the present invention further relates
to a centrifugal platter, labelable zona anchor MEMS device
platforms and labelable zona anchor MEMS device platform
holders.
[0004] The present invention also relates to microelectromechanical
system arrays and devices useful for cell culture. In particular,
the present invention relates to single layer culture arrays,
multi-layer culture arrays, multi-layer culture array environmental
controllers, multi-compartment, multi-modal incubators, and
environmental control instruments.
[0005] The present invention also relates to methods of using the
MEMS devices and kits of the present invention.
[0006] The present invention further relates to methods of making
the MEMS devices of the present invention.
BACKGROUND OF THE INVENTION
[0007] Microelectromechanical Systems (MEMS) are machines
fabricated on a microscopic scale using surface micromachining or
LIGA processes. MEMS devices can include moveable members (e.g.,
gears, rotors, linkages, levers, hinges and mirrors) for
applications including sensing (e.g., acceleration or chemicals),
switching (electrical or optical signals) and optical display
(e.g., moveable mirrors) functions. MEMS devices can further
include actuators or motors for driving gear trains to perform
various functions including coded locks and self-assembling
structures.
[0008] In recent years, the design possibilities of
microelectromechanical systems (MEMS) have expanded as the field
has further matured. Recent advances in single crystal silicon
wafer manipulation, the addition of integrated circuits as a
practical modality for controlling these microstructures as well as
other associated technologies has widened the horizon of possible
uses (Senturia, S. D., et al., (1992) "A Computer-Aided Design
System for Microelectromechanical Systems (MEMCAD)" Journal of
Microelectromechanical Systems 1(1):3; Clerc, P-A., et al., (1998)
"Advanced deep reactive ion etching: a versatile tool for
microelectromechanical systems" J. Micromech. Microeng.
8(4):272-278; Petersen, K. E., (1998) "Toward Next Generation
Clinical Diagnostic Instruments Scaling and New Processing
Paradigms" Biomedical Microdevices 1 (1):71-79). One of the most
promising novel aspects of this field is the design of MEMS which
modulate and manipulate the small scale world of individual cells,
thus facilitating, for the first time, an actual hands-on method
for addressing biological issues at the level of the most basic
unit of order in multicellular organisms.
[0009] Whereas many cells in the body are of a size on the order of
a few microns, there is a special class of cells, the female gamete
called the oocyte, which is far larger, on the order of 100
microns. Further, these cells, in many animals from sea urchins to
mammals, are surrounded by a five to twenty micron thick
selectively permeable glycoprotein coat called the Zona
Pellucida.
[0010] The modification of the surface of the glycoprotein coating
of oocytes and embryos is a desirable operation in endeavors such
as the labeling of a great many of oocytes and embryos in the
animal husbandry industry.
[0011] Further, the delivery of and removal of materials into and
out of the cytoplasm of oocytes is a desirable operation in
endeavors such as the generation of transgenic animals,
intracytoplasmic sperm injection, assisted hatching, enucleation,
nuclear transfer, and cytoplasmic transfer. At present the outcome
of these procedures, being technically demanding and relatively
novel and as such, not optimized, is very poor. The generation of
transgenic animals born by way of microinjection of pronuclei
offers very low percentages of actual transgenic animals but the
applications for transgenic animals offers great promise (Wagner J,
et al., (1995) "Transgenic animals as models for human disease"
Clin Exp Hypertens 1995 May; 17(4):593-605; Woychik R P, and
Alagramam K, (1998) "Insertional mutagenesis in transgenic mice
generated by the pronuclear microinjection procedure" Int J Dev
Biol 42(7 Spec No):1009-17; Ebert K. M., (1998) "The use of
transgenic animals in biotechnology" Int J Dev Biol 1998; 42(7 Spec
No): 1003-8). The use of intracytoplasmic sperm injection (ICSI),
the placement of a sperm into the cytoplasm of an oocyte using a
microinjection pipette, can be found in both animal husbandry as
well as in human assisted reproduction (Joris H, et al. (1998)
"Intracytoplasmic sperm injection: laboratory set-up and injection
procedure" Hum Reprod 13 Suppl 1:76-86). Being a relatively new
procedure, not all human assisted reproduction clinics offer ICSI
as an option but studies have shown that it can offer significant
advantages to those couples suffering from male factor infertility
(Palermo G. D., et al. (1996) "Intracytoplasmic sperm injection: a
powerful tool to overcome fertilization failure" Fertil Steril
65(5):899-908).
[0012] Additionally, many assisted reproduction clinics have found
that the use of assisted hatching, the removal of a portion of the
glycoprotein coating to facilitate embryo escape from the
glycoprotein coat, offers the chance of a positive reproductive
outcome to those women who produce embryos with impaired zona
pellucidas (Meldrum D R, et al. (1998) "Assisted hatching reduces
the age-related decline in IVF outcome in women younger than age 43
without increasing miscarriage or monozygotic twinning." J Assist
Reprod Genet 15(7):418-21; Magli M C, et al. (1998) "Rescue of
implantation potential in embryos with poor prognosis by assisted
zona hatching" Hum Reprod 13(5):1331-5).
[0013] More recent developments in the animal husbandry field
report that somatic cell nuclei can be used as nuclear donors in
nuclear transfer (Campbell, K. H. S. et al. (1996) "Sheep cloned by
nuclear transfer from a cultured cell line" Nature 380, 64-66;
Heyman Y, et al. (1998) "Cloning in cattle: from embryo splitting
to somatic nuclear transfer." Reprod Nutr Dev 38(6):595-603; Loi P,
et al. (1998) "Embryo transfer and related technologies in sheep
reproduction." Reprod Nutr Dev 38(6):615-28). The technique of
nuclear transfer includes several demanding aspects, two of which
are the enucleation, or removal, of the genetic material from the
recipient oocyte and the deposition of a donor nucleus in the
enucleated oocyte.
[0014] Recent early stage research has shown that infertility for
some women can be ameliorated by the transfer of a small quantity
of cytoplasm taken from a donor oocyte from another woman,
presumably one without any cytoplasmic deficiencies (Lanzendorfise;
Mayer J F; Toner J, Oehningers, Saffan D S, Muashers (1999)
"Pregnancy following transfer of ooplasm from cryopreserved-thawed
donor oocytes into receipient oocytes" Fertility and Sterility
74(3):575-7).
[0015] The rigors of the physical manipulation of these cells
during the generation of transgenic animals, intracytoplasmic sperm
injection, assisted hatching, enucleation, nuclear transfers and
cytoplasmic transfer as well as the sheer enormity of the demand
that these procedures place on technical staff represents two of
the main reasons for failure. Thus, any improvements to these
procedures which result in higher rates of success as well as
increased capacity for processing is of great value.
SUMMARY OF THE INVENTION
[0016] The present invention provides for MEMS devices useful in
the labeling and manipulation of oocytes or embryos including: (1)
Cell Labeling MEMS Devices, (2) Labelable Zona Anchor MEMS Devices,
(3) Microinjection MEMS Devices; (4) ICSI MEMS Devices; (5) Zona
Coring MEMS Devices; (6) Enucleation MEMS Devices; (7)
Enucleation/Nuclear Transfer MEMS Devices; and (8) Cytoplasmic
Transfer MEMS Devices. The present invention also provides for kits
comprising the above devices.
[0017] The present invention also relates to devices or articles of
manufacture to be used for the manipulation and use of the MEMS
devices including centrifugal platters, Labelable Zona Anchor MEMS
device platforms, Labelable Zona Anchor MEMS device platform
holders; Automated Multi-Compartment, Multi-Modal Incubators;
Single Layer Culture MEMS Arrays, Multi-layer Culture MEMS Arrays;
Multi-layer Culture Array Environmental Controllers, and Automated
Environmental Instruments.
[0018] Also, the present invention provides for methods of using
the above-identified devices, arrays, controllers and
instruments.
[0019] Further, the present invention provides for methods of
making the above-identified MEMS devices and kits.
[0020] The present invention is based, at least in part, on the
novel application of microelectromechanical systems to the
modification, immobilization, translocation, and modulation of
cells or groups of cells such as culture cells, oocytes, embryos,
and sperm.
[0021] Whereas many cells in the body are of a size on the order of
a few microns, there is a special class of cells, the female gamete
called the oocyte, which is far larger, on the order of 100
microns. Further, these cells, in many animals from sea urchins to
mammals, are surrounded by a five to twenty micron thick
selectively permeable glycoprotein coat called the "Zona Pellucida"
or "Zona". The modification of the surface of the glycoprotein
coating of oocytes and embryos is a desirable operation in
endeavors such as the labeling of a great many of oocytes and
embryos in the animal husbandry industry.
[0022] Further, the ability to manipulate and confine oocytes and
embryos to a specific location for the culture before, during, and
after assisted reproduction procedures is necessary and quite time
consuming as well as requiring dedicated and highly trained
technical staff.
[0023] The rigors of the physical manipulation of these cells
during assisted reproduction procedures as well as the sheer
enormity of the demand that these procedures place on technical
staff represents two of the main reasons for failure. Any
improvements in terms of efficiencies to these procedures which
result in higher rates of success as well as increased capacity for
processing is of great value.
[0024] Methodologies for the tracking of oocytes and embryos during
isolation and manipulation for purposes such as animal husbandry,
embryo tagging, and tracking of manipulated or treated oocytes over
time and through space are rudimentary at present. Current
protocols utilize the segregation of oocytes and embryos into
individual wells or drops of culture medium entrapped under inert
oil layers. The viability of cultured oocytes and embryos is
enhanced by the co-culture of several oocytes or embryos within a
relatively small volume. A device which would facilitate the
identification of an individual oocyte or embryo distinctly from
other oocytes or embryos would allow the co-culture of many
differently treated or coded oocytes or embryos in one volume of
media. Further, the ability to permanently label a particular
oocyte or embryo in such a way as to allow coding of the treatment
of each oocyte or embryo into the label is desirable.
[0025] It is an object of the invention to provide MEMS devices,
kits, and methods of uses thereof, to enable rapid and easy
manipulation of cells or groups of cells including but not limited
to culture cells, oocytes, embryos, or sperm. More specifically,
the present invention allows for an automated method of
manipulating cells which does not rely heavily upon the ability of
the person performing the manipulation. Further, the present
invention allows for the manipulation of many cells
simultaneously.
[0026] More particularly, it is an object of the invention to
provide MEMS devices, and kits, for labeling cells or groups of
cells, in particular oocytes and embryos for easy identification
and ease of manipulation. It is another object of this invention to
provide MEMS devices, kits and methods of use for the following:
[0027] 1. microinjection of material such as a nucleus or cytoplasm
into a donor cell or group of cells; [0028] 2. intracytoplasmic
sperm injection to introduce sperm into an oocyte to facilitate
fertilization; [0029] 3. enucleation of a cell or group of cells to
create a recipient cell for nuclear transfer to enable "cloning";
[0030] 4. nuclear transfer to facilitate insertion of a nucleus
from a donor cell into a recipient cell to enable "cloning"; [0031]
5. cytoplasmic transfer for the transfer of cytoplasmic material
from one cell to another to reduce infertility of oocytes or
embryos; [0032] 6. zona coring for introducing holes or taking
cores of the zona pellucida to improve fertility; and [0033] 7.
cell culture of cells or groups of cells especially in the field of
in vitro fertility methods and implantation.
[0034] It is another object of the invention to provide methods of
making the MEMS devices and kits.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The various figures are schematic and not drawn to
scale.
[0036] FIG. 1 is a perspective view of a first silicon wafer to be
used in a Cell Labeling MEMS Device.
[0037] FIG. 2A is a perspective view of a base silicon wafer to be
used in a Cell Labeling MEMS Device showing the channels in the
wafer.
[0038] FIG. 2B is a perspective view of second silicon wafer
showing the regions etched/deposited or otherwise modified.
[0039] FIG. 2C is a side view and partial see-through view of a
Cell Labeling MEMS device showing the cell wells and the labelable
zona anchor MEMS device at the base of the well.
[0040] FIG. 2D is a perspective view and partial cross-sectional
view of the Cell Labeling MEMS device showing the cell wells and
labelable zona anchor MEMS device within.
[0041] FIG. 2E is a top view of a number of MEMS devices attached
to a centrifugal platter next to Cell Labeling MEMS devices with
oocytes or embryos loaded thereto.
[0042] FIG. 2F is a side cross-sectional view of a cell well of a
labelable zona anchor MEMS device showing a label being attached to
a cell.
[0043] FIG. 2G are schematic drawings of labeled cells with
Labelable Zona Anchor MEMS devices attached thereto.
[0044] FIG. 3A is a side edge view of a docking MEMS for docking
labeled cells having a labelable zona anchor MEMS device.
[0045] FIG. 3B is an end view of a docking MEMS device with cells
having labelable zona anchor MEMS devices attached to the platform
of the docking MEMS device.
[0046] FIG. 3C is a side edge view of a male docking MEMS device
showing cells with labelable zona anchor MEMS devices attached
thereto positioned in a platform channel.
[0047] FIG. 3D is an end view of a male docking MEMS device with
labeled cells attached in platform channels.
[0048] FIG. 4A is a side cross-sectional view of a MEMS platform
holder.
[0049] FIG. 4B is a side view of the plunger during transport of
the cells.
[0050] FIG. 4C is a side view of the plunger of the platform holder
during intrauterine deposit of oocytes/embryos.
[0051] FIG. 5A is a side cross-sectional view of the automated
multi-compartment multi-modal incubation device.
[0052] FIG. 5B is a side cross-sectional view of the platform
holding device holding a platform.
[0053] FIG. 5C is a side cross-sectional view of a platform holding
device holding a platform.
[0054] FIG. 5D is a cross sectional view of a compartment of an
automated multi-compartment multi-modal incubator device containing
a docking MEMS.
[0055] FIG. 6A is a top view of a single layer culture MEMS
array.
[0056] FIG. 6B is a close-up top view of main culture manifold
units of a multi-layer culture MEMS array useful for in vitro
maturation (IVM), in vitro fertilization (IVF) and in vitro culture
(IVC).
[0057] FIG. 7 is a cut-away view of a multi-layer culture array
environmental controller.
[0058] FIG. 8 is a perspective see-through view of an automated
environmental instrument.
[0059] FIG. 9 is a perspective view of a channel etched plate of a
microinjection MEMS device.
[0060] FIG. 10 is a perspective view of a mega-laminate of a
microinjection MEMS device where the plane of the slicing action is
indicated.
[0061] FIG. 11 are side views of a mega-laminate wafer being bonded
to a channel etched plate of a microinjection MEMS device.
[0062] FIG. 12 is perspective view of the first mask (hatched area)
of a microinjection MEMS.
[0063] FIG. 13 is a perspective view of the masks for the
fabrication (hatched area) of a microinjection MEMS.
[0064] FIG. 14 is a perspective view of microinjection MEMS with a
cross-sectional view to show interior detail of the cell wells.
[0065] FIG. 15 is a top schematic view of a channel etched base
plate with piezoelectric pump manifold array MEMS.
[0066] FIG. 16 is perspective view of a centrifugal platter for
receiving a MEMS device.
[0067] FIG. 17 is a top cross-sectional view of a microinjection
MEMS device.
[0068] FIG. 18 is a side cross-sectional view of the dynamic
hydropressure column of a microinjection MEMS device.
[0069] FIG. 21A is a perspective view of the first mask of a zona
coring MEMS device in the cell well.
[0070] FIG. 21B is a perspective view of a cell well of a zona
coring MEMS device showing the zona coring MEMS structure and
barbed penetrating member.
[0071] FIG. 19 is a cut-away view of an ICSI MEMS device.
[0072] FIG. 20A is a representation of a bimorphic sperm guillotine
gating mechanism.
[0073] FIG. 20B is a side cross-sectional view of the ISCI MEMS
channel and bimorph guillotine/gate.
[0074] FIGS. 22A-F are representations of a series of masks
(hatched area) for the manufacture of an enucleation MEMS
device.
[0075] FIG. 23A is a side and cross-sectional view and 23B is a top
view of an enucleation guillotine MEMS coupled to an enucleation
MEMS device.
[0076] FIG. 24A is a side view of an enucleation/nuclear transfer
MEMS device.
[0077] FIG. 24B is perspective view of an enucleation/nuclear
transfer MEMS device.
[0078] FIG. 25A is a top cut-away view of an enucleation MEMS
device unit.
[0079] FIG. 25B is a side cross sectional view of a cell well of an
encleation MEMS device.
[0080] FIG. 26A is a cross-sectional view of one unit of an array
of enucleation/nuclear transfer syphon.
[0081] FIG. 26B is a side cross-sectional view of a cell well of an
enucleation/nuclear transfer unit of a MEMS device.
[0082] FIG. 27 is a side cross-sectional view of a single unit of a
cytoplasmic transfer transfer MEMS device.
[0083] FIG. 28 is a top cross sectional view of a base pumping
substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention provides microelectromechanical
systems (MEMS) devices and kits for the manipulation of a cell or
groups of cells, including but not limited to, primary cells,
culture cells, oocytes, embryos or sperm. The cells can be from any
organism. In a preferred embodiment, the cells are from animals. In
a most preferred embodiment, the cells are from humans. The present
invention also provides for methods of using the devices and kits
for manipulation of a cell or group of cells. Further, the present
invention provides methods of making the MEMS devices described
herein.
[0085] The present invention provides cell labeling MEMS devices
and kits useful for labeling individual cells or groups of cells.
The cell labeling MEMS device enables one to label a cell or group
of cells, including but not limited to oocytes or embryos, with a
labelable zona anchor MEMS device. The labeled cells allow one to
easily identify the cells and facilitates further manipulations.
For example, the labeled cells allow for the tracking of oocytes to
prevent in vitro fertilization with the wrong sperm or to prevent
the implantation of the wrong embryos into a patient.
[0086] The labelable zona anchor MEMS devices are attached or
anchored to the cell or group of cells. For example, the labelable
zona anchor MEMS device can be attached or anchored to the zona
pellucida of oocytes or embryos.
[0087] In another aspect, the present invention provides for arrays
and incubation devices useful for cell culture and handling. In
particular, the present invention provides for automated
multi-compartment, multi-modal incubators, single layer and
multi-layer culture arrays, multi-layer culture array environmental
controllers, and environmental control instrument.
[0088] In yet another aspect, the present invention provides for
labelable zona anchor MEMS device platforms and labelable zona
anchor MEMS device platform holders useful for the manipulation and
implantation of labeled cells or groups of cells into an
animal.
[0089] The present invention also provides microinjection MEMS
devices and kits, methods of their use and methods of making the
devices and kits. The microinjection MEMS devices are useful for
the microinjection of material or nuclei into a cell or group of
cells.
[0090] The present invention provides for intracytoplasmic sperm
injection ("ICSI") MEMS devices and kits, methods of their use, and
methods of making the same. The ICSI MEMS devices are useful for
the injection of a sperm into an oocyte.
[0091] The present invention provides for zona coring MEMS devices
and kits, methods of their use, and methods of making the devices.
The zona coring MEMS devices are useful for the creation of "holes"
or "cores" in the zona pellucida of oocytes and embryos to improve
the ability of the embryo to escape the confines of the zona and
implant in the uterine lining.
[0092] The present invention provides for enucleation MEMS devices
and kits, methods of their use and methods of making the devices.
The enucleation MEMS devices are useful for removing the nucleus
from a recipient cell so that genetic material, a nucleus from
another cell, or a cell may be inserted. Additionally, the
enucleation devices are used to obtain genetic material or
nuclei.
[0093] The present invention provides for an enucleation/nuclear
transfer MEMS device and kits, methods of using, and methods of
making the device. The enucleation/nuclear transfer MEMS devices
are useful for performing enucleation of a recipient cell and the
subsequent transfer of a cell or nucleus into the recipient
cell.
[0094] The present invention provides for a cytoplasmic transfer
MEMS device and kits, methods of using same, and methods of making.
Cytoplasmic transfer MEMS devices are useful for the transfer of
cytoplasmic material from a donor oocyte or embryo into a host
oocyte or embryo.
[0095] Lastly, the present invention provides for cell culture MEMS
devices, kits, methods of using same, and methods of making same,
for culturing cells especially in conjunction with the other MEMS
devices and methods described herein.
[0096] Solely for ease of explanation, the description of the
invention is divided into the following sections: (A) Making MEMS;
(B) Cell Labeling MEMS; (C) Labelable Zona Anchor MEMS; (D)
Labelable Zona Anchor MEMS Device Platforms; (E) Labelable Zona
Anchor MEMS Device Platform Holders; (F) Automated
Multi-Compartment, Multi-Modal Incubator; (G) Single Layer Culture
Arrays; (H) Multi-Layer Culture Arrays; (I) Multi-Layer Culture
Array Environmental Controllers and Environmental Controlled
Instruments; (J) Microinjection MEMS; (K) ICSI MEMS; (L) Zona
Coring MEMS; (M) Enucleation MEMS; (N) Enucleation/Nuclear Transfer
MEMS; and (O) Cytoplasmic Transfer MEMS.
A. Making MEMS
[0097] The MEMS devices described herein can be manufactured using
a variety of methods known and used in the art. For example, MEMS
can be made using methods such as silicon bulk micromachining,
LIGA, silicon surface machining, deep silicon reactive ion etching,
dry etching, advanced deep reactive ion etching (ADRIE), or bulk
anisotropic silicon etching. Micromachining methods are described
in a number of references listed in the Background Section supra
and incorporated herein by reference. Also, methods of
micromachining are described in "Micromechanics and MEMS: classic
and seminal papers to 1990," ed. William F. Trimmer (1997) (IEEE
Press, New York), which is incorporated herein in its entirety.
[0098] Basically, MEMS devices are made by micromachining the
components of the device to build, for example, sensors,
micropumps, wells, micromotors, x-y stages and other "smart"
devices. Additionally, components can be deposited on the MEMS
substrate, such as, circuits, and controllers.
[0099] Devices ranging in size from a dozen millimeters to a dozen
microns can be manufactured by using silicon bulk micromachining.
This process uses either etches that stop on the crystallographic
planes of a silicon wafer or etches that act isotropically to
generate mechanical parts.
[0100] As used herein, the word "wafer" refers to a silicon disc
slice from a crystal on which structures are manufactured, and a
"wafer" is also called a "substrate" or "starting material." These
techniques combined with wafer bonding and boron diffusion allows
complex mechanical devices to be fabricated. The LIGA technology
makes miniature parts with spectacular accuracy. Electro Discharge
Machining, EDM, extends conventional machine shop technology to
make sub-millimeter sized parts.
[0101] As used herein, "LIGA" or "Lithographie, Galvanoformung,
Abformung" refers to the process by which polymethyl methacrylate
(PMMA) plastic is exposed to synchrotron radiation through a mask.
Exposed PMMA is then washed away, leaving vertical wall structures
with great accuracy. Structures a third of a millimeter high and
many millimeters on a side are accurate to a few tenths of a
micron. Metal is then plated into the structure, replacing the PMMA
that was Washed away. This metal piece can become the final part,
or can be used as an injection mold for parts made out of a variety
of plastics.
[0102] "Silicon surface micromachining" refers to the process by
which layers of sacrificial and structural material are deposited
on the surface of a silicon wafer. Further, as each layer is
deposited on the wafer, it is patterned, leaving material only
where the designer wishes. When the sacrificial material is
removed, completely formed and assembled mechanical devices
remain.
[0103] "Deep silicon reactive ion etching" or "Deep Si RIE" or
"DRIE" is an art-recognized term and refers to the process by which
highly anisotropic, randomly shaped and located features are
patterned and etched into a single crystal silicon wafer, with only
photoresist as an etch mask. As used herein, "mask" is an art known
term and includes the fabrication process whereby each layer of the
process is photographically transposed onto the wafer so that a
deposition can be accurately placed within selected areas of the
wafer. Deep Si RIE can be used to etch both shallow and deep
features into the front side and back side of a wafer, and can also
be used to etch completely through the wafer, to produce holes,
diaphragms, or suspended structures. Deep Si RIE can also produce
high aspect ratio features.
[0104] Dry etching technology is useful for fabricating
three-dimensional building blocks for MEMS applications. The
fabrication technique of these blocks demand etching processes with
high etch rate and selectivity, both for bulk- and surface
micromachining. Low ion energy prevents substrate damage to
electronics, mask erosion (the selectivity to metal masks is
practically infinite), and makes it easy to change the profile of
the trench.
[0105] Some silicon bulk micromachined devices require
backside-to-frontside photolithographic alignment fabrication
processes. This process is typically used to align cavities etched
from the backside of the wafer to structures located on the front
side of the wafer. Substrates are rendered ultra-flat prior to
bonding. After bonding, chemical/mechanical grinding and polishing,
chemical etching, and plasma assisted chemical etching are used, as
appropriate, for thinning to final dimension.
[0106] The application of ultraviolet sources in photo-assisted
processing affords the ability to use the chemical effects induced
by high energy photons, as opposed to the thermal effects of high
intensity beams, for high specification lithography and
microfabrication processes with minimal damage. The ability of the
deep ultraviolet 193 nm wavelength of the ArF excimer laser to
ablate glass without damage due to thermal stresses below the
surface of the material is well known.
B. Cell Labeling MEMS
[0107] The present invention encompasses cell labeling MEMS devices
comprising a substrate (i.e., silicon wafer, plastic, metal oxides)
which have been manufactured to comprise at least one well or a
plurality of wells for receiving at least one cell or group of
cells, such as but not limited to, an oocyte or embryo, and a
labelable zona anchor MEMS device. The cell labeling MEMS devices
provides a device for attaching or anchoring labelable zona anchor
MEMS devices (labels) into cells or groups of cells.
[0108] In one embodiment, the cell labeling MEMS device
comprises:
[0109] (a) a first composite silicon wafer comprising: [0110] a
plurality of channels along the length of the wafer; and
[0111] (b) a second silicon wafer bonded to the first composite
silicon wafer comprising a plurality of wells wherein the wells
comprise a labelable zona anchor MEMS device. The channels provide
for a non-bonded or empty space behind the labelable zona anchor
MEMS devices to allow for easy detachment from the device so they
will anchor into the cell or group of cells to be labeled.
[0112] In a specific embodiment, the cell labeling MEMS devices of
the present invention comprises a labelable zona anchor MEMS device
attached to the well by a break-away means. The break-away means
includes any material that is continuous with the label and the
rest of the MEMS device that will fail from mechanical stress as
cell cell moves out of the MEMS device cell well, allowing the
label to remain embedded in the zona.
[0113] In other specific embodiments, the channels of the cell
labeling MEMS devices are from about 2 to about 5 microns deep
(more particularly, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, or
about 5 .mu.m) and range from about 90 to about 150 microns long
(more particularly, about 90 .mu.m, about 100 .mu.m, about 110
.mu.m, about 120 .mu.m, or about 150 .mu.M).
[0114] In yet another specific embodiment, the cell labeling MEMS
device comprises channels separated by from about 5 to about 50
microns (i.e., 5 .mu.m, 10 .mu.m, 15 .mu.m, 20 .mu.m, 50
.mu.m).
[0115] In a more specific embodiment, the wells of the cell
labeling MEMS devices are to from about 50 to about 150 microns
wide (more particularly about 50 .mu.m, about 75 .mu.m, about 90
.mu.m, about 100 .mu.m, about 120 .mu.m, or about 150 .mu.m).
[0116] In yet another specific embodiment, the cell labeling MEMS
device further comprises an incomplete circle inscribed about the
labelable zona anchor MEMS device ranging from about 0.5 to about 5
microns in width (more specifically, about 0.5 .mu.m, about 1
.mu.m, about 1.5 .mu.m, about 2 .mu.m, or about 5 .mu.m).
[0117] The present invention also pertains to a method of making a
cell labeling MEMS device comprising at least one labelable zona
anchor MEMS device per well.
[0118] In a preferred embodiment, the method of making a cell
labeling MEMS device is a method wherein a wafer base or substrate
is modified in a way such that a plurality of cell wells are formed
that contain a structure within them that consists of a barb or a
plurality of barbs attached to a planar element. In another
embodiment, said planar element is located within said plurality of
cell walls and attached to a barb or a plurality of barbs, is not
permanently attached to the structure of said cell wells. In
another embodiment, a means is provided within said cell wells for
a controlled burst of gas or fluid to be produced thus facilitating
the evacuation of cells resident in said cell wells.
[0119] The wafer base substrate of the all labeling device can be
made of a variety of materials known in the art.
[0120] In particular preferred embodiments, the wafer or base
substrate of the cell labeling MEMS device is composed silicon,
sapphire, polymer, metallic compounds, or a multi-laminate
material.
[0121] Referring to FIG. 1, there is shown a wafer or base
substrate 1 of a cell labeling MEMS device. Channels 2 of uniform
dimensions and varying from about 0.01 .mu.m to about 5 .mu.m in
depth and varying from about 1 .mu.m to about 200 .mu.m in width
are etched across the length of said base wafer 1. Further, in
referring to FIG. 1, a layer of sacrificial material 3 is deposited
in the channels 2. Sacrificial materials are known in the art and
can be, for example, but not limited to silicon dioxide, deposited
oxide, photoresist, amorphous silicon, polysilicon, or
aluminum.
[0122] Referring to FIGS. 2A-E, there is shown in FIG. 2-A a first
silicon wafer or base substrate 1 (same as in FIG. 1), FIG. 2B
shows a second wafer 11 regions 15 etched/deposited or otherwise
modified (e.g., polymerized), FIG. 2C shows a slice of a cell
labeling MEMS device; FIG. 2D shows a first wafer 1 fused to a
second silicon wafer 11 forming mega-laminate 16; and cell labeling
MEMS device 7 mounted on a centrifugal platter 8. The second
silicon wafer 11, being modified on one surface with etching or
deposition of materials or other modifications, is bonded, (i.e.,
silicon fusion bonded) to the first silicon wafer 1 such that the
modified second silicon wafer 11 top surface 4 is in contact with
the sacrificial material 3 in the channels in the base wafer 1.
This bonding forms a boundary, (i.e., a silicon fusion bonding
interface) 12. After adhesion of the first silicon wafer 1 and the
second silicon wafer 4, wells 5 are etched to an intermediate depth
whereupon masks and etches create a labelable zona anchor MEMS
device 6 within the wells 5. Alternatively the labelable zona
anchor MEMS devices can be made separately and deposited in the
wells. The multi-welled mega-laminate structure so formed is cut
such that a single well or row of wells 7 occurs. This single row
unit 7 is mounted onto a centrifugal platter 8 such that the
opening of the wells 5 face the center of the centrifugal platter
8. As shown in FIG. 2E, upon rotation 9 of the centrifugal platter
8 a centripetal force 10 is exerted perpendicularly from the center
of the centrifugal platter 8 outward. This force 10 exerts
perpendicularly against the far wall 13 of the wells 5. As shown in
FIG. 2F, cells 14 present near the wells 5, upon rotation 9 of the
centrifugal platter 8, are thrust into the well 5 onto the far wall
13 such that the labelable zona anchor MEMS device 6 penetrates the
cell 14 or group of cells.
[0123] In one embodiment, a plurality of cell labeling MEMS devices
are permanently attached to a centrifugal platter for providing
force along the long axis of the wells of the cell labeling MEMS
devices.
[0124] As used herein, the term "centrifugal platter" refers to a
structure which is mainly planar and which has a securing means for
securely attaching to a driver means.
[0125] A centrifugal platter may be composed of a material that is
sufficiently rigid that it will support the affixing of a MEMS
device of the present invention, is non-corrosive, is non-toxic to
cells (e.g., culture cells, oocytes, embryos) and can be sterilized
(e.g., gamma irradiation, autoclaving). Such materials may be
silicon and plastic. A centrifugal platter may also be so
constructed such that there is no material at it's center of
rotation (e.g., analogous to an optical media disk commonly known
as a compact disk).
[0126] A securing means would be an element resident on a spinner
apparatus that momentarily (e.g., not permanently) attaches to the
centrifugal platter (e.g., gripping the inner edge of the center
opening or on the outer edges of the disk) so that the centrifugal
platter is held firmly to the rotating member of the spinner.
[0127] In one embodiment, a centrifugal platter for applying a
centripetal force to a cell or group of cells contained within a
MEMS device comprises a circular disk having a plurality of ports
for holding the MEMS device.
[0128] In another embodiment, the cell labeling MEMS devices are
permanently attached to a centrifugal platter for providing force
along the long axis of the wells present on the wafer or base
substrate and wherein said operating means comprises a centrifugal
platter that is attachable to a driver means. The term "driver
means" or "spinner" includes a platform that serves to securely
hold a centrifugal platter of the present invention and that is
operably attached to an instrument that provides for the rotation
of the driver means (i.e., a centrifuge, a rotating sputterer
instrument as used in semi conductor fabrication).
[0129] In another embodiment, a driver means exerts a force on the
centrifugal platter such that the centrifugal platter revolves and
thereby providing an outward centripetal force to the attached cell
labeling MEMS device and/or the labelable zona anchor MEMS devices.
In yet another embodiment, a centrifugal platter comprises a
plurality of depressions in direct contact with the cell labeling
anchor MEMS devices. In particular, a centrifugal platter comprises
one or more depressions or wells in the immediate vicinity of one
or more affixed MEMS devices of the present invention wherein each
depression or well is in fluid communication with exactly one
affixed MEMS device. These depressions or wells provide for the
placement of only on oocyte or embryo next to each MEMS device.
Further, these depressions or wells restrict the movement of the
oocytes or embryos from one MEMS device to another, avoiding
multiple manipulations to a single oocyte or embryo.
[0130] In a more specific embodiment, the centrifugal platter
comprises a conductive material which serves as a circuit between
the cell labeling MEMS devices and the driver means. The conductive
material of the centrifugal platter enables the rotation of the
platter by the driver means to be controlled. In particular, the
conductive materials (e.g., circuit lead or a strip of conductive
material resident on the centrifugal platter and in communication
with the circuit leads resident on the MEMS devices) are in contact
with a portion of the spinner (driving means) that provides data
transmission and current, thus providing this data transmission and
current, by way of the circuit leads, to the MEMS devices.
[0131] In the present invention, upon application of centripetal
force to the cell labeling MEMS devices, the labelable zona anchor
MEMS device contacts and anchors into the cells or groups of cells.
In a specific embodiment, the labelable zona anchor MEMS device
anchors into and modifies the surface of the zona pellucida
surrounding oocytes and embryos.
[0132] In the preferred embodiment, upon application of centripetal
force, the labelable zona anchor MEMS device penetrates the zona
pellucida but does not pass through the zona pellucida.
[0133] The present invention also encompasses a cell labeling MEMS
device kit comprising:
[0134] a) a centrifugal platter for applying a centripetal force to
a cell or group of cells contained within a MEMS device wherein the
centrifugal platter comprises a circular disk, a plurality of ports
for affixing the MEMS devices, and a securing means for securing
the centrifugal disk to a spinner or driving means; and
[0135] b) at least one cell labeling MEMS device.
[0136] In another preferred embodiment, a cell labeling MEMS kit
for applying a labelable zona anchor MEMS device to a cell or group
of cells comprising: [0137] (a) a centrifugal platter having an
outer edge and a plurality of grooves, the grooves having an inner
and outer surface, arranged in a concentric pattern on the surface
of the centrifugal platter; and [0138] (b) at least one cell
labeling MEMS device of claim 1 or 19; wherein the cell labeling
MEMS device is attached to the outer edge of the compact
centrifugal platter in an orientation such that the long axis of
each of the wells of the cell labeling MEMS device is horizontal to
plane of the centrifugal platter and the inner surface of the
grooves forming divided chambers, the chamber containing a single
well, which restrict the movement of materials from chamber to
another such chamber.
[0139] The present invention also provide for a method of using a
cell labeling MEMS kit above comprising: [0140] (a) filling the
grooves of the centrifugal platter with a fluid; [0141] (b) loading
the fluid within the grooves of the centrifugal platter with at
least one cell or group of cells; and [0142] (c) applying
centripetal forces to the centrifugal platter by rotation such that
the cell or group of cells is thrust against the wall of well such
that the embedding means of the labelable zona anchor MEMS device
penetrates the surface of the cell.
[0143] In a preferred embodiment, the cell labeling MEMS kit
comprising:
[0144] (a) at least one cell labeling MEMS device; and
[0145] (b) a centrifugal platter having an outer edge and a
plurality of grooves, the grooves having an inner and outer
surface, arranged in a concentric pattern on the surface of the
centrifugal platter; wherein the cell labeling MEMS device is
attached to the outer edge of the centrifugal platter in an
orientation such that the long axis of each of the wells of the
cell labeling MEMS device is horizontal to plane of the centrifugal
platter and the inner surface of the grooves forming divided
chambers, the chamber containing a single well, which restrict the
movement of materials from chamber to another such chamber.
[0146] The present invention further encompasses a method of using
a cell labeling MEMS kit comprising:
[0147] (a) filling the grooves of the centrifugal platter with a
fluid;
[0148] (b) loading the fluid within the grooves of the centrifugal
platter with at least one cell wherein the cell has a zona
pellucida; and
[0149] (c) rotating the kit using a driver means such that
centripetal forces are applied to the centrifugal platter such that
the cell is thrust against the wall of the well such that the
embedding means of the labelable zona anchor MEMS device penetrates
the surface of the zona pellucida of the cell.
[0150] In a more specific embodiment, the cell labeling MEMS device
is permanently affixed to a centrifugal platter.
[0151] The present invention further provides for methods of using
the cell labeling MEMS devices and kits wherein a plurality of cell
labeling MEMS devices have been attached temporarily or permanently
to a centrifugal platter forming a cell labeling MEMS device kit,
and comprising the steps of securing the centrifugal platter to a
driving means and further whereby liquid is placed in the
depressions present in said centrifugal platter directly next to
said labelable zona anchor MEMS devices such that said liquid is in
contact with the wells of the labelable zona anchor MEMS devices
and further cells (e.g., oocytes, embryos) are placed in said
liquid and subsequently said driver means provides a centripetal
force to said centrifugal platter, cell labeling MEMS devices, and
cells such that the liquid and cells migrate towards the outer
margin of the centrifugal platter and, as such, into the wells of
the cell labeling MEMS devices. In a specific embodiment, the
anchor or anchors is a barb or a plurality of barbs.
[0152] In another embodiment, the zona pellucidas of the cells,
upon migration into the cell labeling MEMS devices, are penetrated
by the anchor or anchors of the labelable zona anchor MEMS devices
such that the anchor or anchors of the labelable zona anchor MEMS
devices are embedded in the zona pellucidae of said cells.
[0153] Upon completion of using the cell labeling MEMS device, the
labelable zona anchor MEMS devices remain embedded in the zona
pellucidae of said cells upon the cessation of the centripetal
force as applied by the driver means.
[0154] In another embodiment, the kit further comprises a release
means by which a controlled burst of gas or fluid is released
within the wells of the cell labeling MEMS devices such that the
cells with labelable zona anchor MEMS devices embedded within the
zona pellucidae are ejected from cell labeling MEMS devices. The
controlled burst of gas or fluid can be provided through a separate
microfluidics channel in the kit and can be actuated by a pump to a
fluid or gas into the well in order to assist in the release of the
cell or group of cells from the well after the centripetal force
has been applied.
C. Labelable Zona Anchor MEMS
[0155] The present invention also provides labelable zona anchor
MEMS devices, and kits, methods of using same, and methods of
making same.
[0156] In one embodiment, a labelable zona anchor MEMS device
comprises at least one anchor and a labelable surface. As used
herein, the term "labelable zona anchor MEMS device" refers to a
micromechanical device which is so constructed as to provide at
least one anchor which attaches to or anchors in the zona pellucida
of a cell or group of cells, such as an oocyte or embryo and a
labelable surface.
[0157] As used herein, the term "zona pellucida" refers to the
glycoprotein matrix which encases the oocyte and embryo of a wide
range of animal species.
[0158] In particular embodiments, the anchor or anchors of the
labelable zona anchor MEMS are from about 5 to about 15 .mu.m tall.
In more specific embodiments, the anchors are about 5 .mu.m tall,
about 10 .mu.m tall or about 15 .mu.m tall.
[0159] In another embodiment, the labelable surface of the
labelable zona anchor MEMS device is planar and attached to the
anchors. In a preferred embodiment, the labelable surface is any
geometric shape including but not limited to a circle, square,
rectangle, polygon, etc. and can be from about 0.5 to about 30
.mu.m in diameter. More particularly, the labelable surface is
about 0.5, about 1, about 5, about 10, about 20 or about 30 .mu.m
in diameter.
[0160] In the present invention, the labelable zona anchor MEMS
device further comprises on the labelable surface, which is not
directly opposed to the zona pellucida, a label. In yet another
embodiment, the labelable surface further comprises a distinctive
modification that serves as a label. A label can be any item that
serves to identify one labeled cell or group of cells from another.
In a preferred embodiment, there is at least one label on the
labelable surface.
[0161] In one embodiment, the distinctive modification or label on
the labelable surface of the labelable zona anchor MEMS device
comprises a plurality of etched grooves forming a unique etched
grooved pattern, by which a code may be assigned to each unique
etched grooved pattern.
[0162] In another embodiment, the label comprises a plurality of
deposited grooves, forming a unique deposited pattern, by which a
code may be assigned to each unique deposited grooved pattern.
[0163] In yet another embodiment, the label comprises comprised of
a circuit. In a more specific embodiment, the circuit facilitates
the storage of information (i.e., physical parameters, changes in
physical parameters over time, movement through time, movement
through space, origin of cell, ownership of material, certification
of status of the cell or group of cells). In another embodiment,
the circuit functions as a transponder.
[0164] In another embodiment, the surface of the labelable zona
anchor MEMS device comprises of a magnetically-attractive surface
including, but not limited to, a metallic coating.
[0165] In another embodiment, the label comprises a fluorescent
material or fluorophore.
[0166] For example, the fluorescent material includes but is not
limited to rhodamine, fluorescein, Cy3, Cy5, or other such
fluorophores known to those in the art. Examples of such
fluorescent materials or fluorophores include, but are not limited
to, fluorescein, BODIPY.RTM., TRITC, Lissamine.TM., rhodamine,
Texas Red.RTM., Cy-3.18.TM., Cy-5.18.TM., Lucifer Yellow, Lucifer
Yellow, Ethidium bromide, Propidium iodide, Di-I, Calcium
Green.TM., Calcium Orange.TM., Calcium Crimson.TM., SNARF.RTM.-1,
AND SNAFL.RTM.-1. Additional examples of fluorescent materials are
Dabcyl, Cy2 Green, Fluorescein (FITC) Green, FAM
(Carboxyfluorescein) Green, TET (Tetrachlorofluorescein) Orange,
HEX (Hexachlorofluorescein) Pink, TAMRA (Carboxytetramethyl
rhodamine) Rose, Cy3.5 Scarlet, ROX (carboxy-x-rhodamine) Red,
Malachite Green, Far Red, Near-IR (max. abs. 675 or 743), Fluor X
Green, AMCA-S, Cascade Blue, BODIPY FL, CODIPY 530/550, BODIPY
493/503, BODIPY 558/569, BODIPY 654/570, BODIPY 576/589, BODIPY
581/591, BODIPY FL X, BODIPY R6Gx, BODIPY 630/650 X, Marine Blue,
Pregon Green 500 Green, Oregon Green 514 green, Oregon Green 488
green and Pacific Blue.
[0167] In another embodiment, the labelable zona anchor MEMS device
comprises at least two labels in any combination of an etched bar
code, a deposited bar code, an integrated circuit, a magnetically
attractive substance, and a fluorescent marker.
[0168] FIGS. 2G-2K show several embodiments of labelable zona
anchor MEMS devices. Labelable zona anchor MEMS can be variably
labeled, for example, but not limited to, a universal product code
deposited or etched on it's labelable surface 2G, a logo deposited
or etched on it's labelable surface 2H, a circuit 21 deposited or
etched on it's surface, a surface coating 2J applied to it's
labelable surface. An oocyte or embryo with a labelable zona anchor
MEMS anchored in it's zona is also shown 2K.
[0169] FIGS. 2L-2O show how labelable zona anchor MEMS devices can
have a variety of anchors including but not limited to, one barbed
protuberance, two or more barbed protuberances, a serrated blade, a
non-circular labelable zona anchor MEMS with one or more barbed
protuberances.
[0170] The labelable zona anchor MEMS devices are made using
standard manufacturing methods known in the art of MEMS. Methods
used in making MEMS devices are described above in Section A. In
one embodiment, the labelable zona anchor MEMS device is
manufactured at the same time as the cell labeling MEMS device is
produced. Alternatively, labelable zona anchor MEMS devices can be
made independently and inserted into the wells of a cell labeling
MEMS device, or some other cell handling means. For example,
another cell handling means is an enclosed channel comprising
labelable zona anchor MEMS devices attached to the walls of the
enclosed channel, whereby cells or groups of cells are labeled by
passing through the enclosed channel and coming into contact with
the labelable zona anchor MEMS devices.
[0171] In one embodiment, the labelable zona anchor MEMS devices
are made during the same process of making the cell labeling MEMS
devices as described above in section B. In another embodiment, the
labelable zona anchor MEMS device is made independently and
deposited into the well of a cell labeling MEMS device. Methods of
making MEMS devices are discussed in Section A supra.
[0172] In one embodiment, a method of making a labelable zona
anchor MEMS device comprises the steps of: [0173] 1. orienting a
substrate wherein the substrate has a bottom surface and a top
labelable surface; [0174] 2. applying a mask to the bottom surface
of the substrate; [0175] 3. etching the mask to form at least one
anchor; and [0176] 4. applying a label to the top labelable surface
of the substrate.
[0177] In another embodiment, the method of making a labelable zona
anchor MEMS device further comprises the fusion of a channel-etched
plate to the labelable surface.
D. Labelable Zona Anchor MEMS Device Platform
[0178] The present invention encompasses labelable zona anchor MEMS
device platforms for the holding and transporting of labeled cells
or groups of cells, such as oocytes or embryos.
[0179] As used herein, the term "labelable zona anchor MEMS device
platform" (also referred to herein as a "platform") refers to a
structure which is so constructed as to provide support and a
variably attractive attachment substrate for a labelable zona
anchor MEMS device. For example, the platform can be instructed or
induced to cease attraction to the labelable zona anchor MEMS
devices, facilitating the removal of labeled cells from the
platform. Basically, this means that the platform is an inducible
electromagnet. When you want the cells to stick you apply current
to the platform, to remove you shut off the current.
[0180] In one embodiment, the labelable zona anchor MEMS device
platform comprises at least one attaching element for attaching to
a plurality of labelable zona anchor MEMS devices.
[0181] In particular, the attaching element may be a strip of
magnetized material (e.g., a metallic strip) that lies along the
length of the platform that is an inducible magnet. In one
embodiment, that attaching element (e.g., a metallic strip), if
corrosive, is coated with a non-insulating non-corrosive material
(e.g., a plastic coating). Also, the entire platform may have
embedded within it a electromagnetic coil.
[0182] The platforms can be made from a variety of materials known
and available to those in the art. In a specific embodiment, the
labelable zona anchor MEMS device platform is composed of rigid
material. In another specific embodiment, the labelable zona anchor
MEMS device platform is composed of a non-corrosive material. In
yet another specific embodiment, the labelable zona anchor MEMS
device platform is composed of a material which is opaque to
ultrasonographic detection to mediate localization internal to a
recipient uterus during transfer of oocytes and/or embryos.
[0183] In a preferred embodiment, the labelable zona anchor MEMS
device platform is a cylindrical or a rectangular object that can
be made of plastic, metal or other non-corrosive, ultrasound-opaque
material. The platform is preferably about 1 mm to about 20 mm in
length and about 1 mm to about 10 mm wide. In more particular
embodiments, the length of the platform is about 1 mm, about 5 mm,
about 10 mm, about 15 mm or about 20 mm in length. In other
particular embodiments, the platform is about 1 mm, about 5 mm or
about 10 mm in width.
[0184] In a particular embodiment, the labelable zona anchor MEMS
device platform comprises a docking domain on each end which
facilitates the attachment of the platform to a external movable
plunger. More particularly, an "external plunger" may include a
structural element that forms one end of the platform holder that
holds a platform internal to the container and that can be pushed
further into the container thus pushing the docked platform
partially out of the container. This functionality facilitates the
extension of the platform out of the container (e.g., while the
holder is inside the uterus) allowing the cells, when released from
magnetic attraction to the platform, to float free into the
environment surrounding the platform.
[0185] In other embodiments, the platform has two ends that are
tapered for docking into a labelable zona anchor MEMS device
platform holding device. In preferred embodiments, the docking
domains protrude outwards or are "male" docking domains. In another
preferred embodiment, the docking domains recede inwards or are
"female" docking domains.
[0186] FIGS. 3A-3D, show a labeled cell and two different
embodiments of a labelable zona anchor MEMS platform. In FIG. 3A, a
labeled cell 19, being embedded with a labelable zona anchor MEMS
6, is selectively attracted to a labelable zona anchor MEMS
platform 16 (shown here with multiple labeled cells). FIG. 3A shows
the side view of a platform 16 having a male docking domain on each
end. FIG. 3B shows the end view of a platform 16 illustrating the
manner that a labeled cell 19 sits on platform 16. FIG. 3C edge
view of platform 16 illustrating the manner that labeled cells 19
sit in the platform channel 18. FIG. 3C shows the edge view of
platform 16 illustrating that each end of platform 16 has a male
docking domain 17. FIG. 3D shows the end view of a platform 16
illustrating the manner that the labeled cells 19 sit in the
platform channel 17.
[0187] In another preferred specific embodiment, the labeled cells
or groups of cells are reversibly attached to the platform so that
they may be released for further manipulation or for implantation
into an animal.
[0188] The labelable zona anchor MEMS device platform is used by
attaching labeled cells or groups of cells, such as oocytes or
embryos.
[0189] The platform is made attractive by the induction of
magnetism in the selectively magnetic securing means. This
induction can be mediated by providing an electrical current (e.g.,
extremely low current) to the securing element. The securing
element may include an electromagnetic coil.
[0190] The platform is then inserted into a labelable zona anchor
MEMS device platform holder which allows for transporting the
labeled cells without contamination. Then, either the cap is placed
onto the open end of the platform for long term storage. The
platform can be docked in a compartment of an automated
multi-compartment multi-modal incubator (described in Section F
infra) using the plunger to facilitate the engagement of the male
or female docking domain at the platform with the reciprocal female
or male docking domain of the incubator.
[0191] In particular, the plunger, being attached to the platform,
pushes the platform out of the container and pushes the male or
female docking domain into the reciprocal docking domain resident
in the base of the compartment.
[0192] In yet another preferred embodiment, a labelable zona anchor
MEMS device platform for holding cells or groups of cells labeled
with labelable zona anchor MEMS devices comprises a supporting
platform wherein the platform comprises a structural attaching
element to which a plurality of labelable zona anchor MEMS devices
attached to cells may be attached.
E. Labelable Zona Anchor MEMS Device Platform Holder
[0193] The present invention encompasses a labelable zona anchor
MEMS device platform holder for transporting the labeled cells or
groups of cells that are attached to the platform.
[0194] As used herein, the term "labelable zona anchor MEMS device
platform holder" (also referred to herein as a "holder") refers to
a structure that provides support for a labelable zona anchor MEMS
device platform so that the platform can be transported and that
such transport may include the co-transport of liquid surrounding
the labelable zona anchor MEMS device platform and the labeled
oocytes or embryos.
[0195] In a preferred embodiment, a labelable zona anchor MEMS
device platform holder for holding a labelable zona anchor MEMS
device platform comprises: [0196] a) an inner cylinder comprising a
securing means for securing a labelable zona anchor MEMS device
platform to the holder; [0197] b) a retractable outer cylinder for
containing the inner cylinder; and [0198] c) a plunger mechanism
for moving the inner cylinder and platform.
[0199] In one embodiment, the labelable zona anchor MEMS device
platform holder comprises a docking domain for securely attaching a
labelable zona anchor MEMS device platform. In preferred
embodiments, the docking domains are a male or female docking
domain.
[0200] In another embodiment, the labelable zona anchor MEMS device
platform holder further comprises a container for containing a
volume of liquid surrounding said labelable zona anchor MEMS device
platform.
[0201] In one specific embodiment, the labelable zona anchor MEMS
device platform holder further comprises a material which is
disposable. In another specific embodiment, the holder comprises a
material which is a polymer or a plastic. In yet another specific
embodiment, the holder comprises a material that can be
sterilized.
[0202] In another embodiment, the labelable zona anchor MEMS device
platform holder further comprises a plunger by which the entire
holder is manipulated either by hand or robotic device.
[0203] FIGS. 4A-4C show a labelable zona anchor MEMS platform
holder in a partial side cross-sectional view, a side
cross-sectional view, and schematic drawing of the device in-use,
respectively. The labelable zona anchor MEMS platform holder 20 has
an inner cylinder 21 with a female platform docking domain 22 that
is attached to a plunger mechanism 23. This assembly is contained
within a retractable outer cylinder or container 24. As seen in
FIG. 4B, the labelable zona anchor MEMS platform holder has a cap
25 and is used during transporting in order to retain a fluid or
culture media around a mounted platform within it. FIG. 4C,
illustrates how the labelable zona anchor MEMS platform holder 20
is inserted into the vaginal vault 26, through the cervix 27, into
the interior of the uterus 28, where the reversibly attached
oocytes or embryos are released.
[0204] FIGS. 5B and C show preferred embodiments of the platform
and holder. FIG. 5B shows the labelable zona anchor MEMS platform
holder during transport and has a female docking domain 22 resident
on plunger 23 that receives and docks with the male docking domain
17 of the platform 16 which has a plurality of labeled cells
attached thereto. FIG. 5C shows a platform 16 with labeled cells 19
within a compartment 29, its second male docking domain 17 being
docked with the female docking domain 22 of the incubator
compartment 29, wherein the compartment 29 has an input port 30 and
an output port 31.
F. Automated Multi-Compartment, Multi-Modal Incubator
[0205] The present invention also pertains to an automated
multi-compartment, multi-modal incubator. As used herein, the term
"automated multi-compartment, multi-modal incubator" (also referred
to herein as an "incubator") refers to a device that regulates and
modulates the environment within a plurality of compartments which
may contain a labelable zona anchor MEMS device platform which can
contain at least one or a plurality of labeled cells or groups of
cells such as oocytes and/or embryos that are attached to the
labelable zona anchor MEMS device platform by way of zona-embedded
labelable zona anchor MEMS devices.
[0206] In one embodiment, the automated multi-compartment,
multi-modal incubator comprises a block comprising a plurality of
compartments and a controlling means for regulating the environment
within said compartments. In another embodiment, the automated
multi-compartment, multi-modal incubator further comprises an
incubator docking domain for attaching a labelable zona anchor MEMS
device within each compartment. In specific embodiments, the
incubator docking domains can be male or female docking
domains.
[0207] In another specific embodiment, the controlling means of the
automated multi-compartment, multi-modal incubator regulates
conditions such as, but not limited to, compartment temperature,
pH, the flow rate of input fluids or the flow rate of compartment
output fluids. In more specific embodiments, the input or output
fluids are culture media, a cell suspension, or a sperm
suspension.
[0208] FIG. 5A shows an automated multi-compartment, multi-modal
incubator 33, containing a labelable zona anchor MEMS platform
holder during transport, which has a block 34 comprising one or
more compartments 25. Each compartment 25 has an input port 36 and
an output port 37, a female docking domain 38, and a cap 39 with
input 40 and output ports 41.
[0209] FIG. 5C shows a multi-modal incubator 33 and a
cross-sectional view of a labelable zona anchor MEMS platform
holder 16 within an automated multi-compartment, multi-modal
incubator 33.
[0210] FIG. 5D shows a side cross-sectional view of a compartment
29 with a cap 32 showing a platform 16 docked and the compartment
sealed with the cap 32.
[0211] In one embodiment, an automated multi-compartment,
multi-modal incubator comprises: [0212] (a) a block comprising a
plurality of compartments; [0213] (b) reagent reservoirs for
containing a fluid reagent; [0214] (c) fluid handling means
communicating between compartments and reagent reservoirs; [0215]
(d) at least one environmental controlling means for regulating the
environment within said compartments; [0216] (e) a reagent input
controlling means for regulating input of the fluid reagent from
the reagent reservoirs into the compartments; and [0217] (f) a
fluid output controlling means for regulating output of fluids from
compartments.
[0218] In yet another embodiment, an automated multi-compartment,
multi-modal incubator for incubating cells or groups of cells that
are attached to a labelable zona anchor MEMS device platform
comprises: [0219] 1. a block comprising a plurality of compartments
wherein each compartment receives a labelable zona anchor MEMS
device platform [0220] 2. a plurality of reagent reservoirs for
containing a fluid reagent; [0221] 3. a fluid handling means
communicating between the compartments and the reagent reservoirs;
[0222] 4. at least one environmental controlling means for
regulating the environment within said compartments; [0223] 5. a
reagent input controlling means for regulating input of the fluid
reagent from the reagent reservoirs into the compartments; and
[0224] 6. a fluid output controlling means for regulating output of
fluids from compartments.
[0225] The automated multi-compartment, multi-modal incubator is
used as follows: the incubator is filled with appropriate fluids
(i.e. culture media) and the incubator controller CPU (Central
Processing Unit) is coded with desirable environmental parameters
(i.e., temperature, pH, flow rate of fluids, in and out of
compartments) and the system is allowed to reach the desired
environmental parameters. At this point platforms (with labeled
cells) are introduced into the incubator compartments by way of the
platform holders described above. Once platforms are docked within
the compartments, the compartments are sealed with caps. The
incubator controller CPU is provided with desirable culture
conditions and rates of change in those conditions over time. The
incubator of the present invention allows for the culture and
manipulation of labeled cells.
G. Single Layer Culture MEMS Array
[0226] The present invention also encompasses single layer culture
MEMS arrays for culturing cells or groups of cells.
[0227] As used herein, the term "single layer culture MEMS arrays"
(also referred to herein as "single layer arrays") refers to a
layer wherein materials within the single layer array moves in an
x-y axis. Such single layer arrays are constructed to allow the
communication of culture materials between a plurality of single
layer arrays such that materials move in an x-y-z axis. Further,
the single layer arrays comprise a substrate or wafer (e.g., made
of plastic, silicon, ceramic) on which is resident one or more
enclosed channels that, in turn, each contains one or more movement
tracks.
[0228] A single layer culture array of the present invention
further comprises one or more collecting domains, one or more
router elements resident on one or more movement tracks, and one or
more main culture compartments. In another embodiment, the main
culture compartment of the single layer culture array of the
present invention comprises one or more movement tracks, and one or
more router elements being resident on one or more movement tracks.
In a more specific embodiment, the singe layer culture array of the
present invention further comprises one or more enclosed input
channels in fluid communication between a main culture compartment
and an input fluid handling means of a controller unit.
[0229] As used herein, the term "enclosed channels" refers to a
completely or partially enclosed open spaces within a single layer
array through which materials such as oocytes, embryos and culture
media may travel.
[0230] As used herein, the term "movement tracks" refers to a strip
of material which is selectively attractive to labelable zona
anchor MEMS devices and which is deposited onto a surface within an
enclosed channel, collecting manifold or other surface. The
movement tracks allow for the guided movement of the labeled cells
through the array.
[0231] As used herein, the term "routing elements" refers to a
portion of a movement track which is selectively attractive to
labelable zona anchor MEMS devices and which can mediate a change
in the heading of a labelable zona anchor MEMS device, e.g., move
the labelable zona anchor MEMS device onto another track.
[0232] As used herein, the term "collecting manifolds" refers to a
region of a single layer array which is larger than an enclosed
channel. Further, collecting manifolds may include a region of a
single layer array which is larger than an enclosed channel and
which is open on one side. The collecting manifold is for the
introduction or removal of cells from the MEMS devicesThe culture
manifold is an enclosed area or widened channel or channel that
provides a location where a number of cells or groups of cells can
be cultured together. More particularly, oocytes and embryos often
survive or perform better when cultured together rather than
individually. The culture manifold allows for such co-culture.
[0233] According to the present invention, a culture manifold
comprises: [0234] (a) an enclosed channel or a plurality of
enclosed channels for receiving fluids or cells; and [0235] at
least one movement track traversing through the culture manifold
for allowing movement of cells into the culture manifold. In a
specific embodiment, a culture manifold comprises a plurality of
movement tracks for transporting or allowing movement of a
plurality of cells through the manifold.
[0236] In another specific embodiment, a culture manifold further
comprises at least one input enclosed channel for introducing cells
into the culture manifold.
[0237] In another embodiment, a culture manifold further comprises
at least one output enclosed channel for removing cells from the
culture manifold.
[0238] In yet another embodiment, the culture manifold further
comprises at least one router element resides on a movement
track.
[0239] As used herein, the term "externally communicating input and
export channels" refers to a plurality of enclosed channels which
lie above the plane of the single layer array, communicate with
collecting manifolds and/or enclosed channels, may not contain
movement tracks and which facilitate the movement of fluid into and
out of the collecting manifolds or enclosed channels.
[0240] FIGS. 6A and B show a single layer culture array and a
close-up cross-sectional view of the main culture manifold. The
drawing of the single layer culture array 43 shows a loading
compartment 44 for receiving oocytes or embryos with embedded
labelable zona anchor MEMS that are attracted to the movement
tracks 45 of the single layer culture array. Enclosed channels 42
with movement tracks 45 are in communication with the loading
compartment 44 as well as removal compartment 46 and a main culture
collecting manifold 47 wherein enclosed input 48 and export
channels 49 provide for the introduction and removal of fluids. On
the movement tracks 45 can be found router elements 50. Enclosed
channels 42 are in communication with the main culture manifold 47
and removal compartments 46. FIG. 6B shows the main culture
manifold being made up of a plurality of enclosed channels 42 each
having its own input 48 and export 49 channels. [0241] 1. In one
embodiment, a single layer culture array comprises: a
multi-laminate planar layer comprising; [0242] b) at least one
loading compartment; c) at least one enclosed channel; d)
[0243] a movement track attractive to a labelable zona anchor MEMS
on the floor of enclosed channel; at least one removal compartment;
and e) at least one circuit lead communicating between the movement
track and the controller unit in a preferred embodiment, a
single-layer MEMS culture array for culturing cells or a group of
cells comprises: [0244] (a) at least one loading compartment for
loading cells or groups of cells or fluids into the device; [0245]
(b) at least one enclosed channel in fluid communication with the
loading compartment and wherein the enclosed channel allows for the
passage of cells; [0246] (c) at least one movement track attractive
to labelable zona anchor MEMS attached to the enclosed channel;
[0247] (d) at least one removal compartment for the removal of
cells or groups of cells; and [0248] (e) at least one circuit lead
providing communication between at least one movement track and a
controller unit.
[0249] In a specific embodiment, a single layer cell culture MEMS
array further comprises at least one router element which resides
on a movement track
[0250] In yet another specific embodiment, the single layer cell
culture MEMS array has at least one enclosed channel with movement
track is in fluid communication with a culture manifold for the
transport a cell or group of cells and fluid through the cell
culture device.
[0251] In a specific embodiment, the single layer array has at
least one router element resident on a movement track.
[0252] In another specific embodiment, the single layer array has
at least one enclosed channel with movement track is in
communication with a main culture compartment.
[0253] For example, the single layer array is used for the culture
of oocytes and/or embryos. Further, the single layer culture array
is used such that the enclosed channels and other interior cavities
are filled with an appropriate culture medium (i.e. Hams-F10,
Dearles, M199, DMEM) with appropriate amendments (i.e., hormones,
serum, chemicals, nutrients). The filled array is placed into the
controller unit and the controller, having been stocked with
desired reagents and other fluids in its holding tanks, is provided
desired culture and environmental conditions as well as any active
process needed over time (i.e., addition and removal of fluids from
the array, introduction of sperm, determination of conditions in
the interior of the array, i.e. pH, temperature). Oocytes and/or
embryos, with labelable zona anchor MEMS devices that are
attractive to magnetic media embedded in their zonas, are placed
into a loading compartment either by mouth pipette, by a robotic
means or other automated manner. The labelable zona anchor MEMS
device, being a large object in relation to the oocyte or embryo
will orient itself to the bottom of the compartment and, in doing
so, come in contact with and attach to the movement track resident
in the loading compartment. The movement track provides a forward
heading for an attached cell, moving it into the enclosed channel.
When the cell reaches a router sitting at the union between two
differently oriented movement tracks, the cell is switched
(shunted) to the desired track (that switch being mediated by the
controller CPU). Upon reaching the main culture collecting
compartment the movement tracks cease providing a forward movement
and cells are retained in the main culture compartment for a period
consistent with the culture needs (i.e. minutes, hours, days)
desired as provided by the controller CPU. Upon instruction from
the controller CPU (mediated by circuit leads communicating between
the movement tracks and the controller CPU) the movement tracks
provide forward movement to move the cells out of the main culture
compartment to another main culture compartment or to a removal
compartment by way of the enclosed channels.
[0254] The present invention also provides for a single layer
culture array with a plurality of main culture compartments. For
example, a single layer culture array of the present invention may
have two or more main culture compartments laying in tandem such
that cells will move sequentially from a first main culture
compartment to a subsequent main culture compartment.
[0255] In another embodiment, the present invention provides for a
visual image capture device to be resident on the single layer
culture array. This a visual image capture device captures an image
of cells within the single layer culture array and communicates it
(e.g., fiber optic transmission) to an image collection and
modification device that is not resident on the single layer
culture array. The capture of images in this situation is important
for the real-time assessment of the quality of oocytes and embryos
over time. This assessment provides the ability to cull out
non-viable oocytes or embryos from the array so that resources may
be focused only on the highest quality oocytes or embryos as wells
as conferring the ability to detect problems that may cause a loss
of all oocytes or embryos during culture (e.g., contamination, pH
instability).
[0256] As used herein, the term "visual image capture devices"
refers to a means by which an image may be collected of material
within an enclosed channel, collecting manifold, or any other
portion of the single layer culture array, e.g., fiber optic video
camera leads.
[0257] In a specific embodiment, the single layer culture arrays
described above further comprises a visual image capture device for
visualizing the cells within the array.
[0258] The present invention provides for methods of making single
layer culture arrays. In one embodiment, the method of making a
single layer culture array comprises the modification of a
substrate material (i.e. silicon wafer, plastic, metallic oxide,
other etchable and depositable material) using etching and
depositing modifications (i.e. LIGA, DRIE, silicon fusion bonding,
laser etching, laser-mediated and directed substrate
polymerization) such that channels and collection compartments are
made in the substrate. The movement tracks, circuit leads in
communication with the movement tracks, the router elements, and
circuit leads in communication with the router elements are
deposited onto the previously modified substrate. A second
substrate with channels similarly created is bonded on top of the
first substrate such that the channels of the second substrate are
in communication with the main culture compartment of the first
substrate, forming the input and output channels as well as
enclosing the open domain or compartment of the first
substrate.
H. Multi-Layer Culture Array
[0259] Further, the present invention provides for a multi-layer
culture MEMS arrays into which said labelable zona anchor MEMS
device is attracted and controllably actuated. The multi-layer
culture MEMS arrays serve to facilitate the wholesale movement of a
plurality of labeled oocytes and/or embryos throughout a variably
determinable array of precisely regulated variable culture and
treatment environments.
[0260] As used herein, the term "multi-layer culture MEMS array"
(herein also referred to as a "multi-layer array") refers to a
collection of two or more single layer culture arrays in fluid
communication by way of one or more enclosed channel bridging
elements and provide variably controlled environments for a
labelable zona anchor MEMS device. A multi-layer culture array
provide different single layer arrays for performing different
purposes and activities in each single layer array. For example, a
multi-layer array can be made comprising an in vitro maturation
array, an in vitro fertilization array and an in vitro culture
array.
[0261] As used herein, the term "enclosed channel bridging element"
refers to a portion of an enclosed channel in one single layer
array which is continuous with another section of another single
layer array (e.g., two or more single layer culture arrays in a
multi-layer culture array). The enclosed channel bridging element
"bridges" or connects between separate single layer arrays of a
multi-layer array.
[0262] In one embodiment, the multi-layer array comprises two or
more single layer culture arrays in which the environment (e.g.,
culture media, pH, temperature, perfusion rates, input ports for
non-fluidic culture components, such as, but not limited to, sperm)
are precisely and individually controlled.
[0263] FIGS. 7A-B show a multi-layer culture MEMS array and a
detailed side cross-sectional view. FIG. 7A shows three different
layers manufactured in a single unit 54 for providing differing
functions (i.e., in vitro maturation 55, in vitro fertilization 56,
and in vitro culture 57) and enclosed channel bridging element 58.
FIG. 7B shows an enclosed channel bridging element 58. The channel
bridging element is the portion of the channel between two layers
of a multi-layer culture array that permits fluid communication and
cell movement between the layers. The channel bridging element
further comprises one or more movement tracks permitting cells to
be transported between the layers of the array.
[0264] There is shown a first level 6 and a second level 7, the
second level 7 being open 11 to the first level. A movement track 8
on the first level 6 with a cell 12 is continuous with a movement
track on the side wall of the first layer 9 and a side wall of the
second layer 10 that is then continuous with a movement track in
the second level 7. In another embodiment, the present invention
provides for a visual image capture device to be resident on the
multi-layer culture array. This a visual image capture device
captures an image of cells within the multi-layer culture array and
communicates it (e.g., fiber optic transmission) to an image
collection and modification device that is not resident on the
single layer culture array. The capture of images in this situation
is important for the real-time assessment of the quality of oocytes
and embryos over time. This assessment provides the ability to cull
out non-viable oocytes or embryos from the array so that resources
may be focused only on the highest quality oocytes or embryos as
wells as conferring the ability to detect problems that may cause a
loss of all oocytes or embryos during culture (e.g., contamination,
pH instability).
[0265] As used herein, the term "visual image capture devices"
refers to a means by which an image may be collected of material
within an enclosed channel, collecting manifold, or any other
portion of the multi-layer array, e.g., fiber optic video camera
leads. In another specific embodiment, the multi-layer array
further comprises a visual image capture device for visualizing the
cells within the array.
[0266] In another embodiment, the multi-layer culture MEMS array
comprises a plurality of single layer MEMS arrays wherein the
single layer arrays comprise a plurality of enclosed channels
reside wherein the channels comprise movement track which are
selectively attractive to labelable zona anchor MEMS devices on
labeled cells or groups of cells and which provide forward movement
to the cells. The enclosed channels of the multi-layer culture MEMS
array are capable of containing fluids (i.e., culture media).
[0267] In another embodiment, the movement tracks of the single
layer MEMS arrays contain routing elements that provide for a
change in movement direction (i.e., on to another movement track)
of a particular cell or group of cells at a particular portion of
the single layer MEMS array.
[0268] In another embodiment, circuit elements provide signal
transmission from the culture array environmental controller CPU to
the routing elements.
[0269] In a yet another embodiment, the multi-layer culture array
further comprises collecting compartments, communicating with said
enclosed channels, into which oocytes and/or embryos may be tracked
and held at specified physical parameters for a specified time
period, (i.e., in vitro culture).
[0270] In yet another embodiment, the multi-layer culture array
comprises removal compartments, communicating with said enclosed
channels, into which oocytes and/or embryos may be tracked and then
become available for removal from the multi-layer culture
array.
[0271] In one embodiment, one or more single layer culture arrays
of a multi-layer culture array comprise at least one enclosed
channel bridging element. In another embodiment, the enclosed
channel bridging element of the multi-layer culture array forms a
continuity between the enclosed channels of one single layer
culture array and the enclosed channels of another single layer
culture array.
[0272] In one embodiment, the multi-layer culture array comprises a
plurality of externally communicating input and export channels
that lead into said main culture compartments. In another
embodiment, the multi-layer culture array comprises a plurality of
single layer culture arrays which contain enclosed channels that
are continuous and that facilitate the movement of oocytes and/or
embryos from one single layer culture array to another. In yet
another embodiment, the multi-layer culture array has attached to
it, at specific enclosed channels, visual image collection devices,
(i.e., fiber optic video camera leads). In another embodiment, the
multi-layer culture array is composed of clear plastic. In yet
another embodiment, the multi-layer culture array is composed of a
microelectromechanical device. In another embodiment, the
multi-layer culture array is composed of silicon. In another
embodiment, the multi-layer culture array is composed of sapphire.
In another embodiment, the multi-layer culture array is composed of
metalize oxide. In yet another embodiment, the multi-layer culture
array is composed of plastic.
[0273] For example, the multi-layer culture array of the present
invention is used in a substantially similar manner as that
described for the single layer culture array. Further, the enclosed
channel bridging elements are actuated by the controller CPU,
providing the movement of oocytes and/or embryos from are layer to
another layer.
[0274] In particular, the multi-layer culture array is placed into
an environmental controller, the environmental controller is
provided with the desired culture parameters (e.g., length of
culture time, temperature), culture fluids are loaded into the
multi-layer culture array and any bubbles are purged by way of
pressure exerted at any opening to the culture array (e.g., input
and output channels). The labeled oocyte or embryos are placed into
the loading compartment by mouth pipette, robotic pipette or other
automated fluid handling means. Upon the attachment of the label to
the movement track in the loading compartment, the cells are moved
into the culture compartment and provided with the desired culture
conditions (e.g., input and output fluids, temperature) as provided
by the environmental controller.
[0275] In a preferred embodiment, a multi-layer cell culture MEMS
array for culturing a cell or groups of cells, comprising a
multi-laminate planar layer comprises: [0276] (a) at least one
loading compartment for loading cells or groups of cells or fluids
into the device; [0277] (b) at least one enclosed channel in fluid
communication with the loading compartment and wherein the enclosed
channel allows for the passage of cells; [0278] (c) at least one
movement track attractive to labelable zona anchor MEMS attached to
the enclosed channel; [0279] (d) at least one removal compartment
for the removal of cells or groups of cells; and [0280] (e) at
least one circuit lead providing communication between at least one
movement track and a controller unit.
[0281] In a specific embodiment, a multi-layer cell culture MEMS
array further comprises at least one router element which resides
on a movement track
[0282] In another specific embodiment, the multi-layer cell culture
MEMS array has at least one enclosed channel with movement track is
in fluid communication with a culture manifold for the transport a
cell or group of cells and fluid through the cell culture
device.
Multi-Layer Culture Array Environmental Controllers and
Instruments
[0283] The present invention provides for multi-layer culture array
environmental controllers into which a multi-layer culture array is
contained wherein the culture array environmental controller
communicates with said single layer or multi-layer culture array by
way of the input and export enclosed channels and wherein said
culture array environmental controller regulates physical
parameters within said multi-layer culture array, e.g.,
temperature.
[0284] As used herein, the term "multi-layer culture array
environmental controller" refers to a mechanism whereby a
multi-layer culture array may be selectively subjective to specific
environmental conditions and whereby selective materials may be
introduced into and removed from the multi-layer culture array.
[0285] FIG. 8, shows a culture array environmental controller or
instrument 60 for receiving one or more multi-layer culture MEMS
arrays 64. The drawing shows a first holding reservoir 62, a second
holding reservoir 63, an environmentally controlled docking domain
64 for receiving single layer or multi-layer arrays 64, input and
output 65 leads 66 capable of communicating with single layer or
multi-layer culture MEMS arrays, input and output port fluid
handling means in communication with each reservoir 62,63 and the
input and output leads 65, 66, circuit leads in communication
between a controller CPU 68 and a culture MEMS devices or arrays
61.
[0286] As used herein, the term "fluid-handling means" refers to a
series of fluid containing elements, e.g., tubing, which
communicates between the environmentally controlled docking domain
and another compartment within the multi-layer culture array
environmental controller, e.g., a holding tank or reservoir.
[0287] As used herein, the term "holding reservoir" refers to a
compartment within a multi-layer culture array environmental
controller which is constructed so as to hold fluids and maintain
them at desired physical parameters, e.g., temperature, pH.
Further, the holding tank may be connected to the fluid-handing
means such that a selectively permeable barrier lies between the
holding tank and the single layer or multi-layer culture array. Yet
further, the holding tank may be so constructed as to allow the
input of materials separate from the opening for the connection to
the fluid-handling means.
[0288] As used herein, the term "visual image detection device"
refers to a means to collect image data from the visual image
collection devices at the single layer or multi-layer culture array
and either interpret it or output to an interpreting device.
[0289] As used herein, the term "multi-layer culture array
environmental controller CPU" refers to a programmable data
processor that is operable linked to integrated circuit elements on
the single layer or multi-layer culture array. Further, said
multi-layer culture array environmental controller CPU determines
the temporal and spatial positioning of labelable zona anchor MEMS
devices on said single layer or multi-layer culture array.
[0290] As used herein, the term "integrated circuit elements"
refers to a circuit which provides signal transmission to the
routing elements on a planar array of a single layer or multi-layer
culture array.
[0291] As used herein, the term "environmentally controlled docking
domain" may include a compartment within a multi-layer culture
array environmental controller which accepts and secures a single
layer or multi-layer culture array. Further, the environmentally
controlled docking domain has a means by which it regulates the
physical parameters within the single layer or multi-layer culture
array such as temperature. Yet further, the environmentally
controlled docking domain has a means for connecting the externally
communicating input and export channels of a single layer or
multi-layer culture array to a fluid-handling means within the
multi-layer culture array environmental controller.
[0292] The present invention provides for a method of using said
labelable zona anchoring MEMS device in conjunction with said
single layer or multi-layer culture array and multi-layer culture
array environmental controller wherein the labeled cells or group
of cells, such as oocytes and/or embryos are attached to the are
placed into a loading compartment of said single layer or
multi-layer culture array which is in turn placed into a
multi-layer culture array environmental controller which mediates
the introduction, maintenance, and modulation of environmental
conditions over time.
[0293] In another embodiment, the culture array environmental
controller further comprises a fluid-handling means for
communicating with single layer or multi-layer culture array input
and export enclosed channels. In another embodiment, the
fluid-handling means of the environmental controller connects a
holding tank contained within the multi-layer culture array
environmental controller to the input and export enclosed channels
of the culture array. In another embodiment, the physical
environment of the holding tank contained within the multi-layer
culture array environmental controller can be variably maintained.
In another embodiment, the holding tank contained within the
multi-layer culture array environmental controller holds a fluid,
(i.e., culture media) or a cell suspension, (i.e., capacitated
sperm, cumulus cell suspension).
[0294] In another embodiment, the holding tank further comprises a
selective barrier, (i.e., a filter), between the tank contents and
the fluid handling means so as to prevent particulate materials
from being passed into said fluid-handling means. In another
embodiment, the holding tank with the selective barrier contains a
cell culture thus providing to the single layer or multi-layer
culture array a conditioned culture media without a cellular
component.
[0295] In one embodiment, the culture array environmental
controller further comprises a visual image detection device that
communicates with the visual image collection devices, (i.e., fiber
optic video camera leads) contained within the single layer or
multi-layer culture array. In one embodiment, the culture array
environmental controller further comprises a culture array
environmental controller CPU whereby said multi-layer culture array
environmental controller CPU programmably signals, by way of
circuit elements, to the router elements on movement tracks of a
single layer array or a multi-layer culture array such that the
heading of labelable zona anchor MEMS devices or labeled cells
moving on the movement tracks is changeable.
J. Microinjection MEMS Array
[0296] The introduction of small volumes of fluids, suspensions or
materials containing dyes, proteins, DNA molecules, RNA molecules,
viruses, as well as other compounds is important to a wide range of
developing technologies. The introduction of DNAs and RNAs that
modify and even become integrated into the genome of a target cell
is important to biological studies, gene therapy, as well as the
generation of transgenic cells and, in the case of the introduction
of heritable genetic changes in the genome of an oocyte or embryo,
transgenic animals.
[0297] While there are a great many methods for the introduction of
small volumes of fluid into the cytoplasm of culture cells as well
as cells in situ, there are a limited number of ways that the
introduction of small volumes of fluid into oocytes or embryos can
be effected. Currently, these reagents are introduced into oocytes
and embryos by micromanipulation wherein miniature glass needles
are usually manually forced into the cell and pressure is applied
to push the injection fluid or suspension into the cell. A device
that would facilitate the automation and standardization of this
technique would offer significant advantages over the present state
of the art.
[0298] Accordingly, the present invention provides for the
introduction of fluids or suspensions into the cytoplasm or nucleus
of a cell or group of cells such as but not limited to an oocyte or
embryo, using a microinjection MEMS device array, that facilitates
the injection of a small volume of fluid into the cytoplasm or
nucleus of a cell or group of cells such as an oocyte or embryo.
The present invention also provides for microinjection MEMS Device
kits, methods of using the devices and kits and methods of making
the devices.
[0299] The term "microinjection" refers to the process by which
fluids, such as solutions (i.e., DNA, RNA, proteins, organic
compounds), are injected into the interior (i.e., the cytoplasm,
the nucleus) of cells (i.e., cells, oocytes, embryos) using needles
manufactured from glass (i.e., borosilicate). These needles are
actuated by way of micromanipulators that provide controllable
(i.e., by hand, joystick/servo machinery) movement in all three
planes (i.e., x, y, z). Additionally, fluids contained within the
needle can be expelled by way of a pressure system (i.e., hydro,
pneumatic) in communication with the needle.
[0300] In one embodiment, a microinjection MEMS device
comprises:
[0301] (a) a silicon wafer comprising at least one well in fluid
communication with a fluid transfer channel and wherein the well
comprises at least one hollow protuberance; and
[0302] (b) an input manifold in fluid communication with the fluid
transfer channel.
[0303] In another embodiment, the microinjection MEMS device
further comprises a pumping means.
[0304] In a preferred embodiment, a microinjection MEMS device for
injecting a fluid, a suspension or a material into a cell or group
of cells comprises: [0305] (a) a first substrate comprising at
least one well for holding the cell or group of cells and [0306]
wherein the well comprises at least one hollow protuberance for
penetrating the cell or group of cells and [0307] wherein the well
is in fluid communication with a fluid transfer channel wherein the
fluid transfer channels permits the fluid to enter the hollow
protuberance and to then enter the cell; and [0308] (b) a second
substrate comprising an input manifold in fluid communication with
the fluid transfer channel wherein the input manifold allows for
the input of the fluid, suspension or material into the hollow
protuberances.
[0309] In a more specific embodiment, the invention provides a
microinjection MEMS device wherein the well is cube-shaped.
[0310] In yet another embodiment, the microinjection MEMS device
wherein the cube-shaped well is from about 50 .mu.m to about 200
.mu.m in length per side. In another specific embodiment, of the
microinjection MEMS device, the well is conical-shaped.
[0311] In another specific embodiment, the microinjection MEMS
device, the hollow protuberance is a needle, more specifically a
microneedle. In yet another specific embodiment, the hollow
protuberance is from about 0.01 .mu.m to about 10 .mu.m in
diameter.
[0312] In one embodiment, the hollow protuberance may be so
constructed as to act as an emitter. In particular, the hollow
protuberance may act as a wave guide to conduct electromagnetic
signals (e.g., pulses of light of any frequency). Additionally, the
hollow protuberance can provide vibrational energy (e.g., sound
waves, e.g., ultrasonic waves). The hollow protuberance acting as
an emitter facilitates the piercing of the protuberance or
microneedle into the cell with a minimal amount of damage to the
cell.
[0313] In a particular embodiment, the microinjection MEMS device
further comprises a coating. In more specific embodiments, the
coating is a polypeptide, peptide or protein. In another specific
embodiment polypeptide is polylysine.
[0314] The present invention also provides for a method of making a
microinjection MEMS device comprising the steps of:
[0315] (a) etching a plurality of parallel channels on a first side
of a plurality of silicon wafers in which the wafers each have a
second unetched side;
[0316] (b) silicon fusion bonding the unetched side of a plurality
of silicon wafers of step (a) to the etched side of a plurality of
silicon wafers of step (a) such that the etched channels are in
parallel to form a mega-laminate wherein the mega-laminate has a
plurality of holes formed by the channels;
[0317] (c) cutting the mega-laminate at an angle perpendicular to
the long axis of the etched channels thereby forming a slice of the
mega-laminate having a top surface and a bottom surface wherein
each surface exposes an end of the channel;
[0318] (d) silicon fusion bonding the bottom surface of the slice
of the mega-laminate to the etched side of a channel-etched
base-plate wafer;
[0319] (e) depositing a first mask on the top surface of the slice
of the mega-laminate such that a region surrounding each channel
end is free of mask;
[0320] (f) etching the mask to form a plurality of wells;
[0321] (g) depositing a second mask on the mega-laminate top
surface such that a border forms around each channel end such that
material around the channel is not etched;
[0322] (h) etching the second mask thereby forming a plurality of
hollow protuberances within the wells.
[0323] In a specific embodiment, the method of making a
microinjection MEMS device further comprises applying a coating to
the mega-laminate top surface after step (h).
[0324] In yet another specific embodiment, the method of making a
microinjection MEMS device, wherein the coating is a polypeptide,
peptide or protein.
[0325] In a more specific embodiment, the polypeptide is
polylysine.
[0326] The present invention also provides a method of making a
channel-etched base-plate silicon wafer with a pump/valve
comprising the steps of:
[0327] (a) etching a silicon wafer with a plurality of channels
which are in fluid communication within input manifold
reservoir;
[0328] (b) etching the silicon wafer of step (a) whereby a
pump/valve is constructed in each channel; and
c (c) a circuit lead between the pump/valve and a controller is
deposited. d In a specific embodiment, the method of making a
channel-etched base-plate silicon wafer with a pump/valve comprises
a piezeoelectric pump/valve in step (b).
[0329] The present invention also provides for, a microinjection
MEMS device kit comprising:
[0330] (a) at least one microinjection MEMS device of claim 48;
and
[0331] (b) a centrifugal platter for applying a centripetal force
to a cell or group of cells contained within a MEMS device
comprises a circular disk having a plurality of ports for holding
the MEMS device.
[0332] In a preferred embodiment, a microinjection MEMS device kit
for injecting a fluid, a suspension or a material into a cell or
group of cells comprising: [0333] (a) a centrifugal platter for
applying a centripetal force to a cell or group of cells contained
within a MEMS device wherein the centrifugal platter comprises a
circular disk, a plurality of ports for holding the MEMS devices
and a securing means to secure the platter to a spinner or driving
means; and [0334] at least one microinjection MEMS device.
[0335] In a specific embodiment, the microinjection MEMS device kit
comprises the microinjection MEMS device permanently affixed to the
centrifugal platter.
[0336] In another embodiment, the present invention provides the
microinjection MEMS device kit wherein
[0337] (a) the centrifugal platter comprises a plurality of grooves
arranged in a concentric pattern and wherein each groove has an
inner and outer edge;
[0338] (b) at least one microinjection MEMS device is bonded to the
outer edge of a groove in an orientation such that the axis of each
well of the microinjection MEMS device is horizontal to the plane
of the centrifugal platter; and
[0339] (c) the inner edge of the grooves forming divided
compartments comprising a single well that restrict the movement of
materials from one compartment containing a single well to another
compartment.
[0340] The present invention also provides a method of using a
microinjection MEMS device kit of comprising the steps of: [0341]
(a) filling the input manifold of at least one microinjection MEMS
device resident on a centrifugal platter with a fluid; [0342] (b)
loading the fluid-filled wells of step (b) with at least one oocyte
or embryo; [0343] (c) placing the microinjection MEMS/centrifugal
platter into a centrifuge; [0344] (d) rotate said centrifugal
platter thus applying a centripetal force on the microinjection
MEMS/centrifugal platter.
[0345] In another embodiment, a method of using a microinjection
MEMS device kit comprises the steps of: [0346] (a) filling the
grooves of the centrifugal platter with a fluid; [0347] (b) loading
the grooves of the centrifugal platter with at least one oocyte or
embryo; and [0348] (c) applying a centripetal force to the kit
whereby the oocyte or embryo makes contact with the hollow
protuberance of the microinjection MEMS device and the hollow
protuberance penetrates the surface of the oocyte or embryo.
[0349] In a more specific embodiment, a centripetal force on the
microinjection MEMS device kit by rotating the kit using a spinner
or driving means.
[0350] Another specific embodiment describes a method of using a
microinjection MEMS device kit wherein, upon rotation of
centrifugal platter, a volume of fluid is caused to enter the
oocyte or embryo in the cell well through the hollow
protuberance.
[0351] The present invention also provides a microinjection MEMS
device comprising: [0352] (a) a well for accepting one or more
cells comprising a hollow protuberance; [0353] (b) a fluid handling
means in fluid communication with said hollow protuberance; and
[0354] (c) a central fluid loading manifold.
[0355] In a specific embodiment, fluid handling means is a dynamic
hydropressure column.
[0356] The present invention also provides for a microinjection
MEMS array for injection of a fluid, a suspension or a material
into a cell or group of cells comprising: [0357] (a) a first
substrate comprising at least one well for accepting a cell or
group of cells and wherein the well comprises a hollow protuberance
for penetrating the cell or group of cells; [0358] (b) a second
substrate comprising a fluid handling means in fluid communication
with said hollow protuberance; and [0359] (c) a central loading
manifold for loading a fluid into the array.
[0360] In a specific embodiment, in a microinjection MEMS array of
claim the fluid handling means is a dynamic hydropressure
column.
[0361] In another specific embodiment the device is embedded in a
centrifugal platter.
[0362] The invention also provides for a method of using the
microinjection MEMS array comprising: [0363] (a) applying an
inertial force to the device using a centripetal (angular)
acceleration means brought about by rotation of centrifugal
platter
[0364] In yet another embodiment, a microinjection MEMS array for
injecting a fluid, a suspension or a material into a cell or group
of cells comprises: [0365] (a) a central loading manifold for
loading the fluid, suspension or material into the array; [0366]
(b) a plurality of wells for receiving cells; [0367] (c) a hollow
protuberance within each well for penetrating the cell and
injecting the fluid, suspension or material; and [0368] (d) a
plurality of dynamic hydropressure columns in fluid communication
with the central loading manifold and with the hollow protuberances
wherein the dynamic hydropressure columns provide pressure for
forcing the fluid, suspension or material through the hollow
protuberance and into the cell.
[0369] In a more specific embodiment, the microinjection MEMS array
further comprises at least one valve in the dynamic hydropressure
column for modulating fluid flow.
[0370] In yet another specific embodiment, in a microinjection MEMS
array, wherein each valve is in operable communication with a
controller to control the fluid, suspension or material flowing
into the cell.
[0371] The microinjection MEMS device further comprises at least
one valve in the dynamic hydropressure column for modulating fluid
flow.
[0372] Further, a microinjection MEMS device is provided wherein
each valve is in operable communication with a controller to
control the fluid, suspension or material flowing into the
cell.
[0373] Additionally, a microinjection MEMS device is provided
wherein the operable communication is mediated by circuits.
[0374] In another embodiment, a microinjection MEMS device wherein
the hollow protuberance acts as an emitter More specifically, a
microinjection MEMS device is provided wherein the hollow
protuberance, acting as an emitter, emits pulses of light (e.g.,
any frequency). In yet another specific embodiment, a
microinjection MEMS device is provided wherein the hollow
protuberance, acting as an emitter, emits pulses of sound (e.g.,
ultrasonic waves).
[0375] Further, in other embodiments, a microinjection MEMS device
is provided wherein the operable communication is mediated by
electrical or optical circuits.
[0376] A method of using a microinjection MEMS device of
comprising: [0377] 1. loading at least one cell or group of cells
into an injection domain of the microinjection MEMS device; [0378]
2. applying a centripetal force to the microinjection MEMS device
thereby causing penetration of the cell or group of cells by the
hollow protuberance of the microinjection MEMS device; and
deposition of a substance in the cell or group of cells from the
hollow protuberance.
[0379] A method of using a microinjection MEMS device comprising
[0380] (a) loading at least one cell or group of cells into the
injection domain of the microinjection MEMS device; [0381] 1.
rotation of microinjection MEMS device whereby the cell is thrust
upon hollow protuberance resident within injection domain; [0382]
2. simultaneous passive movement of fluid through dynamic
hydropressure columns provides pressure to push fluid into cell;
and [0383] 3. removal of cell from microinjection MEMS device.
[0384] In another embodiment, the method of using the
microinjection MEMS further comprises a gating valve that, being
activated by a controller by way of a circuit, provides for
variable fluid flow from the dynamic hydropressure column into the
cell.
[0385] In another embodiment, the hollow protuberance emits a pulse
of energy (e.g., lights, sound) that provides a means for a focused
disruption of the lipid bi-layer of the cell membrane of the oocyte
or embryo.
[0386] A variety of methods of using the present invention of
microinjection MEMS devices will be apparent to those skilled in
the art.
[0387] In a preferred embodiment, a method of using a
microinjection MEMS device involves the microinjection MEMS device
being affixed to a means for applying centripetal forces to said
microinjection MEMS. The invention further provides for a method of
using a microinjection MEMS device comprising the microinjection
MEMS device being affixed to a means for applying centripetal
forces to said microinjection MEMS further comprising a well for
receiving an oocyte or embryo communicating directly with a
microinjection MEMS such that when a centripetal force is applied
the oocyte or embryo contained within the well communicating with
the microinjection MEMS, the oocyte or embryo is forced against the
microinjection MEMS such that the hollow protuberance of the
microinjection MEMS penetrates through the zona pellucida, through
the oollema, and into the cytosolic or nucleoplasmic compartment of
the oocyte or embryo.
[0388] Alternatively, as the oocyte or embryo is thrust upon the
hollow protuberance, the hollow protuberance emits a pulse of
energy (e.g., lights, sound), introducing a focused opening in the
lipid bi-layer of the plasma membrane of the oocyte or embryo thus
facilitating a tightly focused puncture of the membrane. This is
important for cell viability.
[0389] In another preferred embodiment, the microinjection MEMS
device of the present invention is permanently embedded in a
substrate base. A substrate base (i.e., silicon wafer, plastic
cartridge) includes, but is not limited to, a silicon wafer or
plastic cartridge that comprises a well for receiving a
microinjection MEMS device, upon fixation of which, a depression
remains adjacent to the microinjection MEMS device. This remaining
depression that is adjacent to the affixed microinjection MEMS
device is for receiving an oocyte or embryo. The substrate base
also comprises a lever adjacent to the remaining depression for
thrusting the oocyte or embryo, being placed in the remaining
depression, against the microinjection MEMS device. The substrate
base also comprises a fluid handling and pumping means in fluid
communication with the hollow protuberance of the microinjection
MEMS device.
[0390] In a more specific embodiment, a microinjection MEMS device
kit comprises (a) a microinjection MEMS device; and a base pumping
substrate. The term "base pumping substrate" includes a substrate
base (i.e., silicon wafer, plastic cartridge) that accepts a MEMS
device, and comprises a fluid handling means in fluid communication
with the hollow protuberance or needle of a microinjection MEMS
device, a pumping member on the substrate base, and a lever that
selectively pushes a oocyte or embryo against the MEMS device.
[0391] FIG. 28 illustrates a base pumping substrate 250 with a MEMS
device attached thereto 251 and the base pumping substrate
comprises an input well 252, a lever 253, that swings between the
input well 252 and the MEMS device regulating the movement of the
cell.
[0392] The microinjection MEMS kit operates, for example, as
follows: the kit being filled with fluid, the oocyte or embryo
being loaded into the kit, the lever thrusting against the oocyte
or embryo and thus trusting the oocyte or embryo against the
microinjection MEMS hollow protuberance, upon the penetration of
the oocyte or embryo by the hollow protuberance of the
microinjection MEMS the pump pushes a a small volume of fluid
through the fluid handling means and through the microinjection
MEMS hollow protuberance and into the cytosol or nucleoplasm of the
oocyte or embryo. In another embodiment, as the oocyte or embryo is
being thrust against the hollow protuberance of the microinjection
MEMS device the hollow protuberance emits a pulse of energy (e.g.,
light, sound) and provides a focused opening in the membrane lipid
bi-layer, facilitating the movement of the hollow protuberance
through the membrane.
[0393] The present invention further provides for the maintenance
of a positive pressure in the microinjection MEMS.
[0394] If a centrifugal platter is being used to provide pressure
to the fluid handling means then a positive pressure is present
upon rotation of the kit. If a substrate base with a fluid pumping
means is used to provide for injection then the pump of the
substrate base provides the positive pressure. Positive pressure is
applied to prevent back flush of oocyte or embryo cytosolic
materials into the hollow protuberance. Neutral pressure would
serve an analogous purpose.
[0395] The present invention also provides for a method of
manufacturing a microinjection MEMS device wherein a silicon wafer
is etched by silicon etchant/modifying technologies (e.g., deep
silicon reactive ion etching, silicon surface micromachining,
LIGA). Those of skill in the art of MEMS manufacturing will know of
a variety of methods of making the MEMS microinjection devices of
the present invention.
[0396] A preferred method of making the microinjection MEMS devices
of the present invention are set forth in FIGS. 9-15.
[0397] FIG. 9, shows a silicon wafer etched with channels 71 also
called a "pre-hole" wafer.
[0398] FIG. 10 shows a multi-laminate wafer 72. This structure is
composed of more than one "pre-hole" wafer 70 of FIG. 9, bonded,
(i.e., silicon fusion bonded forming sfb interfaces 73), such that
channels 71 are sealed along the long axis forming holes 75. This
figure also illustrates the cutting plane 4 where the
multi-laminate wafer is cut forming a pre-needle wafer.
[0399] Referring to FIG. 11, there are shown two wafers, a
pre-needle wafer 80, the result of cutting the multi-laminate wafer
FIG. 10, and a channel-etched base-plate wafer 81 with fluid
channels 83. These two wafers are bonded, (i.e., silicon fusion
bonded), and form a sfb interface, (i.e., a silicon fusion) bonding
interface (sfb interface) 82.
[0400] FIG. 12, shows a pre-needle wafer fused to the
channel-etched base-plate wafer of FIG. 8. A mask 80 is applied to
the top surface of the pre-needle wafer 70 such that there are
square unmasked regions 93. The input ports 1 formed when the
pre-needle wafer 70 and the fluid channel wafer 81 were bonded at
the sfb interface 82.
[0401] FIG. 13, there is shown a pre-needle wafer fused to the
channel-etched base-plate wafer of FIGS. 11 and 12. Two more masks
90 and 91 are applied to the surface of the pre-needle wafer 70 as
shown. The first mask 90 maintains the initial upper height of the
pre-needle wafer. The second mask 91 protects material surrounding
the holes resident within the pre-needle wafer 70. the input ports
92 are shown which provide access to the fluid channels 83.
[0402] FIG. 14 shows the pre-needle wafer to the channel-etched
base-plate wafer of FIGS. 11, 12 and 13 after the final etch. Wells
96 are at their final depth and a hollow protuberance (or a
microneedle) 97 is formed within each well 96 in the pre-needle
wafer 70 forming a microneedle wafer 98. Each hollow protuberance
97 is in communication with the fluid channels 83 of the channel
wafer 81.
[0403] FIG. 15 shows one possible embodiment of a channel etched
wafer 81. In this figure the channel etched wafer has a plurality
of fluid channels 83 etched into the top surface in communication
with an input manifold 100. Between the input manifold 100 and the
distal portions of the etched channels 83 there can be a pump 101,
(i.e., a piezoelectric pump) in the channel 82. Additionally, the
pump may be actuated by circuits, or leads 102 i.e., deposited
integrated circuits (not shown), communicate between said pump and
a controller.
[0404] FIG. 16 shows a portion of microinjection MEMS device 110
and a centrifugal platter 111 with a microinjection MEMS device 110
resident on it thereby forming a kit. The microinjection MEMS
device 110 is seen to be composed of a channel wafer 81, a
microneedle wafer 98, a silicon fusion bonding interface 82, wells
96, microneedles 97, and input ports to fluid channels 92 in
communication with the microneedles 97. Further, this figure
illustrates how a microinjection MEMS 110 is placed on a
centrifugal platter 111 that has cell loading regions 112 that
correspond to each well 96 on the microinjection MEMS devices 110.
Upon rotation of the centrifugal platter 111 a centripetal force
113 is generated in a perpendicular direction out from the center
of the centrifugal platter 111.
[0405] FIG. 17, shows an embodiment of a microinjection MEMS array.
There is a central loading manifold 120 wherein injectant material
is loaded, cell loading regions 121, dynamic hydropressure columns
122, valves on dynamic hydropressure columns 123, microinjection
needles 124. Upon rotation of this array, fluids in the central
loading manifold 120 will migrate into and through the dynamic
hydropressure columns 122, through the microinjection needles 124
and into the cells 125. The direction of the fluid movement is
pointed out by arrows.
[0406] FIG. 18 shows a single microinjection MEMS unit of the array
shown in FIG. 17. This unit comprises a dynamic hydropressure
column 123, a valve 123, a circuit lead 127 that actuates the valve
123, a controller 128 to which the circuit 127 lead communicates, a
well 126, a hollow protuberance or microinjection needle 124, and a
cell 125 to be injected. Upon rotation of the microinjection MEMS
array of which this unit is a part of, a force, the centripetal
force 129, is exerted on the fluid in the dynamic hydropressure
column 122, forcing fluid into the cell 125.
[0407] An alternative method of making the microinjection MEMS
devices of the present invention is the modification of a substrate
base (i.e., silicon wafer, plastic, metallic oxide or other
etchable and depositable material) using etching and deposition
modification (i.e., LIGA, DRIE, silicon fusion bonding, laser
etching, laser mediated and directed substrate polymeritation) such
that desired structures are formed on and in the substrate base. In
a preferred embodiment, the method of making the microinjection
MEMS devices of the present further comprises the formation of the
following structures: a central loading manifold, cell wells with a
hollow protuberance, dynamic hydropressure columns in communication
between the central loading manifold and the hollow protuberance
resident in the cell well and cell loading region. This method
further comprises a gate present on the dynamic hydropressure
column to provide for variable fluid flow, and a circuit lead in
communication with the gate and a controller.
[0408] This method further comprises the deposition of circuit
leads in communication with the hollow protuberance providing
transmission of current and data for the facilitation of hollow
protuberance function as an emitter (e.g., light, sound pulses).
These circuit leads communicate with a controller.
[0409] Another alternate method of making the microinjection MEMS
devices of the present invention is substantially similar to the
method described immediately above wherein the structures formed
are: a cell loading region, an injection fluid loading region, cell
well with a hollow protuberance in fluid communication with a fluid
handling means that is in fluid communication with the injection
fluid loading region and a pump situated on the fluid handling
means between the hollow protuberance and the injection fluid
loading region.
ICSI MEMS
[0410] The introduction of small volumes of fluid containing dyes,
proteins, DNA molecules, RNA molecules, viruses, sperm cells, as
well as other compounds is important to a wide range of developing
technologies.
[0411] While there are a great many methods for the introduction of
small volumes of fluid into the cytoplasm of culture cells or cells
in situ, there are a limited number of effective methods for
introducing sperm cells and small volumes of fluid into individual
cells or groups of cells such as oocytes or embryos. Devices and
methods to facilitate the automation and standardization of ICSI
techniques would offer significant advantages over the present
state of the art.
[0412] Male factor infertility, where sperm are incapable of
penetrating the zona pellucida and oollemma of an oocyte in such a
way that fertilization occurs, has been ameliorated only recently
by the advent of the use of a technique called IntraCytoplasmic
Sperm Injection (ICSI). ICSI involves the micromanipulation of both
the oocyte and sperm such that an oocyte is immobilized, a
micropipette is used to sever the tail of a candidate sperm, the
micropipette is used to pick up the severed sperm head, and the
micropipette is used to inject the sperm head into either the
perivitelline space (the space between the zona pellucida and the
oollemma) or directly into the cytoplasm of an unfertilized oocyte.
By this means, motility-impaired sperm have given rise to
successful pregnancies. This technique represents a significant
investment in highly specialized equipment, extensive training, and
scarce gamete resources. Further, pregnancy outcomes are highly
dependent on the skill of each individual performing the ICSI. A
device that would facilitate the automation and standardization of
this technique would offer significant advantages over the present
state of the art.
[0413] The term "Intracytoplasmic Sperm Injection" or "ICSI" refers
to the process by which a capacitated sperm, usually with the tail
removed, is injected, using needles and handling system similar to
those described for microinjection, into the interior of an oocyte
thereby fertilizing the oocyte and potentially forming an
embryo.
[0414] The present invention provides for ICSI MEMS devices and
kits, that facilitate the injection of a sperm into the cytoplasm
of a cell such as an oocyte. The present invention also provides
for methods of using ICSI MEMS devices and methods of making the
devices.
[0415] In one embodiment, a IntraCytoplasmic Sperm Injection (ICSI)
MEMS device comprises: [0416] (a) at least one well for accepting
cells wherein the well comprises a hollow protuberance; [0417] (b)
a sperm handling manifold; [0418] (c) at least one fluid handling
means in fluid communication between the hollow protuberance and
the sperm loading manifold; and [0419] (d) a sperm guillotine in
communication with the fluid handling means.
[0420] In a more particular embodiment of the ICSI MEMS device, the
fluid handling means is a dynamic hydropressure column.
[0421] In another embodiment, the ICSI MEMS device further
comprises at least one gating valve also called a guillotine gate
(hereinafter referred to as a "gate"). In a specific embodiment,
each gate is in operable communication with a controller. In yet
another specific embodiment, the operable communication is mediated
by circuits, and more specifically, the operable communication is
mediated by Electro-optical circuits.
[0422] In another preferred embodiment, a IntraCytoplasmic Sperm
Injection (ICSI)
[0423] MEMS array for injecting a sperm into a cell comprises:
[0424] (a) a substrate comprising at least one well for accepting
cells wherein the well comprises a hollow protuberance for
penetrating the cell to inject the sperm; [0425] (b) a sperm
handling manifold for loading the sperm into the array; [0426] (c)
at least one fluid handling means in fluid communication between
the hollow protuberance and the sperm loading manifold for
delivering the sperm to the array; and [0427] (d) a sperm
guillotine in communication with the fluid handling means wherein
the sperm guillotine severs the tail from the sperm.
[0428] In a specific embodiment, in the ICSI MEMS array of, the
fluid handling means is a dynamic hydropressure column.
[0429] In yet another specific, an ICSI MEMS array kit comprises at
least one ICSI MEMS array affixed to a centrifugal platter for
applying a centripetal force to a cell or group of cells contained
within a MEMS device wherein the centrifugal platter comprises a
circular disk, a plurality of ports for holding the MEMS devices
and a securing means to secure the platter to a spinner or driving
means.
[0430] In another embodiment, the ICSI MEMS array further
comprising; [0431] (a) at least one valve residing in the dynamic
hydropressure column for regulating the flow of the fluid,
suspension or material; and [0432] (b) a sperm guillotine for
severing the tail from the sperm.
[0433] More particularly, each valve of the array is in operable
communication with a controller.
[0434] More particularly, the sperm guillotine for severing the
tail from the head of a sperm comprises: [0435] (a) an enclosed
sperm channel for containing a sperm having a head and a tail; (b)
a first guillotine gate capable of sliding through a first end of
the enclosed channel and capable of halting the forward movement of
the sperm; for [0436] (c) a second guillotine gate capable of
sliding through a second end of the enclosed channel and capable of
severing the tail from the sperm [0437] (d) a controller for
controlling the sliding motion of the guillotine gates; and [0438]
(e) a circuit lead communicating between each gate and the
controller, wherein the circuit lead enables the controller to
direct the movement of each gate.
[0439] Further, the present invention provides a method of making a
sperm guillotine comprising the steps of: (a) depositing a first
mask that inscribes a channel and at least one guillotine gate;
[0440] (a) etching of the first mask to form the channel and the
guillotine gates; depositing circuit leads between each gate and a
controller
[0441] FIG. 20A shows two views of a sperm guillotine. The top view
shows a sperm channel 133, a first and a second guillotine gate
138, circuit lead 139 communicating between guillotine gate 138 and
controller 139, illustrating how a sperm head 140 is separated from
the sperm tail 141. FIG. 20B shows a side cut-away view
illustrating the position of the guillotine gate 138 in the sperm
channel 133. The circuit lead 139 is shown in communication with
the guillotine gate 138 and the controller.
[0442] The operation of the gate is as follows: the first gate
opens while the second gate remains closed, a sperm swims into the
guillotine (the size of the guillotine, being on the order of
approximately 1 micron wide and the gates being approximately 5
microns long, allows for a single sperm to engage the guillotine
and the span between the first gate and second gate equals
approximately the length of the head, ensuring that the tail will
be cut with very little remaining behind the sperm head), the first
gate shuts closed, severing the tail, the second gate opens. If
centrifugal force is being used then the head will continue in the
dynamic hydropressure column to the hollow protuberance. If a
substrate base is being used then the pumping means facilitates the
movement of the sperm head into the hollow protuberance. The gates,
being bimorphic, respond to controlled inputs (e.g., heat,
electrical current) by changing their conformation, closing or
opening.
[0443] In a specific embodiment, the ICSI MEMS device further
comprises a coating to the mega-laminate after the final etching.
The coating is to prevent the cells from adhering or sticking to
the elements of the device. Preferably, the coating is a
polypeptide, and more preferably, the polypeptide is
poly-lysine.
[0444] The present invention also provides for an ICSI MEMS array
or kit comprising at least one ICSI MEMS device affixed to a
centrifugal platter. In a more specific embodiment, the ICSI MEMS
device is permanently affixed to the centrifugal platter.
[0445] FIG. 19 shows a preferred embodiment of an IntraCytoplasmic
Sperm Injection (ICSI) MEMS array. This ICSI MEMS array comprises
ICSI devices affixed to a centrifugal platter comprising a cell
loading region 130, a sperm loading bay 131, and an opening in the
sperm loading bay 131, a sealed or enclosed sperm channel 133 in
fluid communication between the sperm loading bay 131 and the
dynamic hydropressure columns 134, sperm guillotines 135 resident
on each dynamic hydropressure column 134, microinjection needle
136, and well 137
[0446] In another embodiment, an ICSI MEMS device kit comprises an
ICSI MEMS device permanently fixed onto the surface of a
centrifugal platter and wherein: [0447] (a) the centrifugal platter
having a plurality of grooves arranged in a concentric pattern on
the centrifugal platter; [0448] (b) the ICSI MEMS device is bonded
to the outer edge of said groove in an orientation such that the
long axis of the ICSI MEMS device is horizontal to the plane of the
centrifugal platter and directed towards the center of said
platter; and [0449] (c) the inner surface of the grooves forming
divided chambers which restrict the movement of materials from one
compartment containing a single ICSI MEMS device to another such
compartment.
[0450] In yet another embodiment, a method using an ICSI MEMS
device comprises the steps of: [0451] (a) loading a fluid in the
fluid handling means; loading sperm into the sperm loading
manifold; loading a fluid into the wells; [0452] (b) loading at
least one cell into each well; [0453] (c) applying an inertial
force to the ICSI MEMS device by way of centripetal acceleration
brought about by rotation of the centrifugal platter; [0454] (d)
removing the distal portion of the sperm tail from the sperm using
the sperm guillotine; [0455] (e) providing a variable fluid flow
from the dynamic hydropressure column into the cell by operating a
gating valve controlled by a circuit. [0456] a) and forcing the
sperm head and fluid through dynamic hydropressure columns provides
pressure to push sperm head and fluid into the cell.
[0457] Additionally, upon the oocyte or embryo being thrust upon
the ICSI MEMS device, the ICSI MEMS device hollow protuberance,
being in circuit lead communication with a controller, provides a
pulse of energy (e.g., light, sound). The emission facilitates a
focused opening in the lipid bi-layer of the oocyte or embryo
plasma membrane for hollow protuberance penetration.
[0458] In one embodiment, the present invention provides for an
ICSI MEMS device kit wherein [0459] (a) the centrifugal platter
comprises a plurality of grooves arranged in a concentric pattern
and wherein each groove has an inner and outer edge; [0460] (b) at
least one ICSI MEMS device is bonded to the outer edge of a groove
in an orientation such that the axis of each well of the ICSI MEMS
device is horizontal to the plane of the compact cassette; and
[0461] (c) the inner edge of the grooves forming divided
compartments comprising a single well which restrict the movement
of materials from one compartment containing a containing a single
well to another compartment.
[0462] In a more specific embodiment, a method of using an ICSI
MEMS device kit of comprises the steps of: [0463] (a) filling the
input manifold of at least one ICSI MEMS device with a fluid;
[0464] loading the fluid-filled wells of step (b) with at least one
oocyte or embryo; [0465] (b) placing the ICSI MEMS/centrifugal
platter into a centrifuge; and [0466] (c) applying a centripetal
force on the ICSI MEMS/centrifugal platter.
[0467] In another specific embodiment, a method of using an ICSI
MEMS device kit comprises the steps of: [0468] (a) filling the
grooves of the centrifugal platter with a fluid; [0469] (b) loading
the grooves of the centrifugal platter with at least one oocyte or
embryo; [0470] (c) applying a centripetal force to the kit whereby
the oocyte or embryo makes contact with the protuberance of the
ICSI MEMS device and the protuberance penetrates the surface of the
oocyte or embryo; and [0471] (d) severing the sperm head from the
tail by sperm guillotine and positioning the sperm head near tip of
hollow protuberance. [0472] depositing one sperm head in the oocyte
or embryo. The present invention also provides for methods of
making ICSI MEMS devices as described in Section A supra.
[0473] In one embodiment, a method of making an ICSI MEMS device
comprises; [0474] (a) etching a plurality of channels in parallel
on a silicon wafer; [0475] (b) bonding of said first wafer to the
unetched side of a second identically made etched wafer such that
all etched channels are in parallel; [0476] (c) repeat step (b)
until desired number of wafers have been fused to form a
mega-laminate; [0477] (d) said mega-laminate is cut at an angle
perpendicular to the axis of said etched channels; [0478] (e) the
slice of mega-laminate formed in (d) is bonded to the etched side
of a channel-etched base-plate wafer; [0479] (f) a first mask is
deposited on the surface of mega-laminate, the side not being
bonded to the channel-etched base-plate wafer, such that a square
region surrounding each square center void region is free of mask;
[0480] (g) said mega-laminate surface with mask of (f) being etched
such that the mega-laminate wafer material is removed to form a
plurality of wells in the mega-laminate wafer; [0481] (h) a mask is
deposited on the surface of said mega-laminate which is not bonded
to the channel-etched base-plate wafer such that the mask forms a
border surrounding each well in the mega-laminate; [0482] (i) said
mega-laminate surface with the mask of (h) is etched such that the
mega-laminate wafer material is removed to a depth wherein material
remains above the channel-etched base-plate wafer, forming a
plurality of hollow protuberances of a certain height and
width.
[0483] In a specific embodiment, the method of making an ICSI MEMS
device further comprises applying a coating to the mega-laminate
after the final etching. Preferably, the coating is a polypeptide,
and more preferably, the polypeptide is poly-lysine.
[0484] The present invention provides for a method of using an ICSI
MEMS device. The invention further provides for a method of using
an ICSI MEMS device comprising the ICSI MEMS device being affixed
to a means for applying centripetal forces to said ICSI MEMS
device. The invention further provides for a method of using an
ICSI MEMS device comprising the ICSI MEMS device being affixed to a
means for applying centripetal forces to said an ICSI MEMS device
further comprising a well for receiving an oocyte communicating
directly with an ICSI MEMS device such that when a centripetal
force is applied the oocyte contained within the well communicating
with the ICSI MEMS device is forced against the ICSI MEMS device
such that the ICSI MEMS device hollow protuberance penetrates
through the zona pellucida, through the oollema, and into the
cytosolic compartment of the oocyte.
[0485] The invention further provides that upon the penetration of
the oocyte by the ICSI MEMS device, a sperm, having been
decapitated, travels through the ICSI MEMS device and into the
cytosolic compartment of the oocyte. The present invention further
provides for the maintenance of a positive pressure in the ICSI
MEMS device.
[0486] The present invention provides for a method of manufacture
of an ICSI MEMS device wherein a silicon wafer is modified by
silicon etchant/modifying technologies (e.g., deep silicon reactive
ion etching, silicon surface micromachining, LIGA).
[0487] After alternative method of making the ICSI MEMS devices of
the present invention is substantially similar to the method
described immediately above wherein the structures formed are:
sperm bay, enclosed channel in fluid communication between the
sperm loading bay and the central manifold that is in turn in fluid
communication with the dynamic hydropressure column, sperm
guillotine/gating element on each dynamic hydropressure column and
also in fluid communication with the hollow protuberance in the
cell well, and a cell loading region.
[0488] Alternatively, the sperm guillotine can be located between
the sperm loading bay and the central manifold and a gating element
on each dynamic hydropressure column to allow only one sperm to
enter or oocyte.
[0489] In another embodiment, the present invention provides for an
ICSI MEMS device kit for the injection of a sperm into a cell
comprising:
[0490] (a) a pumping/sperm guillotine base providing support and a
pump; and
[0491] (b) at least one ICSI MEMS device affixed to the base.
[0492] Yet another method of making an ICSI MEMS is substantially
similar to that method of making a microinjection MEMS device as
recited above with the following modifications: the hollow
protuberance is at least 1.5 .mu.m in width and as wide as 7.5
.mu.m; a sperm guillotine/gate element is constructed in the fluid
handling means between a sperm loading bay and the hollow
protuberance.
Zona Coring MEMS
[0493] Infertility can arise from a great many different factors,
one of which is the hardening of the zona pellucida of the oocyte
in some women. While the developing embryo is normally capable of
moving through and out of the zona pellucida after a certain number
of divisions (hatching), it is the case that, with a hardened zona
pellucida, the embryo is trapped and can not escape, leading to a
failure to implant and a failure of pregnancy. To elevate this
condition a technique known as "assisted hatching" has been
developed in which a portion of the surface of the zona pellucida
is eroded to such an extent that the developing embryo can hatch.
This technique represents a significant investment in highly
specialized equipment, extensive training, and scarce gamete
resources. Further, pregnancy outcomes are highly dependent on the
skill of each individual performing the assisted hatching. A device
which would facilitate the automation and standardization of this
technique would offer significant advantages over the present state
of the art.
[0494] The present invention further provides for zona coring MEMS
devices and kits that remove a small portion of the zona pellucida
and thus facilitate assisted hatching of an embryo.
[0495] In one embodiment, a zona coring MEMS device comprises a
silicon wafer comprising a plurality of wells wherein each well
comprises at least one coring member. A coring member is for
creating the holes or cores in the zona.
[0496] The present invention also provide for a zona coring MEMS
device kit comprising: (a) at least one zona coring MEMS device of
claim 1; and (b) a centrifugal platter having an outer edge and a
plurality of grooves, the grooves having an inner and outer
surface, arranged in a concentric pattern on the surface of the
centrifugal platter; wherein the zona coring MEMS device is
attached to the outer edge of the centrifugal platter in an
orientation such that the long axis of each of the cell wells of
the zona coring MEMS device is horizontal to plane of the
centrifugal platter and the inner surface of the grooves forming
divided chambers, the chamber containing a single cell well, which
restrict the movement of materials from chamber to another such
chamber.
[0497] In a particular embodiment, a zona coring MEMS device for
forming one or more cores in the zona pellucida of a cell
comprising a substrate wherein the substrate comprises a plurality
of wells and wherein each well comprises coring member
[0498] In another embodiment, a zona coring MEMS device kit for
forming one or more cores in the zona pellucida of a cell
comprises: [0499] (a) a centrifugal platter having an outer edge
and a plurality of grooves, the grooves having an inner and outer
surface, arranged in a concentric pattern on the surface of the
centrifugal platter [0500] wherein the centrifugal platter is for
applying a centrifugal force to the; and [0501] (b) at least one
zona coring MEMS device of claim 1.
[0502] The present invention also provides for a method of using
the zona coring MEMS device kit comprising the steps of: [0503] 1.
filling the grooves of the centrifugal platter with a fluid; [0504]
2. loading the fluid in the grooves of the centrifugal platter with
at least one oocyte or embryo; [0505] (c) applying centripetal
forces to the kit such that the oocyte or embryo makes contact with
the coring member of the zona coring MEMS device and the coring
member penetrates the zona of the oocyte or embryo forming at least
one zona fragment; and [0506] (d) cessation of centripetal
force;
[0507] In another embodiment, the method of using a zona coring
MEMS device comprises the zona coring MEMS device being affixed to
a centrifugal platter and applying centripetal force to said zona
coring MEMS device. To use the zona coring MEMS device a
centripetal force is applied to the oocyte or embryo contained
within the well the device thereby communicating with the zona
coring MEMS device, the embryo is forced against the zona coring
MEMS device such that the coring means penetrates and attaches onto
the zona pellucida of the oocyte or embryo and creates an opening
in the zona.
[0508] The invention further provides that upon the penetration of
the zona pellucida of the oocyte or embryo by the zona coring MEMS
device and upon the termination of the centripetal force, the
portion of the zona pellucida penetrated by and attached to by the
coring means remains attached to the zona coring MEMS device upon
removal of the oocyte or embryo from the well.
[0509] The present invention provides for a method of making a zona
coring MEMS device wherein a silicon wafer is modified by silicon
etchant/modifying technologies (i.e., deep silicon reactive ion
etching, silicon surface micromachining, LIGA). A variety of
methods of making MEMS structure known to those skilled in the art.
We describe below preferred methods of making the zona coring
devices.
[0510] In other embodiments, a method of using the zona coring MEMS
device kit comprises the steps of: [0511] (a) filling the grooves
of the centrifugal platter with a fluid; [0512] (b) loading the
fluid in the grooves of the centrifugal platter with at least one
oocyte or embryo; and [0513] (c) applying centripetal forces to the
kit such that the oocyte or embryo makes contact with the coring
member of the zona coring MEMS device and the coring member
penetrates the zona of the oocyte or embryo thereby forming a
core;
[0514] The present invention also provides for a method of making a
zona coring MEMS device comprising the steps of: [0515] (a)
applying a first mask layer to a silicon wafer such that a
plurality of wells are inscribed; [0516] (b) etching the first mask
applied in step (a) to form the plurality of wells; [0517] (c)
applying a second mask to the substrate within each etched well
such that a coring member is inscribed within the well; and [0518]
(d) etching the second mask applied in step (c) to form a coring
member.
[0519] In a preferred embodiment, a method of making a zona coring
MEMS device comprises the steps of: [0520] (a) applying a first
mask to a silicon wafer such that a plurality of wells are
inscribed; [0521] (b) etching the first mask applied in step (a) to
form a well; [0522] (c) applying a second mask to the silicon wafer
within each etched cell wells such that a coring member is
inscribed within the well; and [0523] (d) etching the second mask
applied in step (c) to form a coring member.
[0524] A method of making the zona coring MEMS device is
illustrated in FIGS. 21A and B. FIG. 21A shows a first mask 150
being applied to form a zona coring MEMS device and FIG. 21B shows
the results of etching said mask. In FIG. 21A within a well 151 a
mask 150 is deposited to protect a circular structure from etching.
FIG. 21B shows that within a well 151 the coring structure 152
within which there is a coring member 153 such as a barbed coring
member.
Enucleation MEMS
[0525] The technique of nuclear transfer, also known as cloning,
requires the enucleation or removal of the genetic material from
the donor oocyte. Enucleation is commonly performed using a
micropipette by placing the micropipette in the cytoplasm of an
oocyte in a region containing the genetic material or nucleus and
removing it through the micropipette. In most species is it is
difficult to locate the genetic material or nucleus because the
cytoplasm may be relatively opaque or the nuclear membrane may be
relatively translucent.
[0526] Current methods of enucleation are not optimal for removing
the genetic material with great efficiency and often the removal of
excess cytoplasm is unavoidable. This technique represents a
significant investment in highly specialized equipment, extensive
training, and scarce gamete resources. Further, enucleation
efficiencies are highly dependent on the skill of each individual
performing the enucleation. A device that would facilitate the
automation and standardization of this technique would offer
significant advantages over the present state of the art.
[0527] The term "enucleation" refers to the process by which the
nuclear material of an oocyte or early embryo is removed using
needles and handling system similar to those described for
microinjection. Removal of the nucleus creates a recipient cell or
cytoplast for the transplant of a donor cell or nucleus that occurs
during nuclear transfer.
[0528] The present invention further provides enucleation MEMS
devices and kits that are useful in the removal of the genetic
material or nucleus, thereby facilitating enucleation of an oocyte
or embryo. Further, the present invention provides methods of using
the enucleation MEMS devices and kits. Lastly, the present
invention provide for methods of making enucleation MEMS
devices.
[0529] In one embodiment, an enucleation MEMS device for removing
the nucleus from a cell or group of cells comprises a substrate
comprising a plurality of wells for holding a cell or group of
cells to be enucleated, wherein the wells comprise;
[0530] (i) an enucleation penetration member for penetrating a cell
to isolate the nucleus from the cell; and
[0531] (ii) an enucleation pit below the enucleation penetration
member for receiving the nucleus.
[0532] In another embodiment, an enucleation MEMS device further
comprises: [0533] a) a slidable shutter adjacent to the union
between the enucleation penetration member and the enucleation pit
for severing a portion of the cell containing the nucleus; and
[0534] (b) a controller in communication with the slideable shutter
through a circuit lead.
[0535] Another embodiment of the present invention provides for an
enucleation MEMS device kit comprises: [0536] (a) at least one
enucleation MEMS device attached to a top surface of
[0537] (b) a centrifugal platter for applying a centripetal force
to a cell or group of cells contained within a MEMS device and
wherein the centrifugal platter comprises a circular disk, a
plurality of ports for holding the MEMS devices, and a securing
means for attaching to a spinner or driving means.
[0538] In a specific embodiment, the enucleation MEMS device is
permanently affixed to the centrifugal platter.
[0539] In a specific embodiment, the enucleation/nuclear transfer
MEMS device the hollow protuberance is an emitter. Additionally,
the device encompasses when the enucleation penetration member is
an emitter.
[0540] In another embodiment, the enucleation/nuclear transfer MEMS
device kit comprises: [0541] (a) a base substrate comprising an
input well for depositing a cell, a lever element for controlling
the cell apposition and a micropump for handling fluids; and [0542]
(b) an enucleation/nuclear transfer MEMS device.
[0543] In another embodiment, an enucleation MEMS device for
enucleating a cell comprising:
[0544] (a) a base substrate comprising; [0545] (i) an input well to
introduce a cell; [0546] (ii) a lever to control the motion of the
cell; and [0547] (iii) a pump for applying a force to extrude a
portion of the cell; and
[0548] b) an enucleation MEMS device.
[0549] The present invention also provides for a method of making
an enucleation MEMS device comprises the steps of: [0550] (a)
depositing a first mask on the top surface of a substrate
inscribing a square shape; [0551] (b) etching the first mask to
form a plurality of wells; [0552] (c) depositing a second mask in
the wells of step (b) such that an enucleation penetration member
is inscribed at the bottom of each well; [0553] (d) etching the
second mask (g) to form the enucleation penetration member; [0554]
(e) applying a third mask within each well adjacent to the
enucleation penetration member such that an enucleation pit is
inscribed; [0555] (f) etching mask (i) to form the enucleation pit;
[0556] (g) applying a fourth mask such that a slidable shutter is
inscribed; [0557] (h) etching mask (k) to form the slideable
shutter; and [0558] (i) depositing a circuit lead to provide
communication between the shutter and a controller.
[0559] In a particular embodiment the enucleation MEMS device kit
comprises:
[0560] (a) a centrifugal platter comprises a plurality of grooves
arranged in a concentric pattern and wherein each groove has an
inner and outer edge;
[0561] (b) at least one enucleation MEMS device is bonded to the
outer edge of a groove in an orientation such that the axis of each
well of the enucleation MEMS device is horizontal to the plane of
the centrifugal platter; and
[0562] (c) the inner edge of the grooves forming divided
compartments comprising a single well that restricts the movement
of materials from one compartment containing a single well to
another compartment.
[0563] The present invention also provides a method of using the
enucleation MEMS device kit comprising:
[0564] (a) filling the grooves of the centrifugal platter with a
fluid;
[0565] (b) loading the fluid within the grooves of the centrifugal
platter with at least one oocyte or embryo;
[0566] (c) rotating the kit such that centripetal forces are
applied to the centrifugal platter such that the oocyte or embryo
are thrust against the wall of the cell well such that the
enucleation penetration member of the enucleation MEMS device
penetrates the surface of the oocyte or embryo; and
[0567] (d) a portion of the cell contents containing the nucleus
are extruded out of oocyte or embryo into the enucleation pit.
[0568] In a specific embodiment, a method of using the enucleation
MEMS device kit comprises the steps of: [0569] (a) filling the
grooves of the centrifugal platter with a fluid; [0570] (b) loading
the fluid in the grooves of the centrifugal platter with at least
one oocyte or embryo; [0571] (c) applying centripetal forces to the
centrifugal platter by rotating the kit such that the oocyte or
embryo are thrust against the wall of the cell well such that the
enucleation penetration member of the enucleation MEMS device
penetrates the surface of the oocyte or embryo; [0572] (d)
extruding cell contents out of oocyte or embryo into the
enucleation pit; and [0573] (e) severing a remnant of cell that has
extruded into the extrusion pit using a slideable shutter.
[0574] In a more specific embodiment, the enucleation MEMS device
further comprises a slideable shutter element at the union between
the base of the enucleation penetration member and the enucleation
pit.
[0575] Alternatively, an embodiment of the invention provides an
enucleation MEMS device array comprising: a plurality of cell wells
wherein each cell well contains an enucleation penetration member,
each cell well is in fluid communication with an evacuation siphon,
and a compressible substance is contained within the evacuation
siphon.
[0576] In a more specific embodiment, an enucleation MEMS device
further comprises a slidable shutter member between each
enucleation penetration member and each evacuation siphon. The
present invention also provides for an alternative method of using
the enucleation MEMS device comprising:
[0577] (a) filling an input well with a fluid;
[0578] (b) loading the input well with an oocyte or embryo; and
[0579] (c) applying a centrifugal force by rotating the enucleation
MEMS device.
[0580] The present invention also provides for an enucleation MEMS
device kit comprising: [0581] (a) at least one enucleation MEMS
device; and [0582] (b) a centrifugal platter for applying a
centripetal force to a cell or group of cells contained within a
MEMS device and wherein the centrifugal platter comprises a
circular disk having a plurality of ports for holding the MEMS
devices, and a securing means for securing the platter to a spinner
or driving means. In another embodiment, an enculeation MEMS device
kit comprises: [0583] (a) at least one enucleation MEMS device
attached to a top surface of [0584] (b) a substrate base wherein
the substrate base comprises a lever for applying a force to the
oocyte or embryo thrusting it against the enucleation MEMS device
and a fluid handling means with pump in fluid communication with
the enucleation pit and thus providing suction to the enucleation
pit.
[0585] The present invention provides for a method of using an
enucleation MEMS device. The invention further provides for a
method of using an enucleation MEMS device comprising an
enucleation MEMS device being affixed to a means for applying
centripetal forces to said enucleation MEMS device. The invention
further provides for a method of using an enucleation MEMS device
comprising the enucleation MEMS device being affixed to a means for
applying centripetal forces to said enucleation MEMS device further
comprising a well for receiving an oocyte or embryo communicating
directly with an enucleation MEMS device such that when a
centripetal force is applied the oocyte or embryo contained within
the well communicating with the enucleation MEMS device, the oocyte
or embryo is forced against the enucleation penetration member of
the enucleation MEMS device such that the enucleation penetration
member of the enucleation MEMS device penetrates the zona pellucida
and the oollemma of the oocyte or embryo.
[0586] The invention further provides that upon the penetration of
the zona pellucia and oollemma of the oocyte or embryo by the
enucleation penetration member of the enucleation MEMS device the
centripetal force facilitates the migration of the genetic material
through the opening in the oollemma and the zona pellucida and into
the enucleation pit of the enucleation MEMS device. The invention
further provides that upon the termination of the centripetal force
the genetic material, having migrated into the enucleation pit of
the enucleation MEMS device, remains in the enucleation pit of the
enucleation MEMS device upon removal of the oocyte from the pit.
Further, the invention provides for an enucleation guillotine or
enucleation slideable shutter that facilitates the severance of any
connection between the genetic material in the enucleation pit and
the oocyte or embryo. The present invention provides for a method
of manufacture of an enucleation MEMS device wherein a silicon
wafer is modified by silicon etchant/modifying technologies (e.g.,
deep silicon reactive ion etching, silicon surface micromachining,
LIGA). Methods of making MEMS devices are set forth in particular
in Section A Supra.
[0587] FIGS. 22A-F show a series of masks and etches that give rise
to an individual MEMS device of an enucleation MEMS array._In FIG.
22A a first mask 160, is deposited on the surface of a wafer such
that a square shape is inscribed 161. The first mask is etched and
a well 162 is formed. A second mask 163 is deposited to begin
formation of the enucleation penetration member 164. The second
mask 163 is etched a third mask 165 is deposited and etched, a
fourth mask is deposited and etched, and a fifth mask 167 protects
formed enucleation penetration member and allows a central square
to be etched such that an enucleation pit 168 is formed. FIG. 22F
illustrates a side-view of the cell well 162 showing the
enucleation pit 170, and the enucleation penetration member
169.
[0588] FIGS. 23A and B show a perspective and top view of an
enucleation guillotine 180. In the union between the enucleation
penetration member 181 and the enucleation pit 170 of an
enucleation MEMS device there resides a slideable shutter door 182,
and adjacent to the enucleation shutter door 182 is a slideable
shutter 183 with a plane of movement illustrated by an arrow. This
slideable shutter 183 is actuated by a controller 184 in
communication with the shutter 183 by way of a circuit lead 185.
the slideable shutter 183 is operated via a sliding means 186
activated by a gear assembly 187.
[0589] The present invention provides for a method of manufacture
an enucleation MEMS device wherein a silicon wafer is modified by
silicon etchant/modifying technologies (e.g., deep silicon reactive
ion etching, silicon surface micromachining, LIGA). See Section A
above.
[0590] A method of making an enucleation MEMS device comprising the
modification of a substrate (i.e., silicon wafer, plastic, metallic
oxide, other etchable and depositable substrate material) such that
the etching and deposition of material on the substrate (i.e.,
LIGA, DRIE, silicon fusion bonding, laser etching, laser mediated
and directed polymerization of substrate). Using these and other
art-known MEMS fabrication methodologies the substrate is modified
to form a central loading manifold, cell wells, evacuation siphon
that is continuous with the enucleation penetration member
containing in its end distal to the enucleation penetration member
a compressible substance, an enucleation penetration member, and an
enucleation guillotine or slideable shutter at the union between
the enucleation penetration member and the portion of the
evacuation siphon proximal to the enucleation penetration
member.
[0591] In one embodiment, a method of making an enucleation MEMS
device comprising the steps: [0592] (a) etching a plurality of
parallel channels on a first side of a plurality of silicon wafers
in which the wafers each have a second unetched side; [0593] (b)
silicon fusion bonding the unetched side of a plurality of silicon
wafers of step (a) to the etched side of a plurality of silicon
wafers of step (a) such that the etched channels are in parallel to
form a mega-laminate wherein the mega-laminate has a plurality of
channels; [0594] (c) cutting the mega-laminate at an angle
perpendicular to the long axis of the etched channels thereby
forming a slice of the mega-laminate having a top surface and a
bottom surface wherein each surface exposes an end of the channel;
[0595] (d) silicon fusion bonding the bottom surface of the slice
of the mega-laminate to the etched side of a channel-etched
base-plate wafer; [0596] (e) depositing a first mask on the top
surface of the slice of the mega-laminate such that a region
surrounding each channel end is free of mask; [0597] (f) etching
the mask to form a plurality of wells [0598] (g) depositing a
second mask in the wells of step (f) such that an enucleation
penetration member is inscribed at the bottom of each well; [0599]
(h) etching the second mask (g); [0600] (i) applying a third mask
within each well adjacent to the enucleation penetration member
such that an enucleation pit is inscribed; [0601] (j) etching mask
(i); [0602] (k) applying a fourth mask such that a slidable shutter
is inscribed; [0603] (l) etching mask (k) and [0604] (m) depositing
a circuit lead in operable communication between the pump/valve and
a controller
[0605] In a specific embodiment, the method of making an
enucleation MEMS device further comprises the step of applying a
coating to the mega-laminate top surface after step (h). In more
specific embodiments, the coating is a polypeptide, peptide or
protein. In yet a more preferred embodiment, the polypeptide is
polylysine.
[0606] In another embodiment, the method of making an enucleation
MEMS device utilizes a method of making a channel-etched base-plate
silicon wafer with a pump comprising the steps of:
[0607] (a) etching a silicon wafer with a plurality of channels
which are in fluid communication with an input manifold
reservoir;
[0608] (b) etching the silicon wafer of step (a) whereby a
pump/valve is constructed in each channel; and
[0609] (c) depositing a circuit lead in operable communication
between the pump/valve and a controller.
[0610] In a specific embodiment, the method of making a
channel-etched base-plate silicon wafer with a pump further
comprises attaching a piezoelectric pump/valve to the channels.
[0611] In one embodiment, the enucleation penetration member may be
so constructed as to act as an emitter. In particular, the
enucleation penetration member may act as a wave guide to conduct
electromagnetic signals (e.g., pulses of light of any frequency).
Additionally, the enucleation penetration member can provide
vibrational energy (e.g., sound waves, e.g., ultrasonic waves).
N. Enucleation/Nuclear Transfer MEMS
[0612] As discussed in Section M, supra, the technique of nuclear
transfer, also known as cloning, requires the removal of the
genetic material or nucleus from the donor oocytes and the
introduction of the donor nucleus into the recipient enucleated
cell.
[0613] The term "nuclear transfer" refers to the process whereby a
cell or nucleus of a cell is transplanted, using needles and the
handling system similar to those described for microinjection, into
an oocyte from which the nucleus has been removed (i.e., a
recipient cell or cytoplast). This process gives rise to an embryo
that carries the donor nuclei's genetic information.
[0614] Further, the introduction of a donor nucleus into the
cytoplasm of the recipient cell requires either the introduction of
a donor cell into the perivitelline space with a subsequent
electropulse facilitating the fusion of the donor cell with the
oocyte or the direct injection of either a donor cell or a donor
nucleus. These techniques represent a significant investment in
highly specialized equipment, extensive training, and scarce gamete
resources. Further, enucleation as well as nuclear transfer
efficiencies are highly dependent on the skill of each individual
performing the procedures. A device that would facilitate the
automation and standardization of these technique would offer
significant advantages over the present state of the art.
[0615] The present invention provides for the enucleation/nuclear
MEMS devices and kits for the removal of the genetic material of an
oocyte or embryo, facilitating enucleation, as well as the
simultaneous or subsequent injection of a donor cell or donor
nucleus into the cytoplasm of the recipient oocyte or embryo. The
present invention also provides for methods of using the
enucleation/nuclear transfer MEMS devices and kits and methods of
making same.
[0616] FIGS. 24A and B illustrate specific embodiments of the
present invention in a side cross-sectional view and perspective
view a well of an enculeation/nuclear transfer MEMS device. FIG.
24A shows a well 96 with a microneedle or hollow protuberance 97 in
fluid communication with a donor nucleus/cell injection channel
190, an enucleation pit 170, and an enucleation penetration member
169. Additionally, the position of the channel plate 191 and the
silicon fusion bonded (sfb) interface 192 is shown. FIG. 24B shows
the well 96 comprising the microneedle 97 or hollow protuberance,
the enucleation penetration member 169, and the enucleation pit
170.
[0617] FIGS. 25A-B show an enucleation/nuclear transfer MEMS array
with a detailed inset view of an enucleation/nuclear transfer MEMS
device. FIG. 25A shows a central loading manifold 206, a cell 19 in
an input well 200 that is continuous with an enucleation/nuclear
transfer MEMS unit 201 that has evacuation siphons 202. FIG. 525B
shows a well 96, an enucleation siphon proximal portion 203 that
receives an extruded portion of the cell 207 upon rotation of the
array (arrow 204 points out direction of force generated upon
rotation). The evacuation siphon distal portion 205 is filled with
a compressible substance 206 that, upon rotation of the array pulls
a partial vacuum at the proximal portion 203 such that the
extrusion of the cell 19 is facilitated. A slideable enucleation
shutter 183 below the enucleation penetration member 181
facilitates completion of the removal of the extruded portion of
the cell 19.
[0618] FIG. 26A is another view one an enucleation/nuclear transfer
MEMS device of an encleation/nuclear transfer MEMS array a central
loading manifold 220 that is in fluid communication with the
dynamic hydropressure column 221 with a gating element 225 that
injects a donor nucleus or donor cell into the cell 19 and the
extraction siphon 222 facilitates enucleation. FIG. 26B is a side
cross-sectional view of an enucleation/nuclear transfer MEMS unit
showing a cell 19 with its zona 223 in a well 96, an enucleation
penetration member 181, a slideable enucleation shutter 183, an
evacuation siphon 222 that has a compressible substance 206 that
compresses upon rotation of the array, in part facilitating the
extrusion of an extruded portion of a cell with a nucleus 207 into
the enucleation siphon 222. The donor nucleus or donor cell 223 is
simultaneously brought from the central loading manifold 220 (in
FIG. 26A) through the dynamic hydropressure column 122, past a
regulating valve 123, being actuated by a controller 128 in
communication with the valve 123 by way of a lead 127, then
injected through the hollow protuberance or microneedle 124 into
the cell 19.
[0619] In one embodiment, an enucleation/nuclear transfer MEMS
device for the enucleation and transfer of a donor nucleus or donor
cell comprises a substrate comprising: [0620] (a) a central loading
manifold for the loading of a cell or group of cells into the
device; [0621] (b) at least one well for holding a cell during the
enucleation process, wherein the well comprises: [0622] (i) a
hollow protuberance in the well for penetrating the cell to
introduce a donor nucleus; [0623] (ii) an enucleation penetration
member for penetrating a cell to facilitate the removal of a cell
nucleus; [0624] (iii) an enucleation evacuation siphon to provide
suction to remove the nucleus from the cell forming an enucleated
cell; and [0625] (c) a dynamic hydropressure column for providing a
pressurized fluid to introduce the donor nucleus through the hollow
protuberance into the enucleated cell.
[0626] In another embodiment, an enucelation/nuclear transfer MEMS
device further comprises a gate on the dynamic hydropressure column
for the modulation of fluid handling and allowance of a single
donor nucleus or donor cell to pass through to the hollow
protuberance for injection into the cytoplasm of the enucleated
cell, such as an oocyte or embryo.
[0627] In another embodiment, an enucleation/nuclear transfer MEMS
device further comprises a slideable shutter that operates at the
union between the enucleation penetration member and the
enucleation siphon.
[0628] The present invention also provides for a method of using
enucleation/nuclear transfer MEMS device described above comprising
the steps of:
[0629] a) filling the enucleation/nuclear transfer MEMS device
central loading manifold with a fluid;
[0630] b) loading a donor nucleus or donor cell into the central
loading manifold;
[0631] c) loading the well with an oocyte or embryo; and
[0632] d) applying a force to the enucleation/nuclear transfer MEMS
device to facilitate the enucleation of the oocyte or embryo and
the introduction of a donor nucleus or donor cell into the
enucleated oocyte or embryo.
[0633] The present invention provides for a method of using an
enucleation/nuclear transfer MEMS device comprising the
enucleation/nuclear transfer MEMS device being affixed to a means
for applying centripetal forces to said enucleation/nuclear
transfer MEMS device. The invention further provides for a method
of using an enucleation/nuclear transfer MEMS device comprising the
enucleation/nuclear transfer MEMS device being affixed to a means
for applying centripetal forces to said enucleation/nuclear
transfer MEMS device further comprising a well for receiving an
oocyte or embryo communicating directly with an enucleation/nuclear
transfer MEMS device such that when a centripetal force is applied
the oocyte or embryo contained within the well communicating with
the enucleation/nuclear transfer MEMS device, is forced against the
enucleation/nuclear transfer MEMS device such that the enucleation
penetration member of the enucleation/nuclear transfer MEMS device
penetrates the zona pellucida and the oollemma of the oocyte or
embryo.
[0634] The invention further provides that upon the penetration of
the zona pellucia and oollemma of the oocyte or embryo by the
enucleation penetration member of the enucleation/nuclear transfer
MEMS device, the centripetal force facilitates the migration of the
genetic material through the opening in the oollemma and the zona
pellucida and into the enucleation pit of the enucleation/nuclear
transfer MEMS device while a donor nucleus or donor cell travels
through the hollow protuberance of the enucleation/nuclear transfer
MEMS device such that the donor nucleus or donor cell is injected
into the cytoplasm of the enucleated oocyte or embryo. The
invention further provides that upon the termination of the
centripetal force the genetic material, having migrated into the
enucleation well or evacuation siphon of the enucleation/nuclear
transfer MEMS device, remains in the enucleation well or evacuation
siphon of the enucleation/nuclear transfer MEMS device upon removal
of the oocyte or embryo from the pit. The present invention further
provides for the maintenance of a positive pressure in the
microinjection means of the enucleation/nuclear transfer MEMS
device. Further, the invention provides for an enucleation
guillotine or enucleation slideable shutter element that
facilitates the severance of any connection between the genetic
material in the enucleation pit and the enucleated oocyte or
embryo.
[0635] The present invention further provides for
enucleation/nuclear transfer MEMS device kits comprising at least
one enucleation/nuclear transfer MEMS device attached to a
centrifugal platter to provide support and facilitate applying a
centripetal force to the device.
[0636] In one embodiment, the enucleation penetration member may be
so constructed as to act as an emitter. In particular, the
enucleation penetration member may act as a wave guide to conduct
electromagnetic signals (e.g., pulses of light of any frequency).
Additionally, the enucleation penetration member can provide
vibrational energy (e.g., sound waves, e.g., ultrasonic waves).
[0637] In another embodiment, the hollow protuberance may be so
constructed as to act as an emitter. In particular, the hollow
protuberance may act as a wave guide to conduct electromagnetic
signals (e.g., pulses of light of any frequency). Additionally, the
hollow protuberance can provide vibrational energy (e.g., sound
waves, e.g., ultrasonic waves).
[0638] In a more specific embodiment, of the enucleation/nuclear
transfer MEMS device kit, the enucleation/nuclear transfer MEMS
device is permanently affixed to the centrifugal platter.
[0639] Alternatively, the present invention provides for
enucleation/nuclear transfer MEMS device kits comprising at least
one enucleation/nuclear transfer MEMS device attached to a
substrate base to provide support and facilitate applying a force
to the device.
[0640] Alternatively, the enucleation/nuclear transfer MEMS device
of the present invention may be affixed to a substrate base as
described previously for MEMS devices of the present invention
wherein cell apposition to the enucleation/nuclear transfer MEMS
device is mediated by a lever element and fluid handling by
micropumps. Both the micropumps and the lever elements are actuated
by a controller by way of circuit leads in communication between
the micropumps, the lever elements and the controller.
[0641] In yet another more specific embodiment, the present
invention provides for an enucleation/nuclear transfer MEMS device
kit wherein
[0642] (a) the centrifugal platter comprises a plurality of grooves
arranged in a concentric pattern and wherein each groove has an
inner and outer edge;
[0643] (b) at least one enucleation/nuclear transfer MEMS device is
bonded to the outer edge of a groove in an orientation such that
the axis of each well of the enucleation/nuclear transfer MEMS
device is horizontal to the plane of the centrifugal platter;
and
[0644] (c) the inner edge of the grooves forming divided
compartments comprising a single well which restrict the movement
of materials from one compartment containing a containing a single
well to another compartment.
[0645] The present invention also provides for a method of using
the enucleation/nuclear transfer MEMS device kits comprising the
steps of: [0646] 1. filling the input manifold of at least one
enucleation/nuclear transfer MEMS device with a fluid; [0647] 2.
loading the fluid-filled wells of step (b) with at least one oocyte
or embryo; [0648] 3. rotating the kit and thus applying a
centripetal force on the enucleation/nuclear transfer
MEMS/centrifugal platter.
[0649] In another embodiment, a method of using an
enucleation/nuclear transfer MEMS device kit comprises the steps
of: [0650] (a) loading donor nuclei or donor cells into the central
loading manifold; [0651] (b) filling the grooves of the centrifugal
platter with a fluid; [0652] (c) loading the grooves of the
centrifugal platter with at least one oocyte or embryo; [0653] (d)
applying a centripetal force to the kit whereby the oocyte or
embryo makes contact with the hollow protuberance of the
enucleation/nuclear transfer MEMS device and the enucleation
penetration member and thereby facilitating enucleation and nuclear
transfer.
[0654] The present invention provides for a method of manufacture
an enucleation/nuclear transfer MEMS device wherein a silicon wafer
is modified by silicon etchant/modifying technologies (e.g., deep
silicon reactive ion etching, silicon surface micromachining,
LIGA).
[0655] A method of making an enucleation/nuclear transfer MEMS
device comprising the modification of a substrate (i.e., silicon
wafer, plastic, metallic oxide, other etchable and depositable
substrate material) such that the etching and deposition of
material on the substrate (i.e., LIGA, DRIE, silicon fusion
bonding, laser etching, laser mediated and directed polymerization
of substrate). Using these and other art-known MEMS fabrication
methodologies the substrate is modified to form a central loading
manifold, cell wells, dynamic hydropressure columns in fluid
communication between the central loading manifold and the hollow
protuberances in the cell wells, evacuation siphon that is
continuous with the base of the enucleation penetration member
containing in its end distal to the cell well a compressible
substance, an enucleation penetration member, an enucleation
guillotine or slideable shutter at the union between the
enucleation penetration member and the portion of the evacuation
siphon proximal to the enucleation penetration member. Further,
this method of making may include a gate element on the dynamic
hydropressure column that facilitate precise fluid control
temporally and volumetrically, and allows a single donor nucleus or
donor cell to pass through the hollow protuberance and into the
enucleated oocyte or embryo.
[0656] In one embodiment, a method of making an enucleation/nuclear
transfer MEMS device comprising the steps: [0657] (a) etching a
plurality of parallel channels on a first side of a plurality of
silicon wafers in which the wafers each have a second unetched
side; [0658] (b) silicon fusion bonding the unetched side of a
plurality of silicon wafers of step (a) to the etched side of a
plurality of silicon wafers of step (a) such that the etched
channels are in parallel to form a mega-laminate wherein the
mega-laminate has a plurality of channels; [0659] (c) cutting the
mega-laminate at an angle perpendicular to the long axis of the
etched channels thereby forming a slice of the mega-laminate having
a top surface and a bottom surface wherein each surface exposes an
end of the channel; [0660] (d) silicon fusion bonding the bottom
surface of the slice of the mega-laminate to the etched side of a
channel-etched base-plate wafer; [0661] (e) depositing a first mask
on the top surface of the slice of the mega-laminate such that a
region surrounding each channel end is free of mask; [0662] (f)
etching the mask to form a plurality of wells [0663] (g) depositing
a second mask in the wells of step (f) such that an enucleation
penetration member is inscribed at the bottom of each well; [0664]
(h) etching the second mask (g); [0665] (i) applying a third mask
within each well adjacent to the enucleation penetration member
such that an enucleation pit is inscribed; [0666] (j) etching mask
(i); [0667] (k) applying a fourth mask such that a slidable shutter
is inscribed; [0668] (l) etching mask (k) and [0669] (m) depositing
a circuit lead in operable communication between the slideable
shutter and a controller
[0670] In a specific embodiment, the method of making an
enucleation/nuclear transfer MEMS device further comprises the step
of applying a coating to the mega-laminate top surface after step
(h). The coating is to prevent the cells from adhering or sticking
to the elements of the devices. In more specific embodiments, the
coating is a polypeptide, peptide or protein. In yet a more
preferred embodiment, the polypeptide is polylysine.
[0671] In another embodiment, the method of making an
enucleation/nuclear transfer MEMS device utilizes a method of
making a channel-etched base-plate silicon wafer with a pump
comprising the steps of:
[0672] (a) etching a silicon wafer with a plurality of channels
which are in fluid communication with an input manifold
reservoir;
[0673] (b) etching the silicon wafer of step (a) whereby a
pump/valve is constructed in each channel; and
[0674] (c) depositing a circuit lead in operable communication
between the pump/valve and a controller.
[0675] In a specific embodiment, the method of making a
channel-etched base-plate silicon wafer with a pump further
comprises attaching a piezoelectric pump/valve to the channels.
O. Cytoplasmic Transfer MEMS Array
[0676] The present invention provides cytoplasmic transfer MEMS
devices, methods of using same and methods of making same.
[0677] A cytoplasmic transfer MEMS device facilitates the automated
extraction of an aliquot of cytoplasm from a donor oocyte or embryo
and the injection of that cytoplasmic aliquot into a recipient
oocyte or embryo. This cytoplasmic transfer MEMS device can be
fabricated in arrays of more than one cytoplasmic transfer MEMS
device and as such can provide for the transfer of cytoplasm from
many pairs of donor/recipient oocytes or embryos simultaneously.
Cytoplasmic transfer is utilized in human Assisted Reproduction to
ameliorate cytoplasmic insufficiencies in some patient oocytes or
embryos.
[0678] In one embodiment, the present invention provides for a
cytoplasmic transfer MEMS device for the transfer of cytoplasm from
one cell to another comprising:
[0679] (a) a cytoplasmic transfer MEMS device comprising a
substrate comprising: [0680] (i) at least one first well wherein
each first well compromises a hollow protuberance; [0681] (ii) at
least one second well wherein each second well compromises a hollow
protuberance; [0682] (iii) an extraction siphon in fluid
communication with the hollow protuberance in the first well and
with the hollow protuberance in the second well; [0683] (iv) a
supplemental input channel in fluid communication with the
extraction siphon; and
[0684] (b) a centrifugal platter for applying a centripetal force
to a cell or group of cells contained within a MEMS device and
comprises a circular disk having a plurality of ports for holding
the MEMS device.
[0685] In another embodiment, a cytoplasmic transfer MEMS device
further comprises: [0686] a) a first valve on the extraction siphon
proximal to first input well; [0687] b) a supplemental input
channel located between the first and second valves for providing
fluid flow once the first gate closes and the second gate opens;
and [0688] c) a second valve on the extraction siphon distal to the
supplemental input channel.
[0689] In yet another embodiment, the cytoplasmic transfer MEMS
device has a supplemental input channel that enters the extraction
siphon between the first valve and the second valve. Upon operation
of the cytoplasmic transfer MEMS device a portion of the cytoplasm
of the donor oocyte or embryo enters the extraction siphon, passes
the open first valve, then the first valve closes. Isolating the
cytoplasmic aliquot. Then fluid flow through the supplemental input
channel, in fluid communication with the cell well, and the opening
of the second gates allows the cytoplasmic aliquot to travel to and
through the hollow protuberance in the second cell well and into
the cytoplasm of the recipient oocyte or embryo.
[0690] The present invention also provides a method of using a
cytoplasmic transfer MEMS device comprising: [0691] a) loading a
fluid in the extraction siphon; [0692] b) loading a cytoplasmic
donor oocyte or embryo in the first well; [0693] c) loading a
recipient oocyte or embryo in the second well; and [0694] d)
applying a force to the cytoplasmic transfer MEMS device by
rotating the array. FIG. 27 shows a preferred embodiment of a
single cytoplasmic transfer MEMS device. A first well 230, receives
a cytoplasmic donor oocyte or embryo 231 whereby upon application
of a force and or suction to the cytoplasmic transfer MEMS device a
cytoplasmic aliquot 232 is extruded into the extraction
siphon-proximal portion 233 to the first gate 234 where upon the
first gate 234 closes, the cytoplasmic aliquot 232, finding a
second gate 235 closed, is stopped momentarily. Upon opening of the
second gate 235 fluid from the supplemental input channel 236 the
cytoplasmic aliquot 233 proceed through the extraction
siphon-distal portion 237, through a hollow protuberance 12 and
into a recipient oocyte or embryo 239 having been loaded into a
second well 240.
[0695] The present invention provides for a method for making a
cytoplasmic transfer MEMS device wherein a substrate (i.e., silicon
wafer, plastic, metallic oxide, other etchable and depositable
substrate material) is modified by art known methods of MEMS
fabrication (i.e., LIGA, DRIE, silicon fusion bonding, laser
etching, laser mediated and directed polymerization of substrate
surface). These modification are applied to the substrate such that
certain structures are formed and in particular for the cytoplasmic
transfer MEMS device an extraction siphon, a first cell well with a
hollow protuberance in fluid communication with the extraction
siphon, a first gate, a supplemental input fluid handling means in
fluid communication with the cell well, second gate are formed in
the substrate, and a second cell well with a hollow protuberance in
communication with the extraction siphon. These structures may be
built or embedded on a spinnable platter, a centrifugal platter,
for the ability to provide centripetal to the cytoplasmic transfer
MEMS device.
[0696] Alternatively, these structures may be built in or affixed
to a substrate base that provides fluid handling by way of
micropumps and cell apposition to the cytoplasmic transfer MEMS
device by way of a lever element. Further, the micropumps and lever
elements are in communication with a controller by way of circuit
leads.
[0697] The following examples are presented for purposes of
illustration only and are not intended to limit the scope of the
invention in any way.
EXAMPLES
[0698] The following protocols and experimental methods and
materials are employed in the Examples that follow.
Superovulation
[0699] Mice (i.e., RB Swiss, (CBA*C57BL6/J)fl) are given 5 i.u./ml
Pregnant Mares Serum Gonadotrophin (PMSG) interperitoneally (i.p.).
At 46 to 48 hours post injection a second injection is i.p.
administered providing 5 i.u./ml Human Chorionic Gonadotrophin
(hCG) in Phosphate Buffered Saline (PBS). If mating is desired,
females are placed with males immediately.
Blastocyst Flush
[0700] The uterus is removed from 3.5 day pregnant mice and placed
into sterile PBS. Using a sterile fine forceps the mesometrium is
trimmed and the ovaries, oviducts, the utero-tubal junction, and
the cervical bifurcation are dissected from both of the uterine
horns. The uterine horn is flushed using a syringe of DMEM
(Dulbecco's Modification of Eagle's Medium (Mod.) 1.times. (DMEM)
with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate; Fisher
Scientific cat. # MT10013CM) with HEPES with a blunt-ended 23 gauze
needle. Using sterile forceps, a uterine horn is picked up at one
end and the blunt-ended needle is inserted just inside the open end
of the horn. The syringe plunger is pushed and the horn is flushed
with DMEM with HEPES medium into a sterile plastic dish. The horn
is then flushed from other end. The remaining uterine horn is
flushed in the same manner. Flushed blastocysts are now present in
the sterile plastic dish. Using a transfer pipette blastocysts are
collected and transferred into small drops of culture medium under
an overlayment of sterile inert culture medium-equilibrated oil.
Dishes with drops are incubated at 37.degree. C. with 5%
CO.sub.2.
Blastocyst Transfer (Assay for Developmental Fitness of Manipulated
Cells)
[0701] Blastocyst transfer is performed 24 hours after aggregation
when the morulae have become expanded blastocysts and on the same
day as manipulation. Using Rompun/Ketavet at 0.02 ml/g body weight
provide i.p. anesthesia to animals. To the shaved sterilized
abdomen, an incision is made and the uterine horns exteriorized. A
transfer pipette loaded with embryos and DMEM is inserted into the
end of a uterine horn and the contents are expelled into the horn.
After transfer of embryos into second horn the uterus is replaced
into the body cavity, the wound is sealed with clips, and the
animal allowed to recover. Offspring will be born approximately 16
days after transfer.
One-Cell Transfer into Pseudopregnant Recipient Female Mouse
[0702] After checking for cell fitness (i.e., cytoplasmic
condensation, causing the cellular material to become less glossy
and darker in color as the cytoplasm shrinks away from the Zona
Pellucida pellucida, indicating a damaged cell) cells are
transferred into a pseudopregnant recipient female mouse as before.
Offspring will be born approximately 19-21 days after transfer.
Oocyte and Morula Harvest
[0703] After superovulation for oocytes and at 2.5 days p.c. (post
coitus) for morulae, these cells are present in the oviducts. After
animal sacrifice the oviducts are removed to sterile PBS. Oviducts
are flushed with flushed into culture medium and morulae are
collected, placed in drops of culture under oil, and incubated at
37.degree. C. with 5% CO.sub.2.
Post Manipulation Viability Assays
[0704] Oocytes or embryos are placed in vital stain and then
live-mounted onto slides. A microscope is used to determine if the
vital stain has penetrated the cell membrane indicating cell
injury.
[0705] Oocytes are placed in maturation culture and their ability
to mature to the various checkpoints of oocyte maturation (Meiosis
I, Meiosis II) is determined. Additionally, depending on stage of
oocyte at time of manipulation and the type of manipulation, the
oocyte in vitro fertilized to determine fitness for fertilization.
If further early embryonic development is to be assayed the
fertilized embryos are replaced to in vitro culture and extent of
development can be determined. If offspring are desired for the
determination of effect of manipulation on oocytes or embryos the
embryos must be transferred into a recipient animal and allowed to
gestate.
Immunocytochemistry Methanol Fixation
[0706] Cells are extracted in Microtubule Stabilizing Buffer (MTSB;
80 mM PIPES pH 6.8, 1 mM MgCl2, 4 mM EGTA)+0.5% TX-100 for 30
seconds. Cells then are fixed in -20.degree. C. methanol for 1-2.5
minutes, rinsed in TBS 0.15 M NaCl, 0.02 M Tris-Cl pH 7.4),
permeabilized in TBS-0.5% TX for 10 minutes, rinsed in TBS-0.1%
Triton X, and stored in blocking medium.
Immunofluorescence
[0707] Primary antibody diluted in blocking medium is added to the
cells for 1-3 hours. The cells are washed in TBS-0.1%/TX and then
secondary antibody is added to the cells for one hour. The cells
are then washed in TBS-0.1% TX. If another primary antibody is to
be used it can either be added when the first primary antibody was
used or after the first secondary antibody is added and washed. For
nuclear staining the cells are incubated in 1-10 ug/ml DAPI or
Hoescht in blocking medium for 10 minutes. The cells are washed in
TBS-0.1% TX and rinsed in TBS. The cells are then mounted on a
slide with mounting medium (0.5% p-phenylenediamine (Free Base;
Sigma) in 20 mM Tris, pH 8.8, 90% glycerol) and a coverslip is
sealed over top with acrylic.
Example
Use of the Cell Labeling MEMS Device and the Labelable Zona Anchor
MEMS Device
Operation of the Cell Labeling and Labelable Zona Anchor MEMS
Devices
[0708] The cell labeling MEMS device kit (with the centrifugal
platter) is placed onto a spinning device. Cell wells are filled
with loading fluid (i.e., PBS/PVA, M199 media). The labelable zona
anchor MEMS device is spun at 5 g for 10 seconds to purge bubbles.
Oocytes or embryos are placed into cell wells by mouth pipette,
robotic pipette, or other manner. Spinner is rotated at 5 g for 10
seconds and stop. Remove cells to incubated environment.
(Alternatively, CPU controller on spinner provides a ramp up to 5 g
then ramp down, 10 seconds duration).
[0709] Alternatively, the labelable zona anchor MEMS device can be
situated on a substrate base that facilitates close opposition of
the oocyte or embryo with the active domain of the labelable zona
anchor MEMS device by way of a lever element. The lever element is
actuated by a controller being in communication with these elements
by way of circuit leads.
Determination of Labelable Zona Anchor MEMS Device Being Embedded
in Oocyte or Embryo Zona Pellucida
[0710] Cells are observed by microscope to determine rates of
successful attachment of labelable zona anchor MEMS device to the
Zona Pellucida.
Determination of Effect of Labelable Zona Anchor MEMS Device on
Oocyte and Embryo Viability, Developmental Progression and
Cytoskeleton
[0711] Cells are assayed for immediate effect of manipulation on
cell viability (vital staining) as well as for developmental
fitness (either early embryonic development or ability to produce
offspring in recipient animal). Further, cells are processed for
immunofluorescence to visualize microtubule and actin networks to
determine effect of manipulation on the cytoskeleton.
Operation of Labelable Zona Anchor MEMS Device Platform
[0712] Cells that have labelable zona anchor MEMS device embedded
in their Zona Pellucidas are exposed to a labelable zona anchor
MEMS device platform that is in an labelable zona anchor MEMS
device attractive state (i.e., the labelable zona anchor MEMS
device channels of the platform are magnetized and thus attractive
to magnet-attractive labelable zona anchor MEMS devices). The
labelable zona anchor MEMS device platform is observed under a
microscope to determine whether labelable zona anchor MEMS
device/cell conjugates have attracted to the labelable zona anchor
MEMS device platform. The labelable zona anchor MEMS device
platform with attached cells is then attached to a labelable zona
anchor MEMS device platform holder and the holder is also loaded
with culture media. Observation of ability of labelable zona anchor
MEMS device/cells to remain attached to the labelable zona anchor
MEMS device platform is made while the labelable zona anchor MEMS
device platform is situated within the holder. The labelable zona
anchor MEMS device platform/holder assembly is transported to a
receiving docking domain (i.e., automatic multi-compartment
multi-modal incubation device) and the platform is docked to the
receiving docking domain (i.e., automatic multi-compartment
multi-modal incubation device). Further observations for the
determination of ability of labelable zona anchor MEMS device/cells
to remain attached to the labelable zona anchor MEMS device
platform are made at this time. Additionally, the platform holder
with the extended handle in FIG. 5c is docked to a platform that
holds labelable zona anchor MEMS device/cell conjugates. This
labelable zona anchor MEMS device platform/holder is then used to
insert the platform into the interior of an animal uterus (i.e.,
cow, goat, pig) and then the selectively magnetic platform is
provided with input that instructs it to cease magnetic attraction
to the labelable zona anchor MEMS device/cell conjugates. This
procedure is monitored by ultrasound to provide proper positioning
of the labelable zona anchor MEMS device platform/holder within the
uterus. Upon removal of the labelable zona anchor MEMS device
platform/holder from the animal the platform is observed for any
remaining labelable zona anchor MEMS device/cell conjugates. When
embryos have been used in this procedure, the animal is observed
for pregnancy and delivery of young.
Example
The Zona Coring MEMS Device Operation of Zona Coring MEMS
Device
[0713] The Zona coring MEMS device kit with the centrifugal platter
is placed onto a spinning device. Cell wells are filled with
loading fluid (i.e., PBS/PVA, M199 media). The Zona Pellucida
coring MEMS device is spin at high speed (i.e., 5 g, 6 g, 7 g) for
10 seconds to purge bubbles. Oocytes or embryos are placed into
cell wells by mouth pipette, robotic pipette, or other manner.
Spinner is rotated at high speed (i.e., 5 g, 6 g, 7 g) for 10
seconds and stop. Cells are removed to incubated environment.
(Alternatively, CPU controller on spinner provides a ramp up to
desired speed then ramp down).
[0714] Alternatively, the zona coring MEMS device can be situated
on a substrate base that facilitates close opposition of the oocyte
or embryo with the active domain of the zona coring MEMS device by
way of a lever element. The lever element is actuated by a
controller being in communication with these elements by way of
circuit leads.
Determination of Successful Operation of the Zona Pellucida Coring
MEMS Device
[0715] Oocytes or embryos that have undergone manipulation by the
Zona Pellucida coring MEMS device are observed live under a
microscope to determine whether a section of the Zona Pellucida,
having been resected by the zona coring MEMS device, is not
present.
Determination of Effect of Zona Coring MEMS Device on Oocyte or
Embryo Viability and Developmental Fitness
[0716] Cells are assayed for immediate effect of manipulation on
cell viability (vital staining) as well as for developmental
fitness (either early embryonic development or ability to produce
offspring in recipient animal). In particular, embryos that have
undergone this procedure are cultured to observe if the embryo is
able to hatch through the hole in the zona as formed by the Zona
coring MEMS device. Further, cells are processed for
immunofluorescence to visualize microtubule and actin networks to
determine effect of manipulation on the cytoskeleton.
Example
The Microinjection MEMS Device Preparation of Fluids to be
Injected
[0717] All fluids to be injected must be ultra-pure and preferably
centrifuged (i.e., 5 g, 6 g, 7 g) prior to loading into the
microinjection MEMS device.
Preparation of DNA for Injection
[0718] Recombinant plasmid is purified by CsCl gradient (see
Molecular Cloning: A laboratory manual, 2.sup.nd ed. Sambrook, et
al. 1990). Release insert with restriction enzymes. The insert is
separated from the vector on an agarose gel run in
Tris/Acetate/EDTA buffer. Insert is eluted from excised gel slice.
Fragment is recovered by ethanol precipitation and then passed over
an ion exchange column (e.g. Schleicher & Shuell Elutip
columns). DNA is Ethanol-precipitated and resolubilized in
injection buffer (10 mM Tris/0.1 mM EDTA pH 7.5, using Milli-Q
H.sub.2O). DNA concentration is determined. DNA concentration is
adjusted to 1-5 ng/.mu.l with injection buffer.
Operation of Microinjection MEMS Device
[0719] The microinjection MEMS device/centrifugal platter cartridge
is placed onto a spinning device. The central loading manifold is
filled with loading fluid (i.e., PBS/PVA, M199 media, fluorescent
dye, visible dye). The microinjection MEMS device is spun at 5 g
for 10 seconds to purge bubbles and to load fluids throughout the
dynamic hydropressure columns and hollow protuberances. Oocytes or
embryos are placed into cell wells by mouth pipette, robotic
pipette, or other manner. Spinner is rotated at 5 g for 10 seconds
and stopped. Alternatively, the spinner is rotated at a lower speed
(i.e., 1 g, 2 g, 3 g; providing for pronuclear positioning) for 5
seconds, pulsed at a higher speed (i.e., 5 g, 6 g, 7 g, providing
needle penetration and injection of fluid into cell) and then
stopped. Cells are removed to incubated environment.
(Alternatively, CPU controller on spinner provides a ramp up to
desired speed then ramp down).
[0720] Alternatively, the microinjection MEMS device can be
situated on a substrate base that facilitates fluid movements
through micropumping means and the oocyte or embryo can be brought
into close opposition with the active domain of the microinjection
MEMS device by way of a lever element. Both the micropumping means
and the lever element are actuated by a controller being in
communication with these elements by way of circuit leads.
Determination of Successful Operation of the Microinjection MEMS
Device
[0721] Cells injected by the microinjection MEMS device loaded with
fluorescent dye are observed by fluorescence microscopy to
determine the deposition of fluorescent dye into the cytoplasmic
compartment of the cell. Where the injection is pronuclear in
manner fluorescence microscopy to determine the deposition of
fluorescent dye into the nuclear compartment of the cell.
[0722] Cells injected by the microinjection MEMS device loaded with
a reporter DNA construct (i.e., .beta.-gal, luciferase) are
processed to determine gene expression as per instructions by
reporter DNA construct manufacturer.
Determination of Effect of Microinjection MEMS Device on Oocyte or
Embryo Viability and Developmental Fitness
[0723] Cells are assayed for immediate effect of manipulation on
cell viability (vital staining) as well as for developmental
fitness (either early embryonic development or ability to produce
offspring in recipient animal). Further, cells are processed for
immunofluorescence to visualize microtubule and actin networks to
determine effect of manipulation on the cytoskeleton.
[0724] Cells injected by the microinjection MEMS device loaded with
an expression DNA construct (i.e., gene of interest) are processed
to determine gene expression as per instructions by expression DNA
construct manufacturer.
[0725] Further, to determine efficacy of genes injected with the
microinjection MEMS device, embryos modified with this device are
transferred to recipient animals and allowed to gestate to
appropriate stage of development or to birth.
Example
The IntraCytoplasmic Sperm Injection (ICSI) MEMS Device Preparation
of Sperm for Injection into Cells
[0726] Sperm washing removes non-competent cells and
prostaglandins. Sperm are washed with Ham's F (HF)-10 medium to
remove the seminal plasma, centrifuged (200.times.g, 300.times.g,
400.times.g) for a short time (5 minute, 6 minutes, 7 minutes) and
resuspended in less than a milliliter of HF-10 medium.
[0727] The swim-up assay provides for the selection of
non-pathologic morphology and motility. Washed semen is incubated
at 37.degree. C. for 1 hour in HF-10 medium. The uppermost fraction
of tissue culture medium is collected, centrifuged, and resuspended
in a smaller volume of HF-10 medium.
Operation of ICSI MEMS Device
[0728] The ICSI MEMS device/centrifugal platter cartridge is placed
onto a spinning device. The cell loading region and the sperm
loading bay is filled with loading fluid (i.e., Ham's F-10 Medium,
PBS/PVA, M199 media, fluorescent dye, visible dye). The ICSI MEMS
device is spun at high speed (i.e., 4.times.g, 5.times.g,
6.times.g) for 10 seconds to purge bubbles and to load fluids
throughout the cell loading region, sperm loading bay, dynamic
hydropressure columns and hollow protuberances. The sperm loading
bay is seeded with an aliquot of concentrated, washed and
capacitated sperm. Sperm are allowed to diffuse throughout the
sperm loading bay and dynamic hydropressure columns. Oocytes are
placed into cell wells by mouth pipette, robotic pipette, or other
manner. Spinner is rotated at low speed (i.e., 2.times.g,
3.times.g, 4.times.g) for a short period (i.e., 8 seconds, 9
seconds, 10 seconds), simultaneously the sperm guillotine(s) are
operated to isolate a single sperm per dynamic hydropressure
column, sever it's tail and allow it to pass into the dynamic
hydropressure column proximal to the hollow protuberances. After
operation of sperm guillotine(s) the speed of rotation is ramped up
to high speed (i.e., 5.times.g, 6.times.g, 7.times.g) for a short
time (2 seconds, 3 seconds, 4 seconds) and rotation is terminated.
Cells are removed to incubated environment. (Alternatively, CPU
controller on spinner provides a ramp up to desired speed then ramp
down).
[0729] Alternatively, the ICSI MEMS MEMS device can be situated on
a substrate base that facilitates fluid movements through
micropumping means and the oocyte or embryo can be brought into
close opposition with the active domain of the ICSI MEMS MEMS
device by way of a lever element. Both the micropumping means and
the lever element are actuated by a controller being in
communication with these elements by way of circuit leads.
Determination of Successful Operation of the ICSI MEMS Device
[0730] Success of this procedure is measured by the presence of a
single sperm in the cytoplasm of the oocyte. After operation of the
ICSI MEMS device cells are removed to an organ culture dish and
observed under a microscope for the presence of sperm.
Determination of Effect of ICSI MEMS Device on Oocyte or Embryo
Viability and Developmental Fitness
[0731] Embryos generated by the above method are tested for their
ability to progress through early embryonic development. Embryos
are collected at successive days and observed by microscope for
cell division, morula formation and blastocyst development.
Example
The Enucleation MEMS Device Operation of the Enucleation MEMS
Device
[0732] The enucleation MEMS device/centrifugal platter cartridge is
placed onto a spinning device. The central loading manifold is
filled with loading fluid (i.e., Ham's F-10 Medium, PBS/PVA, M199
media, fluorescent dye, visible dye). The enucleation MEMS device
is spun at high speed (i.e., 4.times.g, 5.times.g, 6.times.g) for
10 seconds to purge bubbles and to load fluids throughout the cell
loading region. Oocytes or embryos are placed into cell wells by
mouth pipette, robotic pipette, or other manner. Spinner is rotated
at low speed (i.e., 2.times.g, 3.times.g, 4.times.g) for a short
period (i.e., 8 seconds, 9 seconds, 10 seconds), bringing the cells
into contact with the enucleation penetration member. The speed of
rotation is ramped up to high speed (i.e., 5.times.g, 6.times.g,
7.times.g) for a longer period of time (5 seconds, 10 seconds, 15
seconds) to allow penetration of oocytes or embryos by the
enucleation penetration member, to allow the extrusion of cell
cytoplasm and nucleus, allow the severance of any cell remnant by
the slideable shutter at the base of the enucleation penetration
member, and then rotation is terminated. Cells are removed to
incubated environment. (Alternatively, CPU controller on spinner
provides a ramp up to desired speed then ramp down).
[0733] Alternatively, the enucleation MEMS device can be situated
on a substrate base that facilitates fluid movements through
micropumping means and the oocyte or embryo can be brought into
close opposition with the active domain of the enucleation MEMS
device by way of a lever element. Both the micropumping means and
the lever element are actuated by a controller being in
communication with these elements by way of circuit leads.
Determination of Successful Operation of the Enucleation MEMS
Device
[0734] Success operation of the enucleation MEMS device is
determined by the removal of the nuclear material of the oocyte or
embryo in such a way that the cell is not irreparably damaged
(i.e., lysis, extensive loss of cytoplasm). The condition of the
oocyte or embryo after manipulation by the enucleation MEMS device
is assayed by visual inspection under a microscope.
Determination of Effect of Enucleation MEMS Device on Oocyte or
Embryo Viability and Developmental Fitness
[0735] Further determination of oocyte or embryo quality after
manipulation by the enucleation MEMS device includes: the culture
of the cells for 24 hours post manipulation to determine stability
of cell quality, and the further deposition of a donor nucleus or
cell in the cytoplasm to determine the ability of the enucleated
oocyte or embryo to support development of the cloned embryo.
Example
The Enucleation/Nuclear Transfer MEMS Device Preparation of Cells
to Serve as Donor Cells
[0736] Desirable culture cells (i.e., founder cells, transgenic
culture cells, first generation cloned embryonic stem cells, adult
somatic cells, primary culture of adult somatic cells, long term
culture of adult somatic cells) being of any stage of the cell
cycle (i.e., G1, G2, S, Mitosis, Meiosis, and quiescent) are washed
with PBS, PBS/EGTA and then Trypsin/EDTA 0.25% to detach cells from
substrate. Trypsin is neutrilized by washing several times with
centrifuge/wash steps in culture medium (i.e., HF-10, M199, M19,
DMEM) and the cells are dispersed into a single cell suspension
Operation of Enucleation/Nuclear Transfer MEMS Device
[0737] The enucleation/nuclear transfer MEMS device/centrifugal
platter cartridge is placed onto a spinning device. The central
loading manifold is filled with loading fluid (i.e., Ham's F-10
Medium, PBS/PVA, M199 media, fluorescent dye, visible dye) and
donor nuclei or donor cells. The enucleation/nuclear transfer MEMS
device is spun at high speed (i.e., 4.times.g, 5.times.g,
6.times.g) for 10 seconds to purge bubbles and to load fluids and
donor nuclei or donor cells throughout the cell loading region.
Oocytes or embryos are placed into cell wells by mouth pipette,
robotic pipette, or other manner. Spinner is rotated at low speed
(i.e., 2.times.g, 3.times.g, 4.times.g) for a short period (i.e., 8
seconds, 9 seconds, 10 seconds), bringing the cells into contact
with the enucleation penetration member and for nuclear migration
to commence. The speed of rotation is ramped up to high speed
(i.e., 5.times.g, 6.times.g, 7.times.g) for a longer period of time
(5 seconds, 10 seconds, 15 seconds) to allow penetration of oocytes
or embryos by the enucleation penetration member, to allow the
extrusion of cell cytoplasm and nucleus, allow the severance of any
cell remnant by the slideable shutter at the base of the
enucleation penetration member, to allow the injection of a donor
cell or donor nucleus into the cytoplasm of the enucleated oocyte
or embryo, and then rotation is terminated. Cells are removed to
incubated environment. (Alternatively, CPU controller on spinner
provides a ramp up to desired speed then ramp down).
[0738] Alternatively, the enucleation/nuclear transfer MEMS device
can be situated on a substrate base that facilitates fluid
movements through micropumping means and the oocyte or embryo can
be brought into close opposition with the active domain of the
enucleation/nuclear transfer MEMS device by way of a lever element.
Both the micropumping means and the lever element are actuated by a
controller being in communication with these elements by way of
circuit leads.
Determination of Successful Operation of the Enucleation/Nuclear
Transfer MEMS Device
[0739] Success operation of the enucleation/nuclear transfer MEMS
device is determined by the removal of the nuclear material of the
oocyte or embryo in such a way that the cell is not irreparably
damaged (i.e., lysis, extensive loss of cytoplasm) and the
deposition of one donor nucleus or donor cell into the cytoplasm of
the enucleated oocyte or embryo. The condition of the oocyte or
embryo (i.e., presence of a donor nucleus or donor cell) after
manipulation by the enucleation MEMS device is assayed by visual
inspection under a microscope.
Determination of Effect of Enucleation/Nuclear Transfer MEMS Device
on Oocyte or Embryo Viability and Developmental Fitness
[0740] The determination of oocyte or embryo quality after
manipulation by the enucleation/nuclear transfer MEMS device
includes: the culture of the reconstructed embryos post
manipulation to determine stability of cell quality, the ability of
the embryo to proceed through early embryonic development to the
blastocyst stage, and then the determination of the ability of the
reconstructed embryo to proceed through gestation to live birth
after being transferred into a recipient animal.
Example
The Cytoplasmic Transfer MEMS Device Operation of Cytoplasmic
Transfer MEMS Device
[0741] The cytoplasmic transfer MEMS device kit with a centrifugal
platter is placed onto a spinning device. The cell wells are filled
with loading fluid (i.e., Ham's F-10 Medium, PBS/PVA, M199 media).
The cytoplasmic transfer MEMS device, with both gating elements
open, is spun at high speed (i.e., 4.times.g, 5.times.g, 6.times.g)
for 10 seconds to purge bubbles and to load fluids throughout the
cell loading region, extraction siphons, and supplemental input
channel (being in fluid communication with the fluids in the cell
wells). Oocytes or embryos are placed into cell wells by mouth
pipette, robotic pipette, or other manner. Spinner is rotated at
low speed (i.e., 2.times.g, 3.times.g, 4.times.g) for a short
period (i.e., 8 seconds, 9 seconds, 10 seconds), bringing the
oocyte or embryo into contact with the hollow protuberance. The
speed of rotation is ramped up to high speed (i.e., 5.times.g,
6.times.g, 7.times.g) for a longer period of time (5 seconds, 10
seconds, 15 seconds) to allow penetration of oocyte or embryo by
the hollow protuberance, to allow the extraction of an aliquot of
cytoplasm from the cytoplasmic donor cell, to allow the cytoplasm
aliquot to travel through the extraction siphon to the first gate,
pass through the first gate, the closing of the first gate (being
in communication with a controller by way of a circuit lead), the
opening of the second gate allowing the cytoplasm aliquot and
supplemental input fluid to flow through distal portion of the
extraction siphon, through the hollow protuberance of the cell well
containing the host oocyte or embryo into the host oocyte or embryo
cytoplasm, and then rotation is terminated. Cells are removed to
incubated environment. (Alternatively, CPU controller on spinner
provides a ramp up to desired speed then ramp down).
[0742] Alternatively, the cytoplasmic transfer MEMS device can be
situated on a substrate base that facilitates fluid movements
through micropumping means and the oocyte or embryo can be brought
into close opposition with the active domain of the cytoplasmic
transfer MEMS device by way of a lever element. Both the
micropumping means and the lever element are actuated by a
controller being in communication with these elements by way of
circuit leads.
Determination of Successful Operation of the Cytoplasmic Transfer
MEMS Device
[0743] Successful operation of the cytoplasmic transfer MEMS device
is determined by the removal of a portion of cytoplasmic material
from a cytoplasmic donor oocyte or embryo and the deposition of
this portion into the cytoplasm of the host oocyte or embryo. The
determination of this success requires the visualization of the
donor cytoplasm in the host cytoplasm and can be mediated by the
labeling of the donor cytoplasm (i.e., cell being injected with a
dye, cytoplasmic specific antibodies) prior to manipulation by the
cytoplasmic transfer MEMS device. Additionally, it needs to be
determined whether nuclear material from the donor oocyte or embryo
is transferred with the cytoplasmic portion. This can be determined
by the labeling of the DNA of the donor oocyte or embryo (i.e.,
ethidum bromide, anti-histone antibodies) prior to manipulation by
the cytoplasmic transfer MEMS device. Additionally, it needs to be
determined if the host oocyte or embryo has been irreparably
damaged (i.e., lysis, extensive loss of cytoplasm) by this process.
Microscopic examination for lysis or other hallmarks of cell death
is performed.
Determination of Effect of Cytoplasmic Transfer MEMS Device on
Oocyte or Embryo Viability and Developmental Fitness
[0744] The determination of oocyte or embryo quality after
manipulation by the cytoplasmic transfer MEMS device includes: the
culture of the oocytes or embryos post manipulation to determine
stability of cell quality, the ability of the oocyte to proceed
through fertilization, and the ability of the embryo to proceed
through early embryonic development to the blastocyst stage, and
then the determination of the ability of the reconstructed embryo
to proceed through gestation to live birth after being transferred
into a recipient animal.
Example
The In Vitro Culture Device Operation of In Vitro Culture
Device
[0745] After having been placed in an environmental controlling
instrument of the present invention, the single layer or
multi-layer in vitro culture device is loaded with fluids (i.e.,
culture media), any bubbles being purged by gentle pressure being
applied to the system through the loading/removal compartments or
through the input and output enclosed channels. Oocytes or embryos,
having been attached to labelable zona anchor MEMS devices that are
attractive to the movement tracks of the in vitro culture device,
are added to the loading compartment (i.e., by mouth pipette, by
robotic means, by other cell handling means). The environmental
controlling instrument CPU is provided with the desired culture
conditions, time, and amendment parameters. The environmental
controlling instrument CPU actuates the travel of the cells on the
movement tracks through out the single or multi-layer in vitro
culture device, provides culture amendments (i.e., change in sera
concentration, change in hormone composition or concentration,
change in temperature, change in pH, addition of co-culture
conditioned medium, addition of sperm for IVF), provides for the
observation of physical parameters within the in vitro culture
device as well as for visual inspection of the oocytes or embryos
as they pass the visual inspection devices of the environmental
controlling instrument, provides for labelable zona anchor MEMS
device telemetry, namely the collection of information (i.e.,
specific physical parameters encountered by any single oocyte or
embryo) from the labelable zona anchor MEMS device resident on the
oocyte or embryo, and provides for the positioning of the oocyte or
embryo in a removal compartment for removal from device.
Determination of Successful Operation of the In Vitro Culture
Device
[0746] Successful operation of the in vitro culture device is
measured by determining the ability of the in vitro culture device
to handle the oocytes or embryos in such a way that damage does not
occur (i.e., the in vitro culture device does: not clog with cells;
does not provide adverse physical conditions). Additionally, the
oocytes or embryos are observed to determine if the in vitro
culture device was able to provide the appropriate culture
conditions. For example, if IVF of oocytes was desired the result
must be that an acceptable number of oocytes have been inseminated,
if oocyte maturation was desired then the result must be that and
acceptable number of oocytes have been cultured to the stage
desired such as the Meiosis II block, if in vitro culture of
embryos was desired then an acceptable number of embryos must reach
the stage of early embryonic development desired such as the
blastocyst stage.
[0747] Although the invention described in detail with reference to
specific embodiments thereof, it will be understood that variations
which are functionally equivalent are within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0748] All references and patents cited within are hereby
incorporated by reference in their entirety.
* * * * *