U.S. patent application number 11/582027 was filed with the patent office on 2007-04-26 for microfluidic cell culture device.
Invention is credited to Lourdes Marcella Cabrera, Yun Seok Heo, Gary Daniel Smith, Shuichi Takayama.
Application Number | 20070090166 11/582027 |
Document ID | / |
Family ID | 37963248 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070090166 |
Kind Code |
A1 |
Takayama; Shuichi ; et
al. |
April 26, 2007 |
Microfluidic cell culture device
Abstract
Microfluidic devices for cell culturing and methods for using
the same are disclosed. One device includes a substrate and
membrane. The substrate includes a reservoir in fluid communication
with a passage. A bio-compatible fluid may be added to the
reservoir and passage. The reservoir is configured to receive and
retain at least a portion of a cell mass. The membrane acts as a
barrier to evaporation of the bio-compatible fluid from the
passage. A cover fluid may be added to cover the bio-compatible
fluid to prevent evaporation of the bio-compatible fluid.
Inventors: |
Takayama; Shuichi; (Ann
Arbor, MI) ; Cabrera; Lourdes Marcella; (Ann Arbor,
MI) ; Heo; Yun Seok; (Ann Arbor, MI) ; Smith;
Gary Daniel; (Ann Arbor, MI) |
Correspondence
Address: |
BROOKS KUSHMAN P.C.
1000 TOWN CENTER
TWENTY-SECOND FLOOR
SOUTHFIELD
MI
48075
US
|
Family ID: |
37963248 |
Appl. No.: |
11/582027 |
Filed: |
October 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60727934 |
Oct 18, 2005 |
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60728030 |
Oct 18, 2005 |
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60741665 |
Dec 2, 2005 |
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60741864 |
Dec 2, 2005 |
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60802705 |
May 23, 2006 |
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60812166 |
Jun 9, 2006 |
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Current U.S.
Class: |
228/101 |
Current CPC
Class: |
B01L 3/5027 20130101;
C12M 23/12 20130101; B01L 3/5085 20130101; C12N 5/0075 20130101;
C12M 23/34 20130101; C12M 23/16 20130101 |
Class at
Publication: |
228/101 |
International
Class: |
A47J 36/02 20060101
A47J036/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under at
least one of: Contract No. F012482; Contract No. F008090; Contract
No. N006624; Grant No. HD049607-01 awarded by National Institute of
Health; Contract/Grant No. DAAD19-03-1-0168 awarded by the U.S.
Army Research Laboratory and the U.S. Army Research Office;
BES-0238625 awarded by the National Science Foundation; NNC04AA21A
awarded by the NASA BioScience and Engineering Institute; and USDA
2005-35203-16148 awarded by the United States Department of
Agriculture. The Government has certain rights to the invention.
Claims
1. A microfluidic cell culture device comprising: a substrate; a
reservoir formed in the substrate and having upper and lower
portions, the upper portion being sized to permit the insertion and
removal of a cell mass from the reservoir; and a passage formed in
the substrate and in fluid communication with the reservoir wherein
at least one of the lower portion of the reservoir and the passage
adjacent the lower portion is sized relative to the cell mass to
retain at least a portion of the cell mass within the lower portion
of the reservoir so that the cell mass can be directly removed from
the lower portion through the upper portion.
2. The device of claim 1 wherein the lower portion of the reservoir
is sized relative to the cell mass to retain at least a portion of
the cell mass within the lower portion of the reservoir so that the
cell mass can be directly removed from the lower portion through
the upper portion.
3. The device of claim 2 wherein the lower portion of the reservoir
has a width less than 250 micrometers to retain at least a portion
of the cell mass within the reservoir.
4. The device of claim 2 wherein the lower portion of the reservoir
has a width, wherein the cell mass is a denuded human zygote, and
wherein the width is less than 140 micrometers to retain at least a
portion of the cell mass within the reservoir.
5. The device of claim 2 wherein the lower portion of the reservoir
has a width, wherein the cell mass is a denuded mammalian zygote,
and wherein the width is less than 70 micrometers to retain at
least a portion of the cell mass within the reservoir.
6. The device of claim 2 wherein the lower portion of the reservoir
has a width, wherein the cell mass is a clump of mammalian cells,
and wherein the width is less than 50 micrometers to retain at
least a portion of the cell mass within the reservoir.
7. The device of claim 2 wherein the lower portion of the reservoir
has a width, wherein the cell mass is a single mammalian cell, and
wherein the width is less than 5 micrometers to retain at least a
portion of the cell mass within the reservoir.
8. The device of claim 1 wherein the passage adjacent the lower
portion is sized relative to the cell mass to retain at least a
portion of the cell mass within the lower portion of the reservoir
so that the cell mass can be directly removed from the lower
portion through the upper portion.
9. The device of claim 8 wherein the passage adjacent the lower
portion has a passage width and a passage height and wherein at
least one of the passage height and the passage width is less than
250 micrometers to retain at least a portion of the cell mass
within the reservoir.
10. The device of claim 8 wherein the passage adjacent the lower
portion has a passage width and a passage height, wherein the cell
mass is a denuded human zygote, and wherein at least one of the
passage height and the passage width is less than 140 micrometers
to retain at least a portion of the cell mass within the
reservoir.
11. The device of claim 8 wherein the passage adjacent the lower
portion has a passage width and a passage height, wherein the cell
mass is a denuded mammalian zygote, and wherein at least one of the
passage height and the passage width is less than 70 micrometers to
retain at least a portion of the cell mass within the
reservoir.
12. The device of claim 8 wherein the passage adjacent the lower
portion has a passage width and a passage height, wherein the cell
mass is a clump of mammalian cells, and wherein at least one of the
passage height and the passage width is less than 50 micrometers to
retain at least a portion of the cell mass within the
reservoir.
13. The device of claim 8 wherein the passage adjacent the lower
portion has a passage width and a passage height, wherein the cell
mass is a single mammalian cell, and wherein at least one of the
passage height and the passage width is less than 5 micrometers to
retain at least a portion of the cell mass within the
reservoir.
14. The device of claim 1 wherein a cross-section of the reservoir
has a polygonal shape.
15. The device of claim 1 wherein the reservoir has a
frusta-conical shape.
16. The device of claim 1 wherein an angle defined by opposite
surfaces of the reservoir is between 30 degrees and 160 degrees
inclusive.
17. The device of claim 1 wherein the upper portion has an upper
width and the lower portion has a lower width and wherein the upper
width is greater than the lower width.
18. The device of claim 1 wherein a surface of the reservoir tapers
inwardly from the upper portion to the lower portion.
19. The device of claim 1 wherein the passage is U-shaped.
20. The device of claim 1 wherein the passage has a volume less
than 1 microliter.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the following U.S.
provisional applications: Ser. No. 60/727,934 filed Oct. 18, 2005;
Ser. No. 60/728,030, filed Oct. 18, 2005; Ser. No. 60/741,665,
filed Dec. 2, 2005; Ser. No. 60/741,864, filed Dec. 2, 2005; Ser.
No. 60/802,705, filed May 23, 2006; and Ser. No. 60/812,166, filed
Jun. 9, 2006.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention relates to microfluidic cell culture
devices.
[0005] 2. Discussion
[0006] Microfluidic devices allow a user to work with nano- to
microliter volumes of fluids and are useful for reducing reagent
consumption, creating physiologic cell culture environments that
better match the fluid-to-cell-volume ratios in vivo, and
performing experiments that take advantage of low Reynolds number
phenomenon such as subcellular treatment of cells with multiple
laminar streams. Many microfluidic systems are made of
polydimethylsiloxane (PDMS) because of its favorable mechanical
properties, optical transparency, and bio-compatibility.
[0007] Cell culture is an essential tool in biological science,
clinical science, and biotechnology. Microfluidic cell culture
devices offer the advantages of increased fluid control,
approximated physiologic culture environments, and improved culture
efficiency.
[0008] Microfluidic cell culture devices have been developed for
diverse cell types such as Eukaryotic cells, lung cells, embryonic
stem cells, and mammalian embryos.
[0009] Most microfluidic cell culture devices separate cell loading
zones from designated cell culture zones. This separation requires
additional external forces and elaborate works for the cell in the
loading zone to be transported to the designated culture zone.
Also, the transport processes can put stress on sensitive cells
such as mammalian embryo or embryonic stem cells. In addition, once
the cells reach the designated culture zone, additional design and
fabrications are required for cell confinement to apply diverse
culture conditions with flows.
SUMMARY
[0010] Embodiments of the invention may take the form of a
microfluidic cell culture device. The device includes a substrate
and a reservoir formed in the substrate having upper and lower
portions. The upper portion is sized to permit the insertion and
removal of a cell mass from the reservoir. The device also includes
a passage formed in the substrate and in fluid communication with
the reservoir. At least one of the lower portion of the reservoir
and the passage adjacent the lower portion is sized relative to the
cell mass to retain at least a portion of the cell mass within the
lower portion of the reservoir so that the cell mass can be
directly removed from the lower portion through the upper
portion.
[0011] The lower portion of the reservoir may be sized relative to
the cell mass to retain at least a portion of the cell mass within
the lower portion of the reservoir so that the cell mass can be
directly removed from the lower portion through the upper
portion.
[0012] The lower portion of the reservoir may have a width less
than 250 micrometers to retain at least a portion of the cell mass
within the reservoir.
[0013] The lower portion of the reservoir may have a width, the
cell mass may be a denuded human zygote, and the width may be less
than 140 micrometers to retain at least a portion of the cell mass
within the reservoir.
[0014] The lower portion of the reservoir may have a width, the
cell mass may be a denuded mammalian zygote, and the width may be
less than 70 micrometers to retain at least a portion of the cell
mass within the reservoir.
[0015] The lower portion of the reservoir may have a width, the
cell mass may be a clump of mammalian cells, and the width may be
less than 50 micrometers to retain at least a portion of the cell
mass within the reservoir.
[0016] The lower portion of the reservoir may have a width, the
cell mass may be a single mammalian cell, and the width may be less
than 5 micrometers to retain at least a portion of the cell mass
within the reservoir.
[0017] The passage adjacent the lower portion may be sized relative
to the cell mass to retain at least a portion of the cell mass
within the lower portion of the reservoir so that the cell mass can
be directly removed from the lower portion through the upper
portion.
[0018] The passage adjacent the lower portion may have a passage
width and a passage height and at least one of the passage height
and the passage width may be less than 250 micrometers to retain at
least a portion of the cell mass within the reservoir.
[0019] The passage adjacent the lower portion may have a passage
width and a passage height, the cell mass may be a denuded human
zygote, and at least one of the passage height and the passage
width may be less than 140 micrometers to retain at least a portion
of the cell mass within the reservoir.
[0020] The passage adjacent the lower portion may have a passage
width and a passage height, the cell mass may be a denuded
mammalian zygote, and at least one of the passage height and the
passage width may be less than 70 micrometers to retain at least a
portion of the cell mass within the reservoir.
[0021] The passage adjacent the lower portion may have a passage
width and a passage height, the cell mass may be a clump of
mammalian cells, and at least one of the passage height and the
passage width may be less than 50 micrometers to retain at least a
portion of the cell mass within the reservoir.
[0022] The passage adjacent the lower portion may have a passage
width and a passage height, the cell mass may be a single mammalian
cell, and at least one of the passage height and the passage width
may be less than 5 micrometers to retain at least a portion of the
cell mass within the reservoir.
[0023] A cross-section of the reservoir may have a polygonal
shape.
[0024] The reservoir may have a frusta-conical shape.
[0025] An angle defined by opposite surfaces of the reservoir may
be between 30 degrees and 160 degrees inclusive.
[0026] The upper portion may have an upper width and the lower
portion may have a lower width and the upper width may be greater
than the lower width.
[0027] A surface of the reservoir may taper inwardly from the upper
portion to the lower portion.
[0028] The passage may be U-shaped.
[0029] The passage may have a volume less than 1 microliter.
[0030] While exemplary embodiments in accordance with the invention
are illustrated and disclosed, such disclosure should not be
construed to limit the claims. It is anticipated that various
modifications and alternative designs may be made without departing
from the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is an exploded, perspective view of a microfluidic
cell culture device in accordance with an embodiment of the
invention;
[0032] FIG. 2a is a top view of a substrate of the system of FIG.
1;
[0033] FIG. 2b is a side view, and in cross-section, of the
substrate taken along section line 2b-2b in FIG. 2a;
[0034] FIG. 2c is a bottom view of the substrate of FIG. 2a;
[0035] FIG. 3a is a side view, partially broken-away and in
cross-section, of a membrane of the system of FIG. 1 with a pair of
actuator pins in engagement with a lower surface of the
membrane;
[0036] FIG. 3b is a side view, partially broken-away and in
cross-section, of an alternative embodiment of a membrane of the
system of FIG. 1 with a pair of actuator pins spaced away from the
lower surface of the membrane;
[0037] FIG. 4a is a side view, partially broken-away and in
cross-section, of an alternative embodiment of the substrate of
FIG. 1 and the membrane of FIG. 3b and illustrating two different
heights of a biological fluid in a pair of reservoirs formed in the
substrate;
[0038] FIG. 4b is an enlarged side view, partially broken-away and
in cross-section, of the substrate and membrane of FIG. 4a and
illustrating the relative heights of a cell mass and an end portion
of a passageway;
[0039] FIG. 4c is another enlarged side view, partially broken-away
and in cross-section, of the substrate and membrane of FIG. 4a and
illustrating the angle of the surface which defines a reservoir and
the relative widths of a cell mass and a lower portion of the
reservoir;
[0040] FIG. 5a is an enlarged side view, partially broken-away and
in cross-section, of an alternative embodiment of a substrate and
membrane of FIG. 1 and illustrating a cell mass retained in a lower
portion of the reservoir above the passageway;
[0041] FIG. 5b is another enlarged side view, partially broken-away
and in cross-section, of an alternative embodiment of a substrate
and membrane of FIG. 1 and illustrating a cell mass retained in a
lower portion of the reservoir below the passageway; and
[0042] FIG. 5c is yet another enlarged side view, partially
broken-away and in cross-section, of an alternative embodiment of a
substrate and membrane of FIG. 1 and illustrating a cell mass with
a width smaller than the width of the lower portion of the
reservoir.
DETAILED DESCRIPTION
[0043] FIG. 1 is an exploded, perspective view of microfluidic cell
culture system or device 10. Device 10 includes substrate 12
configured to receive a cellular mass, e.g., an embryo, as
explained in detail below, non-rigid membrane 14, locating block
16, and pin actuating device 18.
[0044] FIG. 2a is a top view of substrate 12. Substrate 12 includes
funnel 22, reservoir 24, and overlay reservoir 26. Bottom portion
28 of funnel 22 is in fluid communication with reservoir 24 via
microchannel 30. Microchannel 30 has a volume less than 1
microliter. Reservoir 24 includes reservoir openings 32 which
provide openings to microchannel 30 such that fluids may travel
between funnel 22 and reservoir 24 as explained in detail
below.
[0045] FIG. 2b is a side view, and in cross-section, of substrate
12 taken along section line 2b-2b in FIG. 2a. A portion of
microchannel 30 is formed in substrate 12 while another portion of
microchannel 30 is formed by membrane 14 as described in detail
below. Microchannel 30, however, may be completely formed in
substrate 12 or in any other suitable fashion. Microchannel 30 may
have a square, circular, bell, or any other suitably shaped
cross-section. Substrate 12 further includes hydrophilic surface 34
to promote fluid retention within overlay reservoir 26.
[0046] Fluid may move between funnel 22 and reservoir 24 via
localized deformation of membrane 14. Fluid may also move between
funnel 22 and reservoir 24 under the influence of gravity as
explained in detail below.
[0047] Substrate 12 may be optically transparent and made from such
materials as plastic, e.g., PDMS, polymethylmethacrylate,
polyurethane, or glass.
[0048] FIG. 2c is a bottom view of substrate 12. Substrate 12
includes female locators 36 which assist in locating substrate 12
relative to membrane 14 as explained in detail below.
[0049] Substrate 12 may comprise a thick, e.g., 8 mm, PDMS slab,
fabricated by using soft lithography. The PDMS slab may be prepared
by casting a prepolymer (Sylgard 184, Dow-Corning) at a 1:10 curing
agent-to-base ratio against positive relief features. Relief
features may comprise SU-8 (MicroChem, Newton, Mass.) and be
fabricated on a thin, e.g., 200 .mu.m, glass wafer by using
backside diffused-light photolithography. The prepolymer may then
cure at 60.degree. C. for 60 minutes, and holes may be punched by a
sharpened 14-gauge blunt needle.
[0050] Substrate 12 may comprise two layers of cured PDMS at a
ratio of 1:10 base to curing agent sealed together irreversibly
using plasma oxidation (SPI supplies, West Chester, Pa.). Funnel 22
and reservoir 24 are formed in the top layer. Microchannel 30 is
formed in the bottom layer using soft lithography. Microchannel 30
faces downward and may be sealed against membrane 14 as explained
in detail below.
[0051] FIG. 3a is a side view, partially broken-away and in
cross-section, of membrane 14 and pins 54 of pin actuating device
18 (FIG. 1). Membrane 14 includes male locators 38 (FIG. 1)
configured to be received by female locators 36 of substrate 12 to
locate membrane 14 relative to substrate 12.
[0052] Membrane 14 is optically transparent and includes top layer
40, upper surface 41, middle layer 42, bottom layer 44, and bottom
surface 45. Top layer 40 and bottom layer 42 comprise PDMS. Middle
layer 42 comprises parylene. Top layer 40 and bottom layer 44,
alternatively, may comprise any suitable non-rigid, bio-compatible
polymer such as a non-rigid plastic, e.g., polyurethane, or a
hyrdrogel, e.g., polyvinylalcohol. Middle layer 42, alternatively,
may comprise any suitable non-rigid polymer such as polyvinylidene
chloride or polyurethane.
[0053] Top layer 40 and bottom layer 44 may have a combined
thickness of less than 1 mm, e.g., 200 .mu.m. Middle layer 42 may
range in thickness from 2-20 .mu.m, e.g., 2-5 .mu.m.
[0054] Pins 54 of pin actuating device 18 may selectively extend
from the position shown into membrane 14 to locally deform membrane
14 such that at least a portion of top layer 40 extends into
microchannel 30 (FIG. 2b). The selective actuation of pins 54 may
move a fluid in microchannel 30 or prevent, or impede, the movement
of the fluid in microchannel 30 as explained in detail below.
[0055] Middle layer 42 minimizes evaporation of a fluid, e.g., a
water based fluid, contained within microchannel 30 to prevent, for
example, undesirable shifts in osmolality of the fluid. Middle
layer 42 is also resistant to the flow of at least one gas, such as
oxygen and carbon dioxide, from microchannel 30 and provides
mechanical durability and stability against cracking caused by the
selective actuation of pins 54. Fatigue from the actuation of pins
54 does not substantially increase middle layer's 42 ability to
substantially reduce the rate at which a fluid from microchannel 30
moves through membrane 14.
[0056] Membrane 14 includes female locators (not shown) which are
used to locate membrane 14 relative to locating block 16 as
explained in detail below.
[0057] Membrane 14 may be prepared by spin-coating PDMS onto a 4''
silanized silicon wafer to a thickness of 100 .mu.m, curing this
layer at 120.degree. C. for 30 minutes, depositing a 2.5 or 5 .mu.m
thick parylene layer, plasma oxidizing the resulting parylene
surface for 90 seconds, spin-coating another 100 .mu.m thick layer
of PDMS, and curing for a total thickness of approximately 200
.mu.m.
[0058] FIG. 3b is a side view, partially broken-away and in
cross-section, of an alternative embodiment of membrane 114 and
pins 154 of pin actuating device 118 (not shown). Membrane 114
includes top layer 140, upper surface 141, bottom layer 142, and
lower surface 145. Top layer 140 comprises PDMS and bottom layer
142 comprises polyvinylidene chloride. Top layer 140,
alternatively, may comprise any suitable non-rigid, bio-compatible
polymer such as a non-rigid plastic, e.g., polyurethane, or a
hyrdrogel, e.g., polyvinylalcohol, whereas bottom layer 142 may
comprise any suitable non-rigid polymer such as polyurethane.
[0059] Top layer 140 and bottom layer 142 may have a combined
thickness of less than 1 mm, e.g., 200 .mu.m.
[0060] Bottom layer 142 minimizes evaporation of a fluid, e.g., a
water based fluid, contained within microchannel 30 to prevent, for
example, undesirable shifts in osmolality of the fluid. Bottom
layer 142 is also resistant to the flow of at least one gas, such
as oxygen and carbon dioxide, from microchannel 30 and provides
mechanical durability and stability against cracking caused by the
selective actuation of pins 154. Fatigue from the actuation of pins
154 does not substantially increase bottom layer's 142 ability to
substantially reduce the rate at which a fluid from microchannel 30
moves through membrane 114.
[0061] Membrane 114 may be prepared by spin-coating freshly mixed
1:10 PDMS onto silanized glass slides (Corning Glass Works,
Corning, N.Y.) to a uniform thickness of either approximately 120
.mu.m or 400 .mu.m, curing overnight at 120.degree. C., and then
adhering polyvinylidene chloride via conformal contact with the
PDMS.
[0062] Referring to FIG. 1, locating block 16 includes pin holes 48
and male locators 50. Pin holes 48 are configured to receive pins
54 of pin actuating device 18. Male locators 50 are configured to
be received by the female locators of membrane 14 to locate
locating block 16 relative to membrane 14. In particular, by
locating block 16 relative to membrane 14, pin holes 48 are aligned
with microchannel 30. Locating block 16 includes female locators
(not shown) which are used to locate locating block 16 relative to
pin actuating device 18 as explained in detail below.
[0063] Locating block 16 is rigid and optically transparent and
made from such materials as polystyrene, cyclic olefin copolymer,
glass, or metal.
[0064] Pin actuating device 18 is a Braille-type actuator as
described in detail below. Pins 54 are actuated with a force of 18
g. Pins 54, however, may be actuated with a force ranging from
approximate 3 g to 300 g. Pins 54 may be actuated, for example, 10
times per second or once a minute. Pins 54 may be actuated for a
period ranging from minutes to weeks. Any suitable tactile device,
however, may be used.
[0065] Pins 54 of pin actuating device 18, when actuated, extend
and press upon membrane 14, restricting or closing microchannel 30.
Pins 54 may be actuated in any suitable fashion such that a fluid
flows between funnel 22 and reservoir 24 via microchannel 30. Pins
54 may also be actuated such that the fluid does not move between
funnel 22 and reservoir 24 via microchannel 30.
[0066] Pin actuating device 18 includes male locators 56. Male
locators 56 are configured to be received by female locators 52 of
locating block 16 to align locating block 16 relative to pin
actuating device 18. By aligning locators 46, 56, pins 54 are
aligned with pin holes 48.
[0067] FIG. 4a is side view, partially broken-away and in
cross-section, of substrate 112 and membrane 114. Reservoir 124 and
funnel 122 are in fluid communication via microchannel 130.
Bio-compatible fluid 158 may be transported between reservoir 124
and funnel 122 via localized deformation of membrane 114 by pin
actuating device 118. D is the difference in height between
bio-compatible fluid 158 in reservoir 124 and funnel 122.
[0068] Funnel 122 and reservoir 124 are further in fluid
communication via upper channel 126. Microchannel 130 has a
resistance to fluid flow greater than upper channel 126. Upper
channel 126 is defined by a hydrophobic surface to, for example,
repel bio-compatible fluid 158.
[0069] Immiscible fluid 160, e.g., an oil having a density lower
than bio-compatible fluid 158, may move between funnel 122 and
reservoir 124 via channel 126. Immiscible fluid 160 reduces
evaporation of bio-compatible fluid 158 and reduces the flow of
oxygen and carbon dioxide into and out bio-compatible fluid 158.
Gravity will act upon immiscible fluid 160 such that the height of
immiscible fluid 160 in funnel 122 will equal the height of
immiscible fluid 160 in reservoir 124 thereby maintaining the
difference in height, D, of bio-compatible fluid 158.
[0070] D' is the desired difference in height between
bio-compatible fluid 158 in funnel 122 and bio-compatible fluid 158
in reservoir 124 after pin actuating device 118, for example, has
been used to move bio-compatible fluid 158 from reservoir 124 to
funnel 122. Such a height may provide a desired amount of fluid in
funnel 122 conducive to cell culturing. As bio-compatible fluid 158
is moved from reservoir 124 to funnel 122, immiscible fluid 160
will flow from funnel 122 to reservoir 124 via channel 126 under
the influence of gravity such that in the absence of deformation of
membrane 114 that would cause, for example, bio-compatible fluid
158 to further move between funnel 122 and reservoir 124 or prevent
bio-compatible fluid 158 from moving between funnel 122 and
reservoir 124, immiscible fluid 160 will substantially maintain the
difference in height D' under the influence of gravity for a
desired period of time, e.g., approximately 30 minutes.
Microchannel 130 and and channel 126 thus from a continuous fluid
path between funnel 122 and reservoir 124.
[0071] Fluid may move between funnel 122 and reservoir 124 in any
number of ways. For example, a pump may pump immiscible fluid 160
from one of funnel 122 and reservoir 124 to the other of funnel 122
and reservoir 124 thereby changing the height of bio-compatible
fluid 158.
[0072] Funnel 122 includes upper portion 164 and lower portion 166.
Surface 168 of funnel 122 tapers inwardly from upper portion 164 to
lower portion 166. Furthermore, upper portion 164 has a width
greater than lower portion 166.
[0073] The shape of funnel 122 facilities the one-step loading and
unloading of cells into and out of lower portion 166. A pipette
holding cells may be inserted into funnel 122 at an angle such that
a user has a substantially unobstructed view of lower portion 166.
Likewise, a pipette may be inserted into funnel 122 to remove cells
from lower portion 166 such that a user has a substantially
unobstructed view of lower portion 166.
[0074] FIG. 4b is an enlarged side view, partially broken-away and
in cross-section, of funnel 122 and microchannel 130. Lower portion
166 of funnel 122 is configured to receive cellular mass 170.
Cellular mass 170 has a cellular height H and microchannel 130 has
a channel height h. Cellular mass 170 may be, for example, a human
zygote, a mammalian zygote, a clump of mammalian cells, or a single
mammalian cell. Microchannel 130 is configured such that cellular
mass 170 will not exit lower portion 166 of funnel 122.
[0075] FIG. 4c is another enlarged side view, partially broken-away
and in cross-section, of funnel 122 and microchannel 130 looking
down the length of microchannel 130. Cellular mass 170 has a
cellular width W and microchannel 130 has a channel width w.
Cellular mass 170 also has a cellular length (not shown).
Microchannel 130 may be configured such that at least one of the
channel height h and the channel width w is less than at least one
of the cellular height H, the cellular width W, and the cellular
length L.
[0076] Angle A is defined by opposite surfaces 168 of funnel 122.
Angle A may range between 30.degree. and 160.degree. inclusive.
[0077] At least one of the channel height h and the channel width w
may be less than 250 .mu.m or the width of human hair. In the case
where cellular mass 170 is a denuded human zygote, at least one of
the channel height h and the channel width w may be less than 140
.mu.m. In the case where cellular mass 170 is a denuded mammalian
zygote, at least one of the channel height h and the channel width
w may be less than 70 .mu.m. In the case where cellular mass 170 is
a clump of mammalian cells, at least one of the channel height h
and the channel width w may be less than 50 .mu.m. In the case
where cellular mass 170 is a single mammalian cell, at least one of
the channel height h and the channel width w may be less than 5
.mu.m.
[0078] FIG. 5a is an enlarged side view, partially broken-away and
in cross-section, of funnel 222 and microchannel 230. Lower portion
266 is sized such that a portion of cellular mass 270 is confined
to lower portion 266.
[0079] Lower portion 266 may have a width less than 250 .mu.m. In
the case where cellular mass 270 is a denuded human zygote, the
width may be less than 140 .mu.m. In the case where cellular mass
270 is a denuded mammalian zygote, the width may be less than 70
.mu.m. In the case where cellular mass 270 is a clump of mammalian
cells, the width may be less than 50 .mu.m. In the case where
cellular mass 270 is a single mammalian cell, the width may be less
than 5 .mu.m.
[0080] FIG. 5b is an enlarged side view, partially broken-away and
in cross-section, of funnel 322 and microchannel 330. Lower portion
366 is sized such that a portion of cellular mass 370 is confined
to lower portion 366. Additionally, microchannel 330 is above lower
portion 366.
[0081] FIG. 5c is an enlarged side view, partially broken-away and
in cross-section, of funnel 422 and microchannel 430. Lower portion
466 and microchannel 430 are sized such that portions of cellular
mass 470 may be in either of lower portion 466 and the portion of
microchannel 430 adjacent lower portion 466.
[0082] Embodiments of the invention may take the form of a
microfluidic device composed of a PDMS slab with bell-shaped
microfluidic channel features, a culture media reservoir, and a
funnel shaped well for culture. The media reservoir and funnel
shaped well are connected with the microfluidic channels. The
funnel shaped well may have an approach angle of approximately
60.degree. to facilitate the one-step loading and unloading of
cells and an approximately 500 .mu.m diameter tip.
[0083] In funnel type wells, cells do not need to be moved to
designated areas. Instead, cells loaded in the funnel remain
stationary. The medium or chemical composition in the funnel can be
gradually changed to mimic conditions cells experience in vivo. In
addition, the dimensions of the channels connected to the funnel
can be controlled through soft-lithography processes such that
cells are confined to the funnel. Cells may then be subjected to
diverse flow conditions.
[0084] PDMS slabs may be prepared by casting prepolymer (Sylgard
184, Dow-Corning) at a 1:10 curing agent-to-base ratio against
positive relief features approximately 30 .mu.m in height and 400
.mu.m in width. The relief features may comprise SU-8 (MicroChem,
Newton, Mass.) and be fabricated on a thin glass wafer,
approximately 200 .mu.m thick, using backside diffused-light
photolithography.
[0085] Embodiments of the invention may include a tapered well
which at its tip has an opening which communicates with one or a
plurality of microchannels. The well and microchannels may be
filled with fluid. One or more cells, e.g., embryos, may be
introduced into the well, for example, by pipet. The cells settle
to the bottom, but are prevented from exiting the well due to them
being larger than the microchannels.
[0086] Fluid may be introduced into the well continuously or
discontinuously. The fluid may contain the necessary growth media
for the cells. In a well with a single hole at the bottom, for
example, fluid may be caused to rise in the well from the
microchannels, introducing extra nutrients, and then to fall,
removing fluid which now contains exogenous substances, e.g.,
waste, via the microchannels.
[0087] Introduction and removal of fluid can be made using
conventional gravity pumps or constant flow gravity driven pumps.
Introduction and removal of fluid can also be made by outside
supplies, such as pumps, or by on-board or "semi-on-board" tactile
actuator-based pumping systems.
[0088] Wells may have inlets at other locations and or heights
rather than at the bottom, so long as the entrance ways are sized
such that cells will not pass into the channels. For example, there
may be an opening at the bottom of a well and an opening near the
middle or top, with fluid being supplied at the bottom and being
removed closer to the top.
[0089] Wells may have a polygonal shape whose walls are inclined,
in either a linear or curved fashion, such that cells added to the
well have a tendency to gravitate toward the bottom and center of
the well.
[0090] The material in which a well is formed may be, for example,
thermosetting resin, thermoplastic, metal, glass, or ceramic.
[0091] Embodiments of the invention may take the form of a
multilayer device. The top layer containing a well, and constructed
of a relatively rigid material so as to provide support for
elastomeric layers or layers of lesser strength or modulus below.
The top layer may comprise a hard transparent material, such as
glass or polymethylmethacrylate. The well may have a low surface
roughness ranging, for example, between 5 .mu.m Ra and 0.1 .mu.m
Ra.
[0092] The well may penetrate through the top layer, thus having an
open, wide-mouthed end on one side of the top layer, and on the
bottom layer, a relatively narrow hole which allows fluid
communication with microchannels in the second layer.
[0093] The microchannels may be positioned closely with respect to
the opening in the well to minimize misalignment. For example,
misalignment should not exceed 50 .mu.m. The second layer may also
constitute the bottom layer, particularly when the microchannels
are substantially on top of the second layer, e.g, abutting the
bottom surface of the top layer.
[0094] Embodiments of the invention may include microchannels that
are, at least in part, along the bottom of the second layer. A
third, or sealing layer may be applied thereto. This sealing layer
may be rather thin, such that braille-type tactile actuators may
act as valves and pumps for the various microchannels. By this
means, for example, fluid can be caused to flow or to be pumped in
one or both directions in a given microchannel depending upon the
valving, whether the valves are on or off, and whether a pump is
pumping one way or the other with respect to the microchannel.
[0095] In use, a well is first filled with fluid, e.g., an embryo
culture medium, and one or more embryos added to the well. An oil
overlay, produced by dropping one or two fine drops of oil onto the
liquid surface in the well, is then provided.
[0096] The oil prevents evaporation of liquid from the well, thus
stabilizing the osmolality, or concentration, of the ingredients
therein. The oil overlay also affects the flow of air, including
specifically oxygen and CO.sub.2 into the fluid, and the release of
these gases from the fluid. The oil may be any compatible oil, for
example, a silicone oil, a paraffin oil, or a polyethylene oligomer
oil. For the same reason, the second or third layers, if present,
may include, for example, parylene, or other materials, which
minimize water loss.
[0097] The second and third layers may be made of cast elastomer,
particularly when the embodiments employ tactile actuators. If
"off-chip" fluid supply or valving is used, however, the use of an
elastomer is not necessary, and other materials, such as cast
epoxy, injection molded thermoplastic, or glass, can be used. The
surface of these materials should be bio-compatible, and if not,
should be coated appropriately.
[0098] Zygotes may be introduced into a well containing a fluid as
is conventionally employed for embryo culture. The fluid in the
well is then covered with oil and incubated at a suitable
temperature. Fluid is directed into and out of the well through
microchannels continuously or discontinuously, e.g., a back and
forth type of fluid supply wherein the fluid level in the well
increases and then decreases cyclically. The growing embryo may be
inspected by conventional optical microscopy methods, and when
judged grown to the proper stage, the embryo is removed from the
well. If the top of the well is larger then the bottom, one-step
removal is particularly easy and the risk of damage to the embryo
is low.
[0099] Embodiments of the invention may contain microchannels whose
flow characteristics are to be actively varied and formed in a
compressible or distortable elastomeric material such as an
organopolysiloxane elastomer. Substrates, however, may be
constructed of hard, e.g., substantially non-elastic material at
portions where active control is not desired.
[0100] Embodiments of the invention may contain at least one active
portion which alters the shape or volume of chambers or passageways
("empty space"). Such active portions include mixing portions,
pumping portions, valving portions, flow portions, channel or
reservoir selection portions, cell crushing portions, and
unclogging portions. These active portions induce some change in
the fluid flow, fluid characteristics, channel, or reservoir
characteristics by exerting a pressure on the relevant portions of
the microfluidic device, and thus alter the shape or volume of the
empty space which constitutes these features. The term "empty
space" refers to the absence of substrate material. In use, the
empty space may be filled with fluid.
[0101] The active portions may be activatable by pressure to close
their respective channels or to restrict the cross-sectional area
of the channels to accomplish the desired active control. To
achieve this purpose, the channels or reservoirs may be constructed
in such a way that modest pressure from the exterior of the
microfluidic device causes the channels or reservoirs
("microfluidic features") to compress, causing local restriction or
total closure of the respective feature.
[0102] Walls surrounding the feature and external surfaces may be
elastomeric such that a minor amount of pressure causes an external
surface and, optionally, the internal feature walls to distort,
either reducing cross-sectional area at this point or completely
closing the feature.
[0103] The pressure required to "activate" the active portion(s) of
the device may be supplied by an external tactile device such as a
refreshable Braille display. The tactile actuator contacts the
active portion of the device, and when energized, extends and
presses upon the deformable elastomer, restricting or closing the
feature in the active portion.
[0104] Dimensions of the various flow channels and reservoirs may
be determined by volume and flow rate properties. Channels which
are designed for complete closure may be of a depth such that the
elastomeric layer between the microchannel and the actuator can
approach the bottom of the channel. Manufacturing the substrate of
elastomeric material facilitates complete closure, in general, as
does also a cross-section which is rounded, particularly at the
furthest corners (further from the actuator). The depth will also
depend, for example, on the extension possible for the actuator's
extendable protrusions, e.g., pins. Thus, channel depths may vary,
for example, from 1 nm to 500 .mu.m.
[0105] Embodiments of the invention may be prepared through the use
of a negative photoresist, for example, SU-8 50 photoresist (Micro
Chem Corp., Newton, Mass.) The photoresist may be applied to a
glass substrate and exposed from the uncoated side through a
suitable mask. Since the depth of cure is dependant on factors such
as length of exposure and intensity of the light source, features
ranging from very thin up to the depth of the photoresist may be
created. The unexposed resist is removed, leaving a raised pattern
on the glass substrate. The curable elastomer is cast onto this
master and then removed.
[0106] The material properties of SU-8 photoresist and the diffuse
light from an inexpensive light source can be employed to generate
microstructures and channels with cross-sectional profiles that are
rounded and smooth at the edges yet flat at the top, e.g,
bell-shaped. Short exposures tend to produce a radiused top, while
longer exposures tend to produce a flat top with rounded corners.
Longer exposures also tend to produce wider channels. These
profiles are ideal for use as compressive, deformation-based valves
that require complete collapse of the channel structure to stop
fluid flow. With such channels, Braille-type actuators produce full
closure of the microchannels, thus producing a very useful valved
microchannel. Such shapes also lend themselves to produce uniform
flow fields, and have good optical properties as well.
[0107] In a typical procedure, a photoresist layer is exposed from
the backside of the substrate through a mask, for example
photoplotted film, by diffused light generated with an ultraviolet
(UV) transilluminator. Bell-shaped cross-sections are generated due
to the way in which the spherical wavefront created by diffused
light penetrates into the negative photoresist. The exposure dose
dependent change in the SU-8 absorption coefficient limits exposure
depth at the edges.
[0108] The exact cross-sectional shapes and widths of the
fabricated structures may be determined by a combination of
photomask feature size, exposure time/intensity, resist thickness,
and distance between the photomask and photoresist. Although
backside exposure makes features which are wider than the size
defined by the photomask and in some cases smaller in height
compared to the thickness of the original photoresist coating, the
change in dimensions of the transferred patterns is readily
predicted from mask dimensions and exposure time.
[0109] The relationship between the width of the photomask patterns
and the photoresist patterns obtained is essentially linear, e.g.,
slope of 1, beyond a certain photomask aperture size. This linear
relationship allows straightforward compensation of the aperture
size on the photomask through simple subtraction of a constant
value. When exposure time is held constant, there is a threshold
aperture size below which incomplete exposure will cause the
microchannel height to be lower than the original photoresist
thickness. Lower exposure doses will make channels with smoother
and more rounded cross-sectional profiles. Light exposure doses
that are too slow or photoresist thicknesses that are too large,
however, are insufficient in penetrating through the photoresist,
resulting in cross-sections that are thinner than the thickness of
the original photoresist.
[0110] The suitability of bell-shaped cross-section microchannels
of 30 .mu.m thickness may be evaluated by exerting an external
force onto the channel using a piezoelectric vertical actuator of
commercially available refreshable Braille display. Spaces may be
left between the membrane and the wall when the channel
cross-section has discontinuous tangents, such as in rectangular
cross-sections. In contrast, a channel with a bell-shaped
cross-section may be fully closed under the same conditions. When a
Braille pin is pushed against a bell-shaped or rectangular-shaped
cross-section microchannel through a 200 .mu.m PDMS membrane, the
bell-shaped channels may be fully closed while the rectangular
channels of the same width may have considerable leakage.
[0111] When used as deformation-based microfluidic valves,
bell-shaped microchannels may show self-sealing upon compression
compared to conventional rectangular or semi-circular cross-section
channels. By way of example, a bell-shaped channel, having a width
and height of 30 .mu.m, may be completely closed by an 18 gf-force
squeeze of a Braille pin.
[0112] Channels that have the bell-shaped cross-sections with
gently sloping sidewalls may not be fabricated by melting resist
technology, one of the most convenient methods to fabricate
photomask-definable rounded patterns, because the profile is
determined by surface tension.
[0113] Bell-shaped channels maximize the cross-sectional area
within microfluidic channels without compromising the ability to
completely close channels upon deformation. Furthermore,
bell-shaped cross-sections provide channels with flat ceilings and
floors, which is advantageous for reducing aberrations in optical
microscopy and in obtaining flow fields with a more uniform
velocity profile across the widths of the channel. These advantages
of microchannels with bell-shaped cross-sectional shapes combined
with the convenient, inexpensive, and commercially available valve
actuation mechanism based on refreshable Braille displays will be
useful for a wide range of microfluidic applications such as
microfluidic cell culture and analysis systems, biosensors, and
on-chip optical devices such as microlenses.
[0114] The extension outwards of tactile actuators should be
sufficient for their desired purpose. Complete closure of a 40
.mu.m deep microchannel, for example, will generally require a 40
.mu.m extension, e.g., pin, or more when a single actuator is used,
and about 20 .mu.m or more when dual actuators on opposite sides of
the channel are used.
[0115] For peristaltic pumping, mixing, and flow regulation, lesser
extensions relative to channel height are useful. The areal size of
the tactile activators may vary appropriately with channel width
and function, and may range from 40 .mu.m to about 2 mm. Larger and
smaller sizes are possible as well.
[0116] Appendix A discloses a handheld recirculation system and
customized media for microfluidic cell culture. Appendix B
discloses a device for embryo culture and use thereof. Appendix C
discloses integrated microfluidic control employing programable
tactile actuators. Appendix D discloses a computerized control
method and system for microfluidics and computer program product
for use therein. Embodiments of the invention may take the form of
embodiments, or portions of embodiments, described in Appendices A,
B, C, and D.
Appendix A
[0117] Many modifications of the present invention will be apparent
to those skilled in the art, and are part of the subject matter
disclosed herein. The clamping mechanism, for example, may be
replaced or augmented by other clamping mechanisms, including
simple clamps which are separate from but engageable with the
fingerplate, or which can span the height of the entire device,
including the braille display module.
[0118] In similar manner, while the transparent heating element is
described as being fabricated on a glass slide, it will be
appreciated that this glass slide may be incorporated into a
disposable device, become an integrated part thereof rather than a
separate device. While less favorable, the heating element may also
be disposed directly on the microfluidics chip. The heating unit
may also be patterned such that only portions of the glass slide or
chip are heated, thus conserving electrical power as well as
avoiding heat in areas where heating is not desired, for example in
fluid storage areas.
[0119] In advanced versions of the present lab-on-chip, it is
desirable to have a battery power supply, either one-time use or
rechargeable, on the chip itself, together with electrical
circuitry for controlled operation of the heater unit, and of the
tactile actuators also, when this is desired. The ability to
divorce the structure from corded power supplies allows the module
to be easily transported to other stations for testing, analysis,
etc., while preserving the microenvironment within the module.
[0120] The subject invention further pertains to PMDS or other
elastomeric silicone structures which incorporate a film, coating,
or membrane over all or only a portion of the module structure,
which serves as a vapor barrier to minimize evaporation of liquids
contained in the channels, reservoirs, etc., of the devices.
Suitable vapor barriers are, in general, relatively pore free,
hydrophobic films, e.g. of parylene. In addition, films which are
resistant to the flow of oxygen, of carbon dioxide, or both these
gases may also be applied to minimize any influence of the ambient
atmosphere on the conditions established within the device. Such
films are well known from the field of plastic, particularly
polyethylene terephthalate, drink containers.
Appendix B
[0121] It has now been surprisingly discovered that embryos may be
grown with good survival rates in an efficient manner by growth at
the bottom of a well which is in communication with a microchannel
device supplying fluid to the well proximate its bottom. The bottom
opening is sized so as not to allow the embryo to enter the
channel.
[0122] The invention may be described with relation to the
accompanying drawings, many of which illustrate the volumes or
hollows, channels, etc. within the microfluidics device rather than
the walls of the device themselves. As illustrated, the best mode
of the device is a generally conical well which at its tip has an
opening which communicates with one or a plurality of
microchannels. The well is filled with fluid, as are the
microchannels, and one or more embryos are introduced into the
well, for example by pipet. The embryos settle to the bottom, but
are prevented from exiting the well due to them being larger than
the holes in the well.
[0123] Fluid may be introduced into the well continuously or
discontinuously, the fluid preferably containing the necessary
growth media for the embryo. For example, in a well with a single
hole at the bottom, fluid may be caused to rise in the well from
the microchannels, introducing extra nutrients, and then to fall,
removing fluid which now contains exogenous substances (waste) via
the microchannels.
[0124] Introduction and removal of fluid can be made using
conventional gravity pumps, or constant flow gravity driven pumps.
Fluid can also be supplied by outside supplies such as pumps, etc.,
or preferably by on-board or "semi-on board" tactile actuator-based
pumping systems.
[0125] The well can also have inlets at other locations and or
heights rather than exclusively at the bottom, so long as the
entrance ways to the channels are sized such that the embryos will
not pass into the channels. For example, there might be an opening
at the bottom of the well and an opening near the middle or top,
with fluid being supplied at the bottom, for example, and being
removed closer to the top.
[0126] The well also need not be entirely conical in shape, but is
preferably shaped such that the walls are inclined, regardless of
whether linear or curved such that the embryo's will have a natural
tendency to gravitate toward the bottom and center of the well. The
material of the well is not overly critical, and may be
thermosetting resin or thermoplastic, metal, glass, ceramic, etc.
In preferred constructions, the device is a multilayer device, the
top layer containing the well, and constructed of relatively rigid
material so as to provide support for elastomeric layers or layers
of lesser strength and/or modulus below.
[0127] Thus, it is preferable that the top layer be of hard
transparent material such as glass, polymethylmethacrylate, etc.
The conical well should have a low surface roughness, preferably
below 5 .mu.m Ra, more preferably less than 1 .mu.m Ra, and yet
more preferably less than 0.1 .mu.m Ra.
[0128] In preferred devices, the conical well penetrates entirely
through the top layer, thus having an open, wide-mouthed end on one
side of the top layer, and on the bottom this layer, a relatively
narrow hole which allows fluid communication with the microchannels
in the second layer. The second layer preferably directly abuts the
first layer, and has one or a plurality of microchannels which are
in fluid communication with the conical well. It is relatively
important that the channels be positioned closely with respect to
the opening in the well. For example misalignment should preferably
be maximized at 50 .mu.m. The second layer may also constitute the
bottom layer, particularly when the microfluid channels are
substantially on top of the second layer, i.e. abutting the bottom
surface of the top layer. However, in preferred devices, the
channels are at least in part along the bottom of the second layer
and a third, or sealing layer is applied thereto. This sealing
layer is preferably rather thin, such that braille-type tactile
actuators may act as valves and pumps for the various
microchannels. By this means, for example, fluid can be caused to
flow or to be pumped in only one direction in a given microchannel,
or can be bidirectional flow, depending upon the valving, whether
the valves are on or off, and whether a pump is pumping one way or
the other with respect to the channel.
[0129] In use, the device is first filled with fluid, for example
an embryo culture medium, and one or more embryos added to the
well, An oil overlay, produced by dropping one or two fine drops of
oil onto the liquid surface in the well is then provided. The oil
prevents evaporation of liquid from the well, thus changing the
osmolality, or concentration, of the ingredients therein. It also
affects the flow of air, including specifically oxygen and CO.sub.2
into the fluid, and the release of these gases from the fluid. The
oil may be any compatible oil, for example a silicone oil, a
paraffin oil, a polyethylene oligomer oil, etc. For the same
reason, portions of the apparatus in the second and/or third layers
may be coated, for example with parylene or other coating which
minimizes, particularly, water loss.
[0130] The second and third layers are preferably made of cast
elastomer, particularly when the valving and pumping embodiments
employing tactile actuators are employed. However, if "off-chip"
fluid supply, valving, etc. is used, then use of an elastomer is
not necessary, and other materials such as cast epoxy, injection
molded thermoplastic, glass, etc., can be used. It is of course
recognized that the surface of these materials should be compatible
with embryo culture, and if not, should be coated
appropriately.
[0131] The process of the subject invention requires introduction
of zygote(s) into the well which contains fluid, preferably a
growth fluid as is conventionally employed for embryo culture. The
fluid in the conical well is then covered with oil, preferably
mineral oil, and the device incubated at a suitable temperature.
Fluid is directed into and out of the well through the
microchannels continuously or discontinuously. For example, a back
and forth type of fluid supply wherein the fluid level in the well
increases and then decreases cyclically has been found most
advantageous. The growing embryo may be inspected by conventional
optical microscopy methods, and when judged grown to the proper
stage, the embryo is removed from the well. Because the top of the
well is larger then the bottom, removal is particularly easy and
the risk of damage is low.
Appendix C
[0132] The microfluidic devices of the present invention contain
microchannels whose flow characteristics are to be actively varied,
formed in a compressible or distortable elastomeric material. Thus,
it is preferred that substantially the entire microfluidic device
be constructed of a flexible elastomeric material such as an
organopolysiloxane elastomer ("PDMS"), as described hereinafter.
However, the device substrate may also be constructed of hard,
i.e., substantially non-elastic material at portions where active
control is not desired, although such construction generally
involves added construction complexity and expense. The generally
planar devices preferably contain a rigid support of glass, silica,
rigid plastic, metal, etc. on one side of the device to provide
adequate support, although in some devices, actuation from both
major surfaces may require that these supports be absent, or be
positioned remote to the elastomeric device itself.
[0133] The microfluidic devices of the present invention contain at
least one active portion which alters the shape and/or volume of
chambers or passageways ("empty space"), particularly fluid flow
capabilities of the device. Such active portions include, without
limitation, mixing portions, pumping portions, valving portions,
flow portions, channel or reservoir selection portions, cell
crushing portions, unclogging portions, etc. These active portions
all induce some change in the fluid flow, fluid characteristics,
channel or reservoir characteristics, etc. by exerting a pressure
on the relevant portions of the device, and thus altering the shape
and/or volume of the empty space which constitutes these features.
The term "empty space" refers to the absence of substrate material.
In use, the empty space is usually filled with fluids,
microorganisms, etc.
[0134] The active portions of the device are activatable by
pressure to close their respective channels or to restrict the
cross-sectional area of the channels to accomplish the desired
active control. To achieve this purpose, the channels, reservoirs,
etc. are constructed in such a way that modest pressure from the
exterior of the microfluidic device causes the channels,
reservoirs, etc. ("microfluidic features") to compress, causing
local restriction or total closure of the respective feature. To
accomplish this result, the walls within the plane of the device
surrounding the feature are preferably elastomeric, and the
external surfaces (e.g., in a planar device, an outside major
surface) are necessarily elastomeric, such that a minor amount of
pressure causes the external surface and optionally the internal
feature walls to distort, either reducing cross-sectional area at
this point or completely closing the feature.
[0135] The pressure required to "activate" the active portion(s) of
the device is supplied by an external tactile device such as are
used in refreshable Braille displays. The tactile actuator contacts
the active portion of the device, and when energized, extends and
presses upon the deformable elastomer, restricting or closing the
feature in the active portion.
[0136] Rather than close or restrict a feature by being energized,
the tactile actuator may be manufactured in an extended position,
which retracts upon energizing, or may be applied to the
microfluidics device in an energized state, closing or restricting
the passage, further opening the passage upon de-energizing.
[0137] The preferred actuators at the present time are programmable
Braille display devices such as those previously commercially
available from Telesensory as the Navigator.TM. Braille Display
with Gateway.TM. software which directly translates screen text
into Braille code. These devices generally consist of a linear
array of "8-dot" cells, each cell and each cell "dot" of which is
individually programmable. Such devices are used by the visually
impaired to convert a row of text to Braille symbols, one row at a
time, for example to "read" a textual message, book, etc. These
devices are presently preferred because of their ready commercial
availability. The microfluidic device active portions are designed
such that they will be positionable below respective actutable
"dots" or protrusions on the Braille display. Braille displays are
available from Handy Tech, Blazie, and Alva, among other
suppliers.
[0138] However, to increase flexibility, it is possible to provide
a regular rectangular array usable with a plurality of
microfluidics devices, for example having a 10.times.10,
16.times.16, 20.times.100, 100.times.100, or other array. The more
close the spacing and the higher the number of programmable
extendable protrusions, the greater is the flexibility in design of
microdevices. Production of such devices follows the methods of
construction known in the art. Addressability also follows from
customary methods. Non-regular arrays, i.e. in patterns having
actuators only where desired are also possible.
[0139] Suitable Braille display devices suitable for non-integral
use are available from Handy Tech Electronik GmbH, Horb, Germany,
as the Graphic Window Professional.TM. (GWP), having an array of
24.times.16 tactile pins. Pneumatic displays operated by
microvalves have been disclosed by Orbital Research, Inc. said to
reduce the cost of Braille tactile cells from 70 $ U.S. per cell to
Ca. 5-10 $/cell. Piezoelectric actuators are also usable where a
piezoelectric element replaces the electrorheological fluid, and
electrode positioning is altered accordingly.
[0140] The microfluidic devices of the present invention have many
uses. In cell growth, the nutrients supplied may need to be varied
to simulate availability in living systems. By providing several
supply channels with active portions to close or restrict the
various channels, supply of nutrients and other fluids may be
varied at will. An example is a three dimensional scaffolding
system to create bony tissue, the scaffolding supplied by various
nutrients from reservoirs, coupled with peristaltic pumping to
simulate natural circulation.
[0141] A further application involves cell crushing. Cells may be
crushed by transporting them in channels through active portions
and actuating channel closure to crush the cells flowing through
the channels. Cell detection may be achieved, for example, by flow
cytometry techniques using transparent microfluidic devices and
suitable detectors. Embedding optical fibers at various angles to
the channel can facilitate detection and activation of the
appropriate activators. Similar detection techniques, coupled with
the use of valves to vary the delivery from a channel to respective
different collection sites or reservoirs can be used to sort
embryos and microorganisms, including bacteria, fungi, algae,
yeast, viruses, sperm cells, etc.
[0142] Growth of embryos generally require a channel or growth
chamber which is capable of accommodating the embryo and allowing
for its subsequent growth. Such deep channels cannot effectively be
closed, however. A microfluidics device capable of embryo growth
may be fabricated by multiexposure photolithography, using two
masks. First, a large, somewhat rectangular (200 .mu.m
width.times.200 .mu.m depth) channel, optionally with a larger 200
.mu.m deep by 300 .mu.m length and 300 .mu.m width growth chamber
at one end is fabricated. Merging with the 200 .mu.m.times.200
.mu.m channel is a smaller channel with a depth of ca. 30 .mu.m,
easily capable of closure by a Braille pin. Exiting the bulbous
growth chamber are one or more thin (30 .mu.m) channels. In
operation, embryo and media are introduced into the large channel
and travel to the bulbous growth chamber. Because the exit channels
from the growth chamber are very small, the embryo is trapped in
the chamber. The merging channels and exit channels can be used to
supply nutrients, etc., in any manner, i.e. continuous, pulsating,
reverse flow, etc. The embryo may be studied by spectroscopic
and/or microscopic methods, and may be removed by separating the
elastomeric layer covering the PDMS body which houses the various
channels.
[0143] Construction of fluidic devices is preferably performed by
soft lithography techniques, as described, for example by D. C.
Duffy et al., Rapid Prototyping of Microfluidic Systems in
Poly(dimethylsiloxane), ANALYTICAL CHEMISTRY 70, 4974-4984 (1998).
See also, J. R. Anderson et al., ANALYTICAL CHEMISTRY 72, 3158-64
(2000); and M. A. Unger et al., SCIENCE 288, 113-16 (2000).
Addition-curable RTV-2 silicone elastomers such as SYLGARD.RTM.
184, Dow Corning Co., can be used for this purpose.
[0144] The dimensions of the various flow channels, reservoirs,
growth chambers, etc. are easily determined by volume and flow rate
properties, etc. Channels which are designed for complete closure
must be of a depth such that the elastomeric layer between the
microchannel and the actuator can approach the bottom of the
channel. Manufacturing the substrate of elastomeric material
facilitates complete closure, in general, as does also a
cross-section which is rounded, particularly at the furthest
corners (further from the actuator). The depth will also depend,
for example, on the extension possible for the actuator's
extendable protrusions. Thus, channel depths may vary quite a bit.
A depth of less than 100 .mu.m is preferred, more preferably less
than 50 .mu.m. Channel depths in the range of 10 .mu.m to 40 .mu.m
are preferred for the majority of applications, but even very low
channel depths, i.e. 1 nm are feasible, and depths of 500 .mu.m are
possible with suitable actuators, particularly if partial closure
("partial valving") is sufficient.
[0145] The substrate may be of one layer or a plurality of layers.
The individual layers may be prepared by numerous techniques,
including laser ablation, plasma etching, wet chemical methods,
injection molding, press molding, etc. However, as indicated
previously, casting from curable silicone is most preferred,
particularly when optical properties are important. Generation of
the negative mold can be made by numerous methods, all of which are
well known to those skilled in the art. The silicone is then poured
onto the mold, degassed if necessary, and allowed to cure.
Adherence of multiple layers to each other may be accomplished by
conventional techniques.
[0146] A preferred method of manufacture of some devices employs
preparing a master through use of a negative photoresist. SU-8 50
photoresist from Micro Chem Corp., Newton, Mass., is preferred. The
photoresist may be applied to a glass substrate and exposed from
the uncoated side through a suitable mask. Since the depth of cure
is dependant on factors such as length of exposure and intensity of
the light source, features ranging from very thin up to the depth
of the photoresist may be created. The unexposed resist is removed,
leaving a raised pattern on the glass substrate. The curable
elastomer is cast onto this master and then removed.
[0147] The material properties of SU-8 photoresist and the diffuse
light from an inexpensive light source can be employed to generate
microstructures and channels with cross-sectional profiles that are
"rounded and smooth" at the edges yet flat at the top (i.e.
bell-shaped). Short exposures tend to produce a radiused top, while
longer exposures tend to produce a flat top with rounded corners.
Longer exposures also tend to produce wider channels. These
profiles are ideal for use as compressive, deformation-based valves
that require complete collapse of the channel structure to stop
fluid flow, as disclosed by M. A. Unger, et al., SCIENCE 2000, 288,
113. With such channels, Braille-type actuators produced full
closure of the microchannels, thus producing a very useful valved
microchannel. Such shapes also lend themselves to produce uniform
flow fields, and have good optical properties as well.
[0148] In a typical procedure, a photoresist layer is exposed from
the backside of the substrate through a mask, for example
photoplotted film, by diffused light generated with an ultraviolet
(UV) transilluminator. Bell-shaped cross-sections are generated due
to the way in which the spherical wavefront created by diffused
light penetrates into the negative photoresist. The exposure dose
dependent change in the SU-8 absorption coefficient (3985 m.sup.-1
unexposed to 9700 m.sup.-1 exposed at 365 nm) limits exposure depth
at the edges.
[0149] The exact cross-sectional shapes and widths of the
fabricated structures are determined by a combination of photomask
feature size, exposure time/intensity, resist thickness, and
distance between the photomask and photoresist. Although backside
exposure makes features which are wider than the size defined by
the photomask and in some cases smaller in height compared to the
thickness of the original photoresist coating, the change in
dimensions of the transferred patterns is readily predicted from
mask dimensions and exposure time. The relationship between the
width of the photomask patterns and the photoresist patterns
obtained is essentially linear (slope of 1) beyond a certain
photomask aperture size. This linear relationship allows
straightforward compensation of the aperture size on the photomask
through simple subtraction of a constant value. When exposure time
is held constant, there is a threshold aperture size below which
incomplete exposure will cause the microchannel height to be lower
than the original photoresist thickness. Lower exposure doses will
make channels with smoother and more rounded cross-sectional
profiles. Light exposure doses that are too slow (or photoresist
thicknesses that are too large), however, are insufficient in
penetrating through the photoresist, resulting in cross-sections
that are thinner than the thickness of the original
photoresist.
[0150] The suitability of bell-shaped cross-section microchannels
of 30 .mu.m thickness to be used as deformation-based valves was
evaluated by exerting an external force onto the channel using a
piezoelectric vertical actuator of commercially available
refreshable Braille displays. Spaces may be left between the
membrane and the wall when the channel cross-section has
discontinuous tangents, such as in rectangular cross-sections. In
contrast, a channel with a bell-shaped cross-section is fully
closed under the same conditions. When a Braille pin is pushed
against a bell-shaped or rectangular-shaped cross-section
microchannel through a 200 .mu.m poly(dimethylsiloxane) (PDMS)
membrane, the bell-shaped channels were fully closed while the
rectangular channels of the same width had considerable
leakage.
[0151] The technique described is cost- and time-effective compared
to other photolithographic methods for generating well defined
rounded profiles such as gray-scale mask lithography, or laser beam
polymerization because there is no need for special equipment such
as lasers, collimated light sources (mask aligner), or submicron
resolution photomasks; it only requires a transilluminator
available in many biological labs. In addition, the backside
exposure technique can generate more profiles compared to other
soft lithography-based patterning methods such as microfluidic mask
lithography and the use of patterned laminar flows of etchant in an
existing microchannel.
[0152] When used as deformation-based microfluidic valves, these
bell-shaped microchannels showed improved self-sealing upon
compression compared to conventional rectangular or semi-circular
cross-section channels as demonstrated by simulations, and by
experiments. A bell-shaped channel (width: 30 .mu.m; height 30
.mu.m) was completely closed by an 18 gf-force squeeze of a Braille
pin. It is notable that channels that have the bell-shaped
cross-sections with "gently sloping" sidewalls cannot be fabricated
by melting resist technology, one of the most convenient methods to
fabricate photomask-definable rounded patterns, because the profile
is determined by surface tension. The bell-shaped channels maximize
the cross-sectional area within microfluidic channels without
compromising the ability to completely close channels upon
deformation. For example, the channel cross-section described here
is larger than previously reported, pneumatically actuated
deformation-based valves (100 .mu.m in width; 201 m in height) and
may be more suitable for mammalian cell culture. Furthermore, the
bell-shaped cross-sections provide channels with flat ceilings and
floors, which is advantageous for reducing aberrations in optical
microscopy and in obtaining flow fields with a more uniform
velocity profile across the widths of the channel. These advantages
of microchannels with bell-shaped cross-sectional shapes combined
with the convenient, inexpensive, and commercially available valve
actuation mechanism based on refreshable Braille displays will be
useful for a wide range of microfluidic applications such as
microfluidic cell culture and analysis systems, biosensors, and
on-chip optical devices such as microlenses.
[0153] The extension outwards of the tactile actuators must be
sufficient for their desired purpose. Complete closure of a 40
.mu.m deep microchannel, for example, will generally require a 40
.mu.m extension ("protrusion") or more when a single actuator is
used, and about 20 .mu.m or more when dual actuators on opposite
sides of the channel are used. For peristaltic pumping, mixing, and
flow regulation, lesser extensions relative to channel height are
useful. The areal size of the tactile activators may vary
appropriately with channel width and function (closure, flow
regulation, pumping, etc.), and may preferably range from 40 .mu.m
to about 2 mm, more preferably 0.5 mm to 1.5 mm. Larger and smaller
sizes are possible as well. The actuators must generate sufficient
force. The force generated by one Braille-type display pin is
approximately 176 mN, and in other displays may be higher or
lower.
[0154] By use of the present invention, numerous functions can be
implemented on a single device. Use of multiple reservoirs for
supply of nutrients, growth factors, etc. is possible. The various
reservoirs make possible any combination of fluid supply, i.e. from
a single reservoir at a time, or from any combination of
reservoirs. This is accomplished by establishing fluid
communication with a reservoir by means of a valved microchannel,
as previously described. By programming the Braille display or
actuator array, each individual reservoir may be connected with a
growth channel or chamber at will. By also incorporating a
plurality of extendable protrusions along a microchannel supply,
peristaltic pumping may be performed at a variety of flow rates.
Uneven, pulsed flow typical of vertebrate circulatory systems can
easily be created. Despite the flexibility which the inventive
system offers, construction is straightforward. The simplicity of
the microfluidics device per se, coupled with a simple,
programmable external actuator, enables a cost-effective system to
be prepared, where the microfluidic device is relatively
inexpensive and disposable, despite its technological
capabilities.
[0155] Combinatorial, regulated flow with multiple pumps and valves
that offer more flexibility in microfluidic cell studies in a
laptop to handheld-sized system are created by using a grid of tiny
actuators on refreshable Braille displays. These displays are
typically used by the visually impaired as tactile analogs to
computer monitors. Displays usually contain 20-80 rows of cells,
each holding 8 (4.times.2) vertically moving pins (.about. 1-1.3
mm). Two pins on the same cell may typically be 2.45 mm apart
center to center and 3.8 mm apart on different cells. Each pin may
have the potential to protrude 0.7.about.1 mm upward using
piezoelectric mechanisms, and may hold up to .about. 15-20 cN.
Control of Braille pins actuators is accomplished by changing a
line of text in a computer program. Unique combinations of Braille
pins will protrude depending on the letters displayed at a given
time. Braille displays are pre-packaged with software, easy to use,
and readily accessible. They are designed for individual use, and
range from walkman to laptop sizes while using AC or battery power.
By using the moving Braille pins against channels in elastomeric,
transparent rubber, it is possible to deform channels and create in
situ pumps and valves.
Appendix D
[0156] Embodiments of microfluidic devices may be suitable for the
culture of a living organism in a fluid. A microfluidic device may
control the flow and composition of fluids provided to the living
organism. The microfluidic device may provide laminar,
pseudo-multiple laminar or non-laminar flows. The microfluidic
device may perform physical operations on the living organism. The
microfluidic device may be used, for example, for general cell
culture including cell washing and detachment, cell seeding and
culture. The microfluidic device may be used as a microreactor, a
tissue culture device, a cell culture device, a cell sorting
device, a cell crushing device, a micro flow cytometer, a motile
sperm sorter, a micro carburetor, a micro spectrophotometer, or a
microscale tissue engineering device. The microfluidic device may
includes sensors to determine states or flow characteristics of
elements of the microfluidic device or the passage of particles in
a channel. The sensors may be, for example, optical, electrical, or
electromechanical sensors.
[0157] In one embodiment, a microfluidic device includes
microchannels having flow characteristics that are actively varied
and formed in a compressible or distortable elastomeric material.
In one embodiment, the entire microfluidic device is constructed of
a flexible elastomeric material, such as an organopolysiloxane
elastomer ("PDMS"), as described hereinafter. However, the device
substrate may also be constructed of hard, e.g., substantially
non-elastic material at portions, where active control is not
desired.
[0158] The microfluidic devices may contain at least one active
portion that alters the shape and/or volume of chambers or
passageways ("empty space"), particularly fluid flow capabilities
of the device. Such active portions include, without limitation,
mixing portions, pumping portions, valving portions, flow portions,
channel or reservoir selection portions, cell crushing portions,
and unclogging portions. These active portions all induce some
change in the fluid flow, fluid characteristics, channel or
reservoir characteristics, by exerting a pressure on the relevant
portions of the device, and thus altering the shape and/or volume
of the empty space which constitutes these features. The term
"empty space" refers to the absence of substrate material. In use,
the empty space is usually filled with fluids or
microorganisms.
[0159] The active portions of the device are activatable by
pressure to close their respective channels or to restrict the
cross-sectional area of the channels to accomplish the desired
active control. To achieve this purpose, the channels, reservoirs,
or other elements are constructed in such a way that modest
pressure from the exterior of the microfluidic device causes the
channels, reservoirs or other elements ("microfluidic features") to
compress, causing local restriction or total closure of the
respective feature. To accomplish this result, the walls within the
plane of the device surrounding the feature are preferably
elastomeric, and the external surfaces (e.g., in a planar device,
an outside major surface) are elastomeric, such that a minor amount
of pressure causes the external surface and optionally the internal
feature walls to distort, either reducing cross-sectional area at
this point or completely closing the feature.
[0160] The pressure used to "activate" the active portion(s) of the
device is supplied by an external tactile device, such as are used
in refreshable Braille displays of the actuator system. The tactile
actuator contacts the active portion of the device, and when
energized, extends and presses upon the deformable elastomer,
restricting or closing the feature in the active portion.
[0161] In some embodiments, rather than close or restrict a feature
by being energized, the tactile actuator may be manufactured in an
extended position, which retracts upon energizing, or may be
applied to the microfluidic device in an energized state, closing
or restricting the passage, further opening the passage upon
de-energizing.
[0162] A significant improvement in the performance, not only of
the subject invention devices, but of other microfluidic devices
which use pressure, e.g., pneumatic pressure, to activate device
features, may be achieved by molding the device to include one or
more voids adjacent the channel walls. These voids allow for more
complete closure or distortion of the respective feature.
[0163] In one embodiment, the actuator system is a programmable
Braille display that includes a plurality of moveable pins that
each engage a corresponding element of the microfluidic device to
perform a fluidic operation. The elements of the microfluidic
device include pumps and valves. The pins may be arranged in a
regular geometric array. Such arrangement maybe used with different
configurations of the microfluidic device. In this arrangement,
some pins may not be used for particular microfluidic devices
because no element in the device corresponds to the pin.
Alternatively the pins may be selected to correspond to elements of
a specific or a group of multifluidic devices. Each pin may be
controlled independently, and individually addressable.
[0164] An example of an actuator system is a Telesensory system
such as the Navigator.TM. Braille Display with Gateway.TM.
software, which directly translates screen text into Braille code.
These devices generally comprise a linear array of "8-dot" cells,
each cell and each cell "dot" of which is individually
programmable. Such devices are used by the visually impaired to
convert a row of text to Braille symbols, one row at a time, for
example to "read" a textual message or book. The microfluidic
device active portions are designed such that they will be
positionable below respective actuable "dots" or protrusions on the
Braille display. Braille displays are available from Handy Tech,
Blazie, and Alva, among other suppliers. As will be described
below, the system may use various software programs for controlling
the pins of the actuator system by allowing the user to select
processes to be performed on the organism, and then executing
processes from a library.
[0165] However, to increase flexibility, it is possible to provide
a regular rectangular array usable with a plurality of microfluidic
devices, for example having a 10.times.10, 16.times.16,
20.times.100, 100.times.100, or other size array. The closer the
spacing and the higher the number of programmable extendable
protrusions, the greater is the flexibility in design of
microdevices. Production of such devices follows the methods of
construction known in the art. Addressability also follows from
customary methods. Non-regular arrays, e.g., in patterns having
actuators only where desired are also possible.
[0166] Devices can also be constructed which integrate the tactile
actuators with the microfluidic device. The actuators are still
located external to the microfluidic device itself, but attached or
bonded thereto to form an integrated whole. Other types of actuator
systems may be used, such as a tactile actuator device, which
employs a buildup of an electrorheological fluid, an
electromechanical Braille-type device employing shape memory wires
for displacement between "on" and "off" portions, devices employing
electrorheologic or magnetorheologic working fluids or gels, a
pneumatically operated Braille device, "voice coil" type
structures, especially those employing strong permanent magnets,
devices employing shape memory alloys and intrinsically conducting
polymer sheets.
[0167] Suitable Braille display devices suitable for non-integral
use are available from Handy Tech Electronik GmbH, Horb, Germany,
as the Graphic Window Professional.TM. (GWP), having an array of
24.times.16 tactile pins. Piezoelectric actuators are also usable
where a piezoelectric element replaces the electrorheological
fluid, and electrode positioning is altered accordingly.
[0168] The microfluidic device has many uses. The software
described herein automates the operation of these uses. In cell
growth, the nutrients supplied may be varied to simulate
availability in living systems. By providing several supply
channels with active portions to close or restrict the various
channels, supply of nutrients and other fluids may be varied at
will. An example is a three dimensional scaffolding system to
create bony tissue, the scaffolding supplied by various nutrients
from reservoirs, coupled with peristaltic pumping to simulate
natural circulation.
[0169] Another application involves cell crushing. Cells may be
crushed by transporting them in channels through active portions
and actuating channel closure to crush the cells flowing through
the channels. Cell detection may be achieved, for example, by flow
cytometry techniques using transparent microfluidic devices and
suitable detectors. Embedding optical fibers at various angles to
the channel can facilitate detection and activation of the
appropriate activators. Similar detection techniques, coupled with
the use of valves to vary the delivery from a channel to respective
different collection sites or reservoirs can be used to sort
embryos and microorganisms, including bacteria, fungi, algae,
yeast, viruses, and sperm cells.
[0170] The software controls the actuator system to control the
pressure and thus the opening and closing of the channel and the
timing. Depending on the processes to be performed, the software
may address the actuators individually or in groups, and in
patterns to provide actions, such as a peristaltic pumping action
or a mixing action with respect to fluid in the channel. The
software may monitor the sensors of the microfluidic device to
selectively control the channel flow.
[0171] As an illustrative example of peristaltic pump formed by
three pins engaging the microfluidic device, a pattern, such as
XXO, OXX, OOX, XOX in repetition, where X is a closed position and
O is an open position, to pump fluid in a channel may be used. The
resultant fluid flow is pulsatile, with transient movements in both
directions. The net movement can be predicted by its linear
relationship to the pattern change frequency, and flow direction
can be switched by reversing the pattern of actuation.
[0172] By use of the present invention, numerous functions can be
implemented on a single device. Use of multiple reservoirs for
supply of nutrients, growth factors, and the like is possible. The
various reservoirs make possible any combination of fluid supply,
e.g., from a single reservoir at a time, or from any combination of
reservoirs. This is accomplished by establishing fluid
communication with a reservoir by means of a valved microchannel,
as previously described. By programming the actuator system, each
individual reservoir may be connected with a growth channel or
chamber at will. By also incorporating a plurality of extendable
protrusions along a microchannel supply, peristaltic pumping may be
performed at a variety of flow rates. Uneven, pulsed flow typical
of vertebrate circulatory systems can easily be created.
Combinatorial, regulated flow with multiple pumps and valves that
offer more flexibility in microfluidic cell studies are created by
using a grid of tiny actuators on refreshable Braille displays and
executed automatically by software in response to user selections
of processes to be performed.
[0173] While embodiments of the invention have been illustrated and
described, it is not intended that these embodiments illustrate and
describe all possible forms of the invention. Rather, the words
used in the specification are words of description rather than
limitation, and it is understood that various changes may be made
without departing from the spirit and scope of the invention.
* * * * *