U.S. patent application number 14/950221 was filed with the patent office on 2016-03-17 for automated vitrification device.
The applicant listed for this patent is University of Kansas. Invention is credited to Sarah L. KIEWEG, Samuel S. KIM, Todd MCDONALD.
Application Number | 20160076982 14/950221 |
Document ID | / |
Family ID | 47629928 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160076982 |
Kind Code |
A1 |
KIM; Samuel S. ; et
al. |
March 17, 2016 |
AUTOMATED VITRIFICATION DEVICE
Abstract
The present application relates to devices for the vitrification
of biological samples, including embryos, oocytes and biological
tissues. Suitably, the devices are automated devices which require
little if any operator intervention or sample handling, thereby
reducing contamination, improving success rates and increasing
efficiency. Also provided are methods of vitrifying a biological
sample, suitably using the disclosed devices.
Inventors: |
KIM; Samuel S.; (Mission,
KS) ; KIEWEG; Sarah L.; (Lawrence, KS) ;
MCDONALD; Todd; (Lenexa, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Kansas |
Lawrence |
KS |
US |
|
|
Family ID: |
47629928 |
Appl. No.: |
14/950221 |
Filed: |
November 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14236418 |
Jun 10, 2014 |
9228925 |
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PCT/US2012/049482 |
Aug 3, 2012 |
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14950221 |
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61515149 |
Aug 4, 2011 |
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Current U.S.
Class: |
435/374 ;
435/307.1; 435/420 |
Current CPC
Class: |
A01N 1/0242 20130101;
A01N 1/0252 20130101; A01N 1/0284 20130101; G01N 1/42 20130101;
A01N 1/0268 20130101 |
International
Class: |
G01N 1/42 20060101
G01N001/42; A01N 1/02 20060101 A01N001/02 |
Claims
1. A container for a biological sample, comprising: a) a support
member having a top surface and a bottom surface; b) a sample
retention area traversing said support member from said top surface
to said bottom surface; and c) a porous mesh positioned in said
sample retention area, wherein said sample retention area allows
for introduction of said biological sample onto said porous mesh
and removal of a fluid from said porous mesh.
2. The container of claim 1, wherein said sample retention area is
a substantially circular hole traversing said support member.
3. The container of claim 2, wherein said sample retention area
comprises an extension surrounding said hole, extending at least
above said top surface of said support member.
4. The container of claim 1, further comprising a frame supporting
said container.
5. A sample handling apparatus, comprising a plurality of
containers for a biological sample, each container comprising: a) a
support member having a top surface and a bottom surface; b) a
substantially circular hole traversing said support member from
said top surface to said bottom surface; and c) a porous mesh
positioned in said substantially circular hole, wherein said
substantially circular hole allows for introduction of said
biological sample onto said porous mesh and removal of a fluid from
said porous mesh, and wherein said plurality of containers are
supported by a frame.
6. The container of claim 5, comprising an extension surrounding
said hole, extending at least above said top surface of said
support member.
7. A method of vitrifying a biological sample, comprising: a)
placing a biological sample in a sample holder; b) placing a
cryo-protectant in a cryo-protectant holder; c) dispensing said
cryo-protectant from said cryo-protectant holder onto said sample;
d) removing excess cryo-protectant from said sample via a drying
mechanism; e) sealing said sample; f) cutting said sealed sample;
and g) transferring said sealed sample into a coolant in a coolant
holder.
8. The method of claim 7, wherein said dispensing, said removing,
said sealing, said cutting and said transferring are automated via
a control module.
9. The method of claim 7, wherein said dispensing comprises
dispensing with a nozzle.
10. The method of claim 7, wherein said sealing comprises sealing
with an adhesive film.
11. The method of claim 7, wherein said cutting comprises cutting
with a first die and a second that align together.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 14/236,418, filed Jun. 10, 2014, which is a U.S. National
Phase of International Patent Application No. PCT/US2012/049482,
filed Aug. 3, 2012, which claims benefit of U.S. Provisional Patent
Application No. 61/515,149, filed Aug. 4, 2011, the disclosures of
each of which are incorporated by reference herein in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present application relates to devices for the
vitrification of biological samples, including embryos, oocytes and
biological tissues. Suitably, the devices are automated devices
which require little if any operator intervention or sample
handling, thereby reducing contamination, improving success rates
and increasing efficiency. Also provided are methods of vitrifying
a biological sample, suitably using the disclosed devices.
[0004] 2. Background of the Invention
[0005] Freezing and preservation of biological samples had
traditionally been carried out using a slow-freezing process. Those
methods were accomplished by tabletop machines that slowly cooled a
sample over the period of several hours to cryogenic temperatures.
These "slow freezing methods" have recently being supplanted by
sample "vitrification," which allows for rapid freezing and storing
of specimens in a matter of minutes instead of hours.
[0006] However, current vitrification devices require significant
operation by a skilled lab technician. Success rates of
vitrification and warming viable samples vary widely depending on
the method used and the skill of the lab technician performing the
procedure. Such methods traditionally require a lab technician to
prepare individual samples for freezing, handle each separately and
carry out specified methods. These methods, however, are not only
time-consuming and inefficient, but can result in variations in
sample vitrification due to differences in technician training and
technique. This makes it extremely difficult to evaluate and
compare various protocols. In addition, many manual vitrification
methods expose the specimen directly to liquid nitrogen (i.e., open
system), which provides rapid cooling (vitrification), but can lead
to introduction of contaminants (fungi, pathogens etc.) to the
specimen, as well as sample loss and damage.
[0007] The inventors have identified a need for protocols and
devices that can be used to repeatedly and efficiently vitrify
biological samples, including for use in fertility preservation and
tissue banking.
SUMMARY OF PREFERRED EMBODIMENTS
[0008] The present application provides automated vitrification
devices, as well as methods of vitrification that meet the needs
identified above.
[0009] In embodiments, automated vitrification devices are
provided. Suitably, such devices comprise a cryo-protectant holder,
a cryo-protectant dispenser, a sample holder oriented to allow a
sample in the sample holder to be contacted with cryo-protectant
from the cryo-protectant dispenser, a sample sealing device, a
coolant holder oriented to allow a sealed sample to be placed in a
coolant in the coolant holder, and a control module operably
connected to the cryo-protectant dispenser, the sample holder and
the sample sealing device.
[0010] Suitably, the cryo-protectant dispenser is operably
connected to a driving mechanism that controls the movement of the
cryo-protectant dispenser. In embodiments, the various components
of the devices are housed within a chamber, the chamber comprising
at least one opening for introduction of the sample to the sample
holder.
[0011] In embodiments, the sample holder is a rotating sample
holder that moves the sample from a position that allows
introduction of the sample, to a position that allows introduction
of the cryo-protectant from the cryo-protectant dispenser.
Suitably, the rotating sample holder further moves the sample from
the position that allows introduction of the cryo-protectant to a
position that allows sealing of the sample by the sample sealing
device.
[0012] In embodiments, the devices further comprise a drying
mechanism for removing excess cryo-protectant from the sample.
Suitably, the sample sealing device comprises a sealing mechanism
and a cutting mechanism for first sealing the sample, then cutting
the sealed sample, and then releasing the sealed sample from the
sample holder into the coolant holder. Suitably, the coolant in the
coolant holder is liquid nitrogen.
[0013] In embodiments, the sealing mechanism comprises an adhesive
film and the cutting mechanism comprises a first die and a second
that align together.
[0014] In further embodiments, automated vitrification devices are
provided that comprise a cryo-protectant holder, a cryo-protectant
dispenser, a sample holder oriented to allow a sample in the sample
holder to be contacted with cryo-protectant from the
cryo-protectant dispenser, a drying mechanism for removing excess
cryo-protectant from the sample, a sample sealing device comprising
a sealing mechanism and a cutting mechanism for first sealing the
sample, then cutting the sealed sample. The devices also comprise a
coolant holder oriented to allow the sealed sample to be placed in
a coolant in the coolant holder and a control module operably
connected to the cryo-protectant dispenser, the sample holder and
the sample sealing device. Suitably, the various device components
are housed within a chamber, the chamber comprising at least one
opening for introduction of the sample to the sample holder.
[0015] Also provided are containers for a biological sample,
comprising a support member having a top surface and a bottom
surface, a sample retention area (e.g., a substantially circular
hole) traversing the support member from the top surface to the
bottom surface and a porous mesh positioned in the sample retention
area. Suitably, the sample retention area allows for introduction
of the biological sample onto the porous mesh and removal of a
fluid from the porous mesh. Suitably, the sample retention area
comprises an extension surrounding said hole, extending at least
above said top surface of said support member. In embodiments, the
containers further comprise a frame supporting the container.
[0016] Also provided are methods of vitrifying a biological sample.
Such methods suitably comprise placing a biological sample in a
sample holder, placing a cryo-protectant in a cryo-protectant
holder, dispensing the cryo-protectant from the cryo-protectant
holder onto the sample, removing excess cryo-protectant from the
sample via a drying mechanism, sealing the sample, cutting the
sealed sample, and transferring the sealed sample into a coolant in
a coolant holder.
[0017] Suitably, the dispensing, removing, sealing, cutting and
transferring are automated via a control module.
[0018] Further embodiments, features, and advantages of the
embodiments, as well as the structure and operation of the various
embodiments, are described in detail below with reference to
accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows an exemplary automated vitrification device as
described herein.
[0020] FIG. 2 shows a cryo-protectant holder and sample holder of
an exemplary automated vitrification device as described
herein.
[0021] FIG. 3 shows a cryo-protectant dispenser, a cryo-protectant
holder and sample holder of an exemplary automated vitrification
device as described herein.
[0022] FIG. 4 shows a drying mechanism of an exemplary automated
vitrification device as described herein.
[0023] FIG. 5 shows a cryo-protectant holder, sample holder and
sample sealing device of an exemplary automated vitrification
device as described herein.
[0024] FIG. 6 shows and alternative view of a cryo-protectant
holder, sample holder and sample sealing device of an exemplary
automated vitrification device as described herein.
[0025] FIG. 7 shows a sealed sample of an exemplary automated
vitrification device as described herein.
[0026] FIG. 8 shows a sealed sample and a coolant holder of an
exemplary automated vitrification device as described herein.
[0027] FIG. 9A shows an alternative view of an exemplary automated
vitrification device as described herein.
[0028] FIG. 9B shows an additional exemplary automated
vitrification device as described herein.
[0029] FIG. 9C shows an alternative view of an exemplary automated
vitrification device as described herein.
[0030] FIG. 10 shows an exemplary sample holder/container for a
biological sample as described herein.
[0031] FIG. 11A shows an additional exemplary sample
holder/container for a biological sample as described herein.
[0032] FIG. 11B shows an additional exemplary sample
holder/container for a biological sample as described herein
[0033] FIG. 12A shows an exemplary control system for use with an
automated vitrification device as described herein.
[0034] FIG. 12B shows an additional exemplary control system for
use with an automated vitrification device as described herein.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0035] It should be appreciated that the particular implementations
shown and described herein are examples and are not intended to
otherwise limit the scope of the application in any way.
[0036] The published patents, patent applications, websites,
company names, and scientific literature referred to herein are
hereby incorporated by reference in their entireties to the same
extent as if each was specifically and individually indicated to be
incorporated by reference. Any conflict between any reference cited
herein and the specific teachings of this specification shall be
resolved in favor of the latter. Likewise, any conflict between an
art-understood definition of a word or phrase and a definition of
the word or phrase as specifically taught in this specification
shall be resolved in favor of the latter.
[0037] As used in this specification, the singular forms "a," "an"
and "the" specifically also encompass the plural forms of the terms
to which they refer, unless the content clearly dictates otherwise.
The term "about" is used herein to mean approximately, in the
region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20%.
[0038] Technical and scientific terms used herein have the meaning
commonly understood by one of skill in the art to which the present
application pertains, unless otherwise defined. Reference is made
herein to various methodologies and materials known to those of
ordinary skill in the art.
[0039] In embodiments, an automated vitrification device 100, for
example as shown in FIG. 1, is provided.
[0040] As used herein "automated" refers to a device that operates,
i.e., proceeds step-by-step through various mechanical movements
and actions, with little or no intervention or control from a human
operator. In general, the automated devices described herein are
controlled via computer or other suitable control module.
[0041] The term "vitrification" as used herein refers to the
solidification of a liquid by an extreme elevation of viscosity as
a result of the rapid cooling of a sample below about -110.degree.
C., suitably below about -150.degree. C., to preserve the sample
and allow it to be stored for extended periods, e.g., days, weeks,
months, years, etc.
[0042] As shown in FIG. 1, automated vitrification device 100
suitably comprises cryo-protectant holder 102. In embodiments,
cryo-protectant holder 102 comprises a base 204 that contains one
or more reservoirs 206 suitable for receiving and holding a
cryo-protectant. Base 204 suitably sits on a rotating platform 202,
which rotates about rotation point 210. As used herein,
"cryo-protectant" or "cryoprotectant" refers to a composition
(suitably a fluid solution) that is used to protect biological
samples from freezing damage due to ice crystal formation.
Exemplary cryo-protectant solutions are known in the art, and
include for example, solutions comprising ethylene glycol, dimethyl
sulfoxide, propane-1,2-diol (PrOH), etc., and mixtures thereof.
Suitably, base 204 comprises multiple (i.e., 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) reservoirs 206 that can be used to bold different
cryo-protectants. As described herein, it is often necessary during
a vitrification procedure to contact a sample with more than one
cryo-protectant (i.e., cryo-protectants having different
compositions).
[0043] Cryo-protectant solutions for use in the devices and methods
described herein suitably comprise ethylene glycol (EG), dimethyl
sulfoxide (DMSO), sucrose and/or trehalose, and serum substitute
supplement (SSS.TM.) in modified human tubal fluid (mHTF) (suitably
from Irvine Scientific, Santa Ana, Calif.). An exemplary
cryo-protectant solution suitably comprises about 2% to about 30%
EG, about 2% to about 20% DMSO, about 0.05 to about 1.0M sucrose
and/or about 0.05 to about 1.0M trehalose, and about 5% to about
30% SSS.TM. in mHTF. More suitably, exemplary cryo-protectants
comprise about 5% to about 20% EG, about 5% to about 10% DMSO,
about 0.1 to about 0.5M sucrose and/or about 0.1 to about 0.5M
trehalose, and about 15% to about 25% SSS in mHTF.
[0044] Exemplary cryo-protectant solutions comprise 1) about 7.5%
EG, about 7.5% DMSO, about 0.1M sucrose and about 20% SSS in mHTF;
2) about 10% EG, about 10% DMSO, about 0.3M sucrose and about 20%
SSS in mHTF; or 3) about 20% EG, about 10% DMSO, about 0.5M
sucrose, about 0.5M trehalose, and about 20% SSS in mHTF. In
embodiments, such cryo-protectant solutions are suitably used for
vitrification of oocytes.
[0045] Additional exemplary cryo-protectant solutions comprise 1)
about 7.5% EG, about 7.5% DMSO, about 0.1M sucrose, and about 20%
SSS in mHTF; 2) about 10% EG, about 10% DMSO, about 0.3M sucrose,
about 20% SSS in mHTF; or 3) about 20% EG, about 10% DMSO, about
0.5M sucrose, about 0.5M trehalose and about 20% SSS in mHTF. In
embodiments, such cryo-protectant solutions are suitably used for
vitrification of blastocysts.
[0046] As used herein, "biological material" and "biological
sample" are used interchangeably and include material obtained from
a human, plant or animal source, and includes for example, tissues,
blood, cells, bone, oocytes, embryos, sperm, eggs, ovarian tissue,
gamete, gonadal tissue, testicular tissue, etc.
[0047] Vitrification device 100 also suitably comprises
cryoprotectant dispenser 104 as shown in FIG. 1. Cryo-protectant
dispenser 104 is suitably oriented above cryo-protectant holder
102, as shown in FIG. 3, however in other embodiments,
cryo-protectant dispenser 104 can be located below cryo-protectant
holder 102, or can be in any other suitable orientation.
[0048] Cryo-protectant dispenser 104 suitably is operably connected
to driving mechanism 302 that controls its movement in any suitable
direction, (i.e., in any x, y or z direction, or combination of
these directions) so as to remove (e.g., suction) cryo-protectant
from cryo-protectant holder 102 via nozzles 304 or other suitable
mechanism.
[0049] Automated vitrification device 100 also comprises sample
holder 208, shown in FIG. 2 and FIG. 3. Sample holder 208 is
suitably oriented so as to allow a sample in the sample holder to
be contacted with one or more cryo-protectants from cryo-protectant
dispenser 104. For example, cryo-protectant that is removed from
reservoirs 206 via nozzles 304 can be administered/delivered to the
sample in sample holder 208. Cryo-protectant dispenser 104 is able
to control the amount of cryo-protectant administered to the sample
and can administer multiple cryo-protectants to a single sample in
any order, at any time, for any duration and in any amount desired
by the operator via a control module as described herein.
[0050] For example, in embodiments, cryo-protectant dispenser
administers cryo-protectants to a biological sample in the amounts
and for the durations indicated below. Drying mechanism 106 is
suitably used to remove excess cryo-protectant at the desired
times/intervals prior to application of the next
cryo-protectant.
[0051] Described below is an exemplary cryo-protectant protocol for
vitrification of oocytes: [0052] A solution comprising about 7.5%
EG+about 7.5% DMSO+about 0.1M sucrose+about 20% SSS in mHTF is
applied to the biological sample for 5 minute; [0053] Excess
solution is removed; [0054] A solution comprising about 1.0%
EG+about 10% DMSO+about 0.3M sucrose+about 20% SSS in mHTF is
applied to the biological sample for about 1 minute; [0055] Excess
solution is removed; [0056] A solution comprising about 20%
EG+about 10% DMSO+about 0.5M sucrose+about 0.5M trehalose+about 20%
SSS in mHTF is applied to the biological sample for about 40
seconds. This 40 second time suitably also Includes sealing the
sample prior to vitrification, as described herein.
[0057] Described below is an exemplary cryo-protectant protocol for
vitrification of blastocysts: [0058] A solution comprising about
7.5% EG+about 7.5% DMSO+about 0.1M sucrose+about 20% SSS in mHTF is
applied to the biological sample for about 10 minutes; [0059]
Excess solution is removed; [0060] A solution comprising about 10%
EG+about 10% DMSO+about 0.3M sucrose+about 20% SSS in mHTF is
applied to the biological sample for about 1 minute; [0061] Excess
solution is removed; [0062] A solution comprising about 20%
EG+about 10% DMSO+about 0.5M sucrose+about 0.5M trehalose+about 20%
SSS in mHTF is applied to the biological sample for about 60
seconds. This 60 second time suitably also includes sealing the
sample prior to vitrification, as described herein.
[0063] As shown in FIGS. 1, 5 and 6, automated vitrification device
100 suitably further comprises sample sealing device 110/108/112,
having upper 110 and lower 112 sections. In embodiments, as shown
in FIGS. 5 and 6, sample sealing device 110/108/112 suitably
comprises a sealing mechanism 108 and a cutting mechanism (602 and
604).
[0064] Automated vitrification device 100 suitably also comprises
coolant holder 114, as shown in FIG. 8. Suitably, coolant holder
114 is oriented to allow a sealed sample 702 to be placed in a
coolant in the coolant holder.
[0065] As discussed herein, the automated vitrification devices
described throughout are suitably under the control of a control
module, suitably a computer or similar device, which is connected
to one or more of the various components of the vitrification
device so as to allow for automation of a vitrification process
carried out by the device. Suitably, a control module (not shown)
is operably connected to cryoprotectant dispenser 104, sample
holder 208 and sample sealing device 110/108/112. In additional
embodiments, a control module can be further connected to drying
mechanism 106, cryo-protectant holder 102, as well as any other
components.
[0066] FIG. 12A shows an exemplary control system 1200 for use with
the automated vitrification devices described herein. In exemplary
embodiments, control system 1200 comprises a National Instruments
USB D/A Board 1202 running a custom program 1204 written in
LabView. As shown in FIG. 12A, the D/A Board suitably controls
three servos, including drying mechanism 106 servo 1206,
cryo-protectant dispenser 104 servo 1208, and cryo-protectant
dispenser suction (not shown) servo 1210 using pulse width
modulation. The D/A Board is also connected stepper motor
controller 1212 using a serial connection. Stepper motor controller
1212 suitably controls four stepper motors; cryo-protectant holder
102 rotation motor 1214, Sample Sealing Device 110/108/112 Motor
1216, Cryo-Protectant Dispenser 104 Positioning Motor 1218, and
Sealant Tape Advancement (not shown) Motor 1220. FIG. 12B shows an
additional exemplary control system 1200' for use with the
automated vitrification devices described herein. Exemplary control
system 1200' comprises components, including motors/servos,
analogous to those disclosed in control system 1200.
[0067] In exemplary embodiments, the various components of
automated vitrification device 100, as described herein, are housed
within chamber 116, as shown in FIGS. 1, 9A, 9B and 9C. In
embodiments, chamber 116 comprises at least one opening (not shown)
that allows for introduction of a sample to sample holder 208.
Suitably, chamber 116 is a metal enclosure that contains the
various device components described herein.
[0068] As described herein, sample holder 208 is suitably a
rotating sample holder attached to base 202 and connected to
rotation point 210, which moves the sample from a position that
allows introduction of the sample, to a position that allows
introduction of the cryo-protectant from cryo-protectant dispenser
104. For example, as shown in FIG. 1, a sample is introduced into
sample holder 208 in a first position. Sample holder 208 is then
rotated to a position below cryo-protectant dispenser 104, in which
cryo-protectant dispenser 104 is also able to remove
cryo-protectant from reservoirs 206 of cryo-protectant holder 102
(see e.g., FIGS. 1, 2 and 3) and then administer the
cryo-protectant to the samples.
[0069] Suitably, cryo-protectant dispenser 104 utilizes a stepper
motor to drive two ACME screws that move the main exposure carriage
(e.g., 302). The dispenser suitably uses a six-bar linkage driven
by a linear solenoid to retrieve cryo-protectant from the
reservoirs and place them on the samples. Each nozzle (suitably 2,
3, 4, 5, 6, 7, 8, 9 10, etc. nozzles) is independently controlled
by its own solenoid, which draws the cryo-protectant into the
nozzles and pushes them out using a suitable device, such as a
syringe.
[0070] The rotating sample bolder is further able to move the
sample from the position where the cryo-protectant is introduced,
to a position that allows sealing of the sample by sample sealing
device 110/108/112, as in FIG. 5. As shown in FIGS. 5 and 6, sample
sealing device suitably comprises sealing mechanism 108. In
exemplary embodiments, sealing mechanism 108 comprises an adhesive
or other suitable film 504, for example an adhesive tape. As shown
in FIG. 5, in embodiments, sealing mechanism 504 is wound over arms
502 that create an envelope or pocket into which sample holder 208
is inserted, as shown in FIG. 5. Sealing mechanism 504 in the form
of adhesive tape can suitably be stored on storage roll 506 (or
multiple storage rolls as shown in FIG. 9B) prior to use in sample
sealing device 110/108/112. As shown in FIG. 5, sample holder 208
is rotated into position with sealing mechanism 504 via a rotation
platform about rotation point 210.
[0071] Sample sealing device 110/108/112 suitably comprises a
cutting mechanism for first sealing the sample (FIG. 5), then
cutting the sealed sample (FIG. 6), and then releasing the sealed
sample 702 from the sample holder into the coolant holder 114.
[0072] In exemplary embodiments, cutting mechanism comprises a
first die 602 (i.e., an upper die 602 on upper portion 110 of
sealing device 110/108/112) and a second die 604 (i.e., a lower die
on lower portion 112 of sealing device 110/108/112) that align
together, as shown in FIG. 6. It should be understood that
orientation of upper and lower die are for illustration purposes,
and any suitable orientation can be used. In addition, other
mechanisms to cut and seal sample 208 can also be used. As shown in
FIG. 6, first die 602 and second die 604 are brought into contact
so as to seal and cut the sample via collapsing arms 512. A person
of ordinary skill in the art will readily be able to envision other
suitable mechanisms for sealing and cutting sample 208.
[0073] Sealing sample utilizing a sealing mechanism such as an
adhesive film or tape provides a mechanism by which the sample can
be vitrified, but without actually directly contacting the sample
with liquid nitrogen, thereby reducing contamination and sample
loss or damage.
[0074] Suitably, a stepper motor is used to drive the movement of
the sealing device 110/108/112, and a slot-and-linkage arrangement
is used to control the movement. The assembly has mating upper 602
and lower 604 dies that press the sealing mechanism, e.g., sealant
tape, onto containers 208, cut the tape, punch out the containers
from their handling frame and tilt to put the containers into
coolant, as described herein.
[0075] The sealing tape is placed in the machine on a roll 506 (or
multiple rolls as shown in FIG. 9B) and is tensioned by a
spring-loaded roller and arm 502 (or multiple arms as shown in FIG.
9B). The tape attaches to a roller, which is driven by a stepper
motor to pull the sealing tape into an envelope-type arrangement.
The specimens are then rotated into the envelope where they are
sealed, punched out and vitrified.
[0076] FIG. 7 shows a sealed sample 702, comprising sealing
mechanism 504 covering and securing sample 208, as well as
individual separated support members 209 of the sample holder 208.
The lower portion 112 of sealing device 110/108/112 suitably
comprises raised portions 510 on platform 508 of second die 604,
see FIGS. 5 and 7.
[0077] As lower die 604 is lowered, it contacts rod 704, which
causes the die to tilt, thereby releasing the sealed sample 702
into the coolant holder 114. Suitably, coolant holder 114 comprises
individual cryo-vials 802, each individually containing a coolant,
suitable for receiving the sealed samples. In exemplary
embodiments, the coolant is liquid nitrogen. Other suitable
coolants include, for example, liquid helium, etc.
[0078] As shown in FIGS. 1 and 4, the automated vitrification
device 100 suitably further comprises a drying mechanism 106, for
removing excess cryo-protectant from the sample. Drying mechanism
106 suitably comprises multiple (i.e., 2 or more, 3, 4, 5, 6, 7, 8,
9, 10 or more) individual drying elements 402 (suitably a cloth or
cotton swab or other suitable adsorbent material) positioned in a
base 404. As shown in FIG. 1, following the addition of
cryo-protectant to the sample 208 with cryo-protectant dispenser
104, the sample is rotated about rotation point 210, so that the
samples are directly above drying elements 402. It should be noted
that this orientation is for illustrative purposes only, and in
other embodiments, the drying mechanism 106 can be positioned above
the sample holder, or any other suitable orientation can be
used.
[0079] Drying mechanism 106 is used to remove excess
cryo-protectant (surplus cryo-protectant that is not necessary for
the vitrification as well as cryo-protectant that may interfere
with additional cryoprotectants) prior to introduction of a
subsequent cryo-protectant to the sample. As described herein,
vitrification methods suitably utilize multiple different
cryo-protectants, each or which requires contact with a sample for
a particular amount of time and in a particular order.
[0080] FIG. 9A shows an additional view of an automated
vitrification device as described herein, showing suitable
orientations of the various components described throughout. FIG.
9B shows an additional automated vitrification device as described
herein, showing suitable orientations of the various components
described throughout. FIG. 9C shows a photograph of an assembled
automated vitrification device as described herein.
[0081] Exemplary materials for use in the various components of the
automated vitrification devices described herein are well known in
the art and include for example, various metals, plastics,
polymers, ceramics and glasses. Methods for mechanically connecting
the various components of the devices herein are also well known
and readily implemented by one of ordinary skill in the art.
[0082] In additional embodiments, automated vitrification device
100 as shown in FIG. 1, FIGS. 9A, 9B and 9C, is provided. The
device suitably comprises cryo-protectant holder 102,
cryo-protectant dispenser 104 (suitably also comprising suction
assembly 902 to provide suction for cryo-protectant dispenser 104,
though the suction assembly can also be integrated into dispenser
104) and sample holder 208 oriented to allow a sample in the sample
holder to be contacted with cryo-protectant from the
cryo-protectant dispenser 104. The device suitably further
comprises drying mechanism 106 for removing excess cryo-protectant
from the sample, sample sealing device 110/108/112 comprising
sealing mechanism 504 and a cutting mechanism 602/604 for first
sealing the sample, then cutting the sealed sample 702. The device
also comprises coolant holder 114 oriented to allow sealed sample
702 to be placed in a coolant in the coolant holder. Suitably, a
control module is operably connected to cryo-protectant dispenser
104, sample holder 208 and sample sealing device 110/108/112. In
embodiments, the various components of the device shown in FIG. 1,
FIGS. 9A, 9B and 9C, are housed within chamber 116, the chamber
comprising at least one opening for introduction of the sample to
the sample holder 208.
[0083] As described herein, suitably cryo-protectant dispenser 104
is operably connected to a driving mechanism that controls the
movement of cryo-protectant dispenser. Suitably, sample holder 208
is a rotating sample holder that moves about rotation point 210, so
that the sample rotates or moves from a position that allows
introduction of the sample, to a position that allows introduction
of one or more cryo-protectants from cryo-protectant dispenser 104.
Suitably, the rotating sample holder further moves the sample about
rotation point 210 from the position that allows introduction of
cryo-protectant to a position that allows sealing of the sample by
sample sealing device 110/108/112. As described herein, suitably
the sealing mechanism 504 comprises an adhesive film, and a cutting
mechanism comprising first die 602 and second die 604 that align
together so as to seal the sample and then cut the sealed
sample.
[0084] Suitably, sealed sample 702 is released from the sample
holder into coolant holder 114, suitably a coolant holder
containing liquid nitrogen, for example, in one or more cryo-vials
802.
[0085] In further embodiments, containers for a biological sample
are provided. The terms "containers for a biological sample,"
"sample holder" and "sample container" are used interchangeably
throughout to refer to a device suitable for receiving a sample and
allowing that sample to be manipulated for vitrification, e.g.,
allows for the introduction and removal of cryo-protectants, allows
for sample sealing and vitrification, and allows for ultimate
storage.
[0086] As shown in FIG. 10, an exemplary container for a biological
sample 208 suitably comprises support member 209 having a top
surface and a bottom surface. Container for a biological sample 208
also suitably comprises sample retention area 1002. Sample
retention area 1002 suitably traverses support member 209 from the
top surface to the bottom surface, thereby forming a bole passing
through support member 209.
[0087] Sample retention area suitably comprises a porous mesh (not
shown) positioned in the sample retention area. As used herein,
"porous mesh" refers to any suitable material, i.e., cloth,
polymer, plastic, metal, organic and synthetic sponges, etc. The
porous mesh provides a surface for retention of the biological
sample on the mesh, was also allowing for removal of a fluid, e.g.,
a cryo-protectant, from the mesh, by allowing the cryo-protectant
to pass through the mesh. Suitably, sample retention area 1002
allows for introduction of a biological sample onto the porous mesh
and removal of a fluid (e.g., cryo-protectant) from the porous
mesh. Suitably, sample retention area 1002 is a substantially
circular hole traversing support member 209.
[0088] As shown in FIG. 11A, in exemplary embodiments, sample
retention area 1002' can comprise an extension surrounding the
hole, extending at least above the top surface of support member
209. This extension allows for the sample retention area to
accommodate larger sample/cryo-protectant volumes as the increased
height of the extension effectively provides a deeper retention
area.
[0089] FIG. 11B shows a further container for a biological sample
208 as described herein.
[0090] As shown in FIGS. 10 and 11A-11B, container for a biological
sample 208 suitably optionally comprises frame 1004 supporting
container(s) 209. Use of frame 1004 allows for the preparation of a
number of individual containers, each separate, but connected via a
single frame surrounding the containers. In this way, multiple
containers can be filled with samples, all of which are held
together via frame 1004, for placement in the automated
vitrification devices described throughout.
[0091] Exemplary materials for preparing the sample
holders/containers described herein are well known in the art, and
include for example various metals, plastics, polymers, ceramics,
glasses, etc. Suitably, the containers are made from a two-part
thermosetting plastic that starts as two liquids and hardens into a
hard plastic. This method allows for casting a metal, plastic or
cloth mesh in the sample retention area 1002. Additional methods
for preparing the containers, include for example, injection
molding.
[0092] In additional embodiments, sample handling apparatuses are
provided. As shown in FIGS. 10 and 11A-11B, suitably such
apparatuses comprise plurality of containers for a biological
sample 208, each container comprising a support member 209 having a
top surface and a bottom surface, a substantially circular hole
traversing the support member from the top surface to the bottom
surface (i.e., sample retention area 1002). Suitably, a porous mesh
is positioned in the substantially circular hole. In exemplary
embodiments, the substantially circular hole allows for
introduction of a biological sample onto the porous mesh and
removal of a fluid from the porous mesh. The plurality of
containers 208 are suitably supported by frame 1004.
[0093] In exemplary embodiments the apparatus further comprises an
extension surrounding the hole, extending at least above the top
surface of support member 209.
[0094] In still further embodiments, methods of vitrifying a
biological sample are provided. Suitably, such methods comprise
placing a biological sample in sample holder 208 and placing a
cryo-protectant in a cryo-protectant holder 102, suitably into
reservoirs 206 of cryo-protectant holder 102. In exemplary
embodiments, the placing of the biological sample in sample holder
208 comprises placing samples in each of the individual sample
retention areas 1002 (e.g., holes) in the support members of a
sample holder. Suitably, placing of the biological sample in the
sample holder is carried out by a human operator, though it can be
an automated process as well. In addition, placing the
cryo-protectant (including multiple cryo-protectants) in the
cryo-protectant holder is also suitably carried out by a human
operator, though in further embodiments, this can also be an
automated process.
[0095] The methods suitably further comprise dispensing the
cryo-protectant from the cryo-protectant holder onto the sample.
Suitably, this dispensing is carried out by cryo-protectant
dispenser 104, for example, via a nozzle 304. The methods further
comprise removing excess cryo-protectant from the sample via drying
mechanism 106.
[0096] The methods suitably further comprise sealing and cutting
the sealed sample. As described herein, suitably such sealing is
carried out with the use of a sample sealing device 110/108/112
comprising sealing mechanism 504 and a cutting mechanism 602/604
for first sealing the sample, then cutting the sealed sample 702.
Suitably, the sealing is carried out with adhesive film. In
exemplary embodiments, the cutting comprises cutting the sealed
sample with first die 602 and second die 604, that align together
to cut the sealed sample.
[0097] Suitably, sealed sample 702 is then transferred into a
coolant in a coolant holder 114, e.g., in a cryo-vial comprising
liquid nitrogen, utilizing the various device components as
described throughout.
[0098] As described herein, suitably the dispensing, removing,
sealing, cutting and transferring that are carried out in the
various methods described herein are automated via a control
module, e.g., via a computer control or similar device.
[0099] Suitable computer control modules and programs for
controlling and manipulating the various components of the devices
as described herein are well known in the art and are readily
determined and implemented by one of ordinary skill in the art.
[0100] In still further embodiments, the devices described herein
can also be utilized for warming or thawing frozen biological
samples (i.e., bringing biological samples to room temperature, or
close to room temperature, so that they can be further utilized as
desired), including for examples biological samples that have been
vitrified utilizing the devices and methods described throughout.
Thus, in embodiments the same device can be used to vitrify a
biological sample and also warm or thaw the biological sample.
[0101] In embodiments, a sealed biological sample in a container
for a biological sample as described herein is unsealed to expose
the biological sample. Unsealing can be performed by an operator or
can be an automated function of the devices described herein.
[0102] Following unsealing of the biological sample, the container
with the biological sample is placed inside of the device, suitably
on a sample holder as described herein. One or more warming
solutions are then added to the biological sample. In embodiments,
the warming solutions are held in the reservoirs of the
cryo-protectant holder prior to being removed by cryo-protectant
dispenser and then administered to the biological samples. The
order, time and duration of administering the warning solutions to
the biological samples is suitably controlled via a control module
as described herein.
[0103] Suitably, the warming solutions are maintained at room
temperature (e.g., about 20-30.degree. C.) prior to administration
to the biological samples, though if desired they can be warmed to
higher temperatures. The warming solutions are utilized to slowly
and safely raise the temperature of the biological samples to about
room temperature prior to additional processing or use as
desired.
[0104] Warming solutions suitably comprise sucrose, trehalose and
SSS in mHTF. Suitable warming solutions comprise about 0.1 to about
1 M sucrose, about 0.05 to about 1 M trehalose and about 10% to
about 30% SSS in mHTF. More suitably, warming solutions comprise
about 0.2 to about 0.5 M sucrose, about 0.1 to about 0.5 M
trehalose and about 15% to about 25% SSS in mHTF.
[0105] Exemplary warming solutions comprise 1) about 0.5M sucrose,
about 0.5M trehalose and 20% SSS in mHTF; 2) about 0.3M sucrose,
about 0.3M trehalose and about 20% SSS in mHTF; or 3) about 0.2M
sucrose, about 0.1M trehalose and about 20% SSS in mHTF. In
embodiments, such warming solutions are suitably used for warming
of oocytes.
[0106] Additional exemplary warming solutions comprise 1) about
0.5M sucrose, about 0.5M trehalose and about 20% SSS in mHTF; 2)
about 0.3M sucrose, about 0.3M trehalose and about 20% SSS in mHTF;
or 3) about 0.2M sucrose, about 0.1M trehalose and about 20% SSS in
mHTF. In embodiments, such warming solutions are suitably used for
warming of blastocysts.
[0107] For example, in embodiments, cryo-protectant dispenser
administers warming solutions to a biological sample in the amounts
and for the durations indicated below. Drying mechanism 106 is
suitably used to remove excess warming solution at the desired
times/intervals prior to application of the next warming
solution.
[0108] Described below is an exemplary warming solution protocol
for warming/thawing of oocytes: [0109] A solution comprising about
0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is applied
to the biological sample for about 1 minute at room temperature;
[0110] Excess solution is removed; [0111] A solution comprising
about 0.3M sucrose+about 0.3M trehalose+about 20% SSS in mHTF is
applied to the biological sample for about 3 minutes at room
temperature; [0112] Excess solution is removed [0113] A solution
comprising about 0.2M sucrose+about 0.1M trehalose+about 20% SSS in
mHTF is applied to the biological sample for about 3 minutes at
room temperature; [0114] Excess solution is removed; [0115] A wash
solution comprising about 20% SSS in mHTF is applied to the
biological sample for about 3 minutes at room temperature; [0116]
Excess solution is removed; [0117] The wash solution is applied
again for about 3 minutes.
[0118] Described below is an exemplary warming solution protocol
for warming/thawing of blastocysts: [0119] A solution comprising
about 0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is
applied to the biological sample for about 2-3 minutes at room
temperature; [0120] Excess solution is removed; [0121] A solution
comprising about 0.3M sucrose+about 0.3M trehalose+about 20% SSS in
mHTF is applied to the biological sample for about 3 minutes at
room temperature; [0122] Excess solution is removed; [0123] A
solution comprising about 0.2M sucrose+about 0.1M trehalose+about
20% SSS in mHTF is applied to the biological sample for about 3
minutes at room temperature; [0124] Excess solution is removed;
[0125] A wash solution comprising about 20% SSS in mHTF is applied
to the biological sample for about 3 minutes at room temperature;
[0126] Excess solution is removed; [0127] The wash solution is
applied again for about 3 minutes.
[0128] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein can be made without
departing from the scope of any of the embodiments.
[0129] It is to be understood that while certain embodiments have
been illustrated and described herein, the claims are not to be
limited to the specific forms or arrangement of parts described and
shown. In the specification, there have been disclosed illustrative
embodiments and, although specific terms are employed, they are
used in a generic and descriptive sense only and not for purposes
of limitation. Modifications and variations of the embodiments are
possible in light of the above teachings. It is therefore to be
understood that the embodiments may be practiced otherwise than as
specifically described.
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