U.S. patent application number 11/389552 was filed with the patent office on 2006-10-19 for method for vitrification of a biological specimen.
This patent application is currently assigned to Vitrolife AB. Invention is credited to Katrina T. Forest, Michelle T. Lane.
Application Number | 20060234204 11/389552 |
Document ID | / |
Family ID | 22299527 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234204 |
Kind Code |
A1 |
Forest; Katrina T. ; et
al. |
October 19, 2006 |
Method for vitrification of a biological specimen
Abstract
The present invention relates to a method of vitrification of a
biological specimen. According to the method of the present
invention, a biological specimen is directly exposed to a freezing
material. Upon exposure to the freezing material, the biological
specimen undergoes vitrification. The biological specimen which has
undergone vitrification may be stored for a period of time, and
then thawed at a later date. The thawed biological specimen remains
viable. Preferred biological specimens according to the present
invention are developmental cells.
Inventors: |
Forest; Katrina T.;
(Madison, WI) ; Lane; Michelle T.; (Littleton,
CO) |
Correspondence
Address: |
FOLEY & LARDNER LLP
150 EAST GILMAN STREET
P.O. BOX 1497
MADISON
WI
53701-1497
US
|
Assignee: |
Vitrolife AB
|
Family ID: |
22299527 |
Appl. No.: |
11/389552 |
Filed: |
March 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10241061 |
Sep 10, 2002 |
7087370 |
|
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11389552 |
Mar 24, 2006 |
|
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09416992 |
Oct 13, 1999 |
6500608 |
|
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10241061 |
Sep 10, 2002 |
|
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60104266 |
Oct 14, 1998 |
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Current U.S.
Class: |
435/1.3 ;
435/2 |
Current CPC
Class: |
A01N 1/0263 20130101;
A01N 1/0268 20130101; A01N 1/02 20130101; A01N 1/0221 20130101;
Y10S 435/975 20130101 |
Class at
Publication: |
435/001.3 ;
435/002 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Statement as to Rights to Inventions Made Under
Federally-Sponsored Research and Development
[0003] Part of the work performed during development of this
invention utilized U.S. Government Funds, specifically the National
Institute of Child Health and Human Development, Grant No. HD22023.
Therefore, the U.S. Government has certain rights in this
invention.
Claims
1. A system for the vitrification of a biological specimen,
comprising: (a) a loop; (b) a base medium comprising a
cryoprotectant held in the loop; and (d) a thaw solution in contact
with the loop.
2. The system of claim 1 wherein the loop holds a volume of 1-5
.mu.l.
3. The system of claim 1 wherein the loop has a width of 20
.mu.m.
4. The system of claim 1 wherein the loop has a diameter of 0.5 mm
to 0.7 mm.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/241,061, filed on Sep. 10, 2002, which is a
divisional of U.S. patent application Ser. No. 09/416,992, filed
Oct. 13, 1999, which claims priority from U.S. Provisional Patent
Application No. 60/104,266, filed
TECHNICAL FIELD
[0004] This invention relates to a method for vitrification of a
biological specimen, such that the biological specimen remains
viable after it is thawed.
BACKGROUND OF THE INVENTION
[0005] The ability to cryopreserve oocytes, embryos, sperm and
other similar biological specimens is critical to the widespread
application of assisted reproductive technologies. However, due to
the large volume of the cells and the high chilling sensitivity of
oocytes and early embryos, cryopreservation techniques are not well
developed in most species.
[0006] Traditionally, embryos are cryopreserved using "slow
freezing techniques". Low concentrations of cryoprotectants and
slow controlled rates of cooling usually in the range of
0.1-0.3.degree. C./min. slowly dehydrate the cell during freezing
to prevent intracellular crystallization. Because of this,
cryopreservation of oocytes, embryos and other developmental cells
using such procedures results in a reduced ability to both
establish and maintain pregnancy following transfer. Oocytes are
particularly susceptible to cryopreservation damage because of
disruption of the metaphase spindle microtubule integrity during
cooling.
[0007] Alternative prior cryopreservation methods have relied on
vitrification with high concentrations of cryoprotectants, which
when rapidly cooled result in a glass-like state. However, a
disadvantage of this vitrification technique is that the
cryoprotectants are very toxic to oocytes, embryos and other
delicate developmental cells. Cryoprotectant toxicity can be
minimized by increasing the cooling rate, which has been
accomplished by plunging oocytes held on electron microscopy grids,
or within thinly walled straws (known as open pulled straw)
directly into liquid nitrogen. However, both of these procedures
are cumbersome and recovery of embryos is problematic.
[0008] Therefore a need remains for a method for the vitrification
of a biological specimen which is able to maximize the cooling rate
of the cells of the specimen; maintain viability of the specimen
during vitrification and subsequent thawing; prevent mechanical
stress to the specimen; and provide ease of manipulations during
cryopreservation and recovery.
SUMMARY OF THE INVENTION
[0009] The present invention relates to a method of vitrification
of a biological specimen. According to the method of the present
invention, a biological specimen is directly exposed to a freezing
material. Upon exposure to the freezing material, the biological
specimen undergoes vitrification. The biological specimen which has
undergone vitrification may be stored for a period of time, and
then thawed at a later date. The thawed biological specimen remains
viable. Preferred biological specimens according to the present
invention are developmental cells.
[0010] The present invention is also drawn to a method of
vitrification of a biological specimen, which includes using a
transfer instrument to place the biological specimen into a
freezing material, such as liquid nitrogen, such that the
biological specimen is directly exposed to the freezing material.
The biological specimen then undergoes vitrification while held by
the transfer instrument, with a loop being a preferred transfer
instrument. The transfer instrument and biological specimen are
then preferably kept within the freezing material, and transferred
into a container which holds a freezing material. The container is
preferably a vial. The vial is then sealed containing the freezing
material, loop and the vitrified biological specimen, and may be
cryopreserved until such time as the biological specimen is
required for further use.
[0011] Another aspect of the present invention is the treatment of
the biological specimen in a cryoprotectant prior to
vitrification.
[0012] The invention also relates to a method for thawing a
biological specimen which has undergone vitrification. The thawing
methodology comprises the removal of the biological specimen from
the freezing material wherein it has been cryopreserved, and
placing the biological specimen in a warmed thaw solution. The thaw
solution may be present in any suitable container, and is
preferably located within a culture dish or a straw.
[0013] A further aspect of the present invention is a method of
vitrification of developmental cells, wherein one or more
developmental cells are placed directly into a freezing material,
such that each developmental cell is directly exposed to the
freezing material thereby undergoing vitrification, wherein the
vitrified developmental cells, when thawed, cultured and implanted
into suitable host organisms, will result in a fertility rate equal
to that of the same developmental cells which had not been
vitrified. Preferably, the developmental cells are contained within
a loop when exposed to the freezing material.
[0014] The present invention also relates to a method of
vitrification of a mammalian blastocyst or mammalian cleavage stage
embryo which comprises placing one or more blastocysts or cleave
stage embryos directly into a freezing material, such that each
blastocyst or cleavage stage embryo is directly exposed to the
freezing material thereby undergoing vitrification, wherein at
least 80 percent, and more preferably, 90 percent, of the vitrified
blastocysts or cleavage stage embryos will be viable after being
thawed and cultured, preferably in the appropriate base medium.
Preferably, the blastocyst or cleavage stage embryo is contained
within a loop when exposed to the freezing material.
[0015] The present invention also relates to a method of
vitrification of a horse embryo or pig embryo which comprises
placing one or more embryos directly into a freezing material, such
that each embryo is directly exposed to the freezing material
thereby undergoing vitrification, wherein at least 25 percent, and
more preferably, 50 percent, of the vitrified embryos will be
viable after being thawed and cultured, preferably in the
appropriate base medium. Preferably, the embryo is contained within
a loop when exposed to the freezing material.
[0016] The present invention also relates to a kit for the
vitrification of a biological specimen. The kit will generally
contain instructions describing the vitrification of a biological
specimen wherein the specimen is directly exposed to a freezing
material. The kit will also include one or more optional
ingredients, including, but not limited to, a transfer instrument,
most preferably a loop, a vial which is of the proper size and
shape to hold the loop and the vitrified specimen it contains, a
base medium, a transfer solution, and a cryoprotectant.
[0017] The present invention is also drawn to biological specimens
which have undergone vitrification by the methods of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic illustrating a method of vitrification
of a biological specimen according to the present invention.
DETAILED DESCRIPTION
[0019] In the present application, the following terms are used
throughout and are defined for the purposes of this application as
follows:
[0020] Base Medium: A solid or liquid preparation made specifically
for the growth, manipulation, transport or storage of the
biological specimen present therein.
[0021] Cryopreservation: The preservation of a biological specimen
at extremely low temperature.
[0022] Developmental Cells: A reproductive body of an organism that
has the capacity to develop into a new individual organism capable
of independent existence. Developmental cells include, but are not
limited to, sperm, oocytes, embryos, morulae, blastocysts, and
other early embryonic cells.
[0023] Directly Exposed: A biological specimen, including
blastocysts and embryos, is "directly exposed" to a freezing
material if the majority of the surface of the biological specimen,
or the medium, solution or material in which the biological
specimen resides, is allowed to come into direct contact with the
freezing material.
[0024] Freezing Material: Any material, including but not limited
to, liquid gases such as liquid nitrogen, liquid propane, liquid
helium or ethane slush, which are capable of causing vitrification
of a biological material.
[0025] Loop: An instrument for the manipulation of small biological
samples, generally consisting of a rod shaped handle which holds a
piece of nylon or metal wire such as platinum or nickel-steel,
etc., formed into a closed loop at the free end.
[0026] Transfer Instrument: An instrument used to manipulate a
biological specimen into a freezing material which is structured in
such a fashion that it encircles and/or holds the biological
specimen, and/or the medium, solution or material containing the
biological specimen, in place during the vitrification process
and/or allows ease of manipulation of the biological specimen
within the freezing material, and wherein the transfer instrument
allows the biological specimen to be directly exposed to the
freezing material. The transfer instrument may be any such
instrument generally known in the art, including, but not limited
to a loop, net with handle, or paddle with handle instrument. The
term "transfer instrument" as defined herein does not include
either electron microscopy grids or straws (including both sealed
straws and open pulled straws).
[0027] Viable: A biological specimen which is able to live and
develop normally for a period of time.
[0028] Vitrification (Vitrify): A phenomenon wherein a biological
specimen is rapidly cooled to very low temperatures such that the
water in the specimen forms a glass-like state without undergoing
crystallization.
[0029] The present invention is directed to a method for the
vitrification of biological specimens, based on U.S. Provisional
Patent Application No. 60/104,266, the entire contents of which is
hereby incorporated by reference.
[0030] According to the method of the present invention, a
biological specimen is placed directly into a freezing material
such that the biological specimen is directly exposed to the
freezing material. Upon exposure to the freezing material, the
biological specimen undergoes vitrification. The biological
specimen which has undergone vitrification may be stored for a
period of time, and then thawed at a later date. The thawed
biological specimen remains viable.
[0031] The present invention therefore has a number of uses. It may
be used for animal husbandry, laboratory research, endangered
species preservation, as well as for human assisted
reproduction.
[0032] The biological specimen of the present invention can be any
sort of viable biological specimen which is a living cell, but is
preferably developmental cells, and more preferably mammalian
developmental cells. Such cells can include, but are not limited
to, sperm, embryos, blastocysts, morulae, and oocytes. Such
preferred cells can be from any desired mammalian source, including
but not limited to: humans; non-human primates such as monkeys;
laboratory mammals such as rats, mice and hamsters; agricultural
livestock such as pigs, sheep, cows, goats and horses; and
zoologically important and/or endangered animals, etc. The use of
other developmental cells from other living creatures are also
within the scope of this invention, such as reptiles, amphibians,
and insects such as Drosophila. Other suitable cells for use with
the present invention include both stem cells, including human stem
cells, and plant tissue cells. The following Examples describe the
use of the present invention with a number of different cell types,
including Hamster embryos, which are extremely sensitive to injury
and therefore make a good model for any cryopreservation technique.
The Examples also show the efficacy of the present invention with
bovine oocytes and embryos which are known in the art to be
extremely sensitive to chilling injury.
[0033] Preferably, the biological specimen is placed on a transfer
instrument prior to vitrification. The transfer instrument can be
any instrument that allows the biological specimen to be
transported into a freezing material, while allowing the biological
specimen to be directly exposed to the freezing material, allowing
the biological specimen to be cooled very quickly, thus allowing
the biological specimen to vitrify rather than form ice crystals
within the cell, which would in turn ultimately disrupt cell walls
and other vital cellular constituents.
[0034] The method of the present invention is in contrast to
previous prior art methods wherein the biological specimen was
enclosed within a container such as a sealed straw or open pulled
straw, rather than being directly exposed to the freezing
material.
[0035] Additionally, the present methodology differs from previous
prior art methods which placed the biological specimen on open
plates such as microscopy grids, which were unable to allow for
facile manipulation of the specimen when contained within the
freezing material, making handling of the specimen difficult and
ultimately resulting in a poor recovery of the vitrified specimen.
The present invention therefore allows better handling of the
biological specimen during the vitrification process, and thereby
solves the problem of specimen recovery known in prior microscopy
grid vitrification methods.
[0036] The transfer instrument according to the present invention
encircles and/or holds the biological specimen in place during the
vitrification process, so that the biological material is not lost
during the process. Therefore, the transfer instrument does not
just allow the biological specimen to rest upon it, as with flat
sheets or microscopy grids, but may actually help keep the specimen
in place, as in the case with a loop via strong adhesion forces
which surround the biological specimen, or medium, solution or
material containing the specimen. Preferred transfer instruments of
the present invention include, but are not limited to, loops, small
nets with an attached handle and small spatulas. The spatulas, nets
or loops may be modified in any way known in the art to help retain
the biological specimen in place, including the placement of extra
polymeric mesh or wire grids within the loop, net or spatula. In a
preferred embodiment, the loop has an open loop and is attached via
the rod-shaped end directly to the inside of a cap of a vial, the
vial having the appropriate size and shape to allow the vitrified
biological specimen and loop to be cryopreserved therein. It has
been surprisingly and unexpectedly discovered that the use of a
loop in the present vitrification methodology allows fast cooling
rates, ease of visualization, facile manipulations and a high
success rate of viability when the vitrified specimen is thawed and
cultured.
[0037] In a preferred embodiment, the biological specimen is
treated with a small amount of a cryoprotectant prior to
vitrification. The methodology of the present invention also allows
for a decrease in the time of exposure of the biological specimen
to the solution phase of the cryoprotectant used, thus lowering the
toxicity of the cryoprotectant to the biological specimen.
Cryoprotectants, such as ethylene glycol, polyethylene glycol,
dimethylsulfoxide, glycerol, propane diol, sugars, and methyl
pentane diol, as well as others well known in the art, can be toxic
to sensitive cells such as oocytes and embryos when used in large
dosages during cryopreservation. The present invention allows for
the use of any optional cryoprotectant to be present in solution
phase in the presence of the biological specimen for shorter time
periods than cryopreservation methods previously described in the
art.
[0038] By allowing for quick cooling times, reduced time of
exposure of solution phase cryoprotectants, and reliable retention
and manipulation of the biological specimen, the present invention
solves a long standing problem in the art of successful
cryopreservation of sensitive biological specimens such as
developmental cells. As further described in the Examples, the
present invention has shown a success rate of vitrifying
blastocysts or cleavage stage embryos such that, when thawed and
cultured in the appropriate base medium, the cryopreserved
blastocysts or cleavage stage embryos have a viability rate of 80
percent, preferably 90 percent, and preferably greater than 90
percent. Moreover, the present invention allows the vitrification
of developmental cells, wherein the vitrified developmental cells,
when thawed, cultured and implanted into suitable host organisms,
will result in a fertility rate equal to that of developmental
cells which are similarly implanted and which have not been
cryopreserved. This helps solve a long-term problem in low
pregnancy rates resulting from the use of cryopreserved
developmental cells.
[0039] Additionally, the present methodology allows for the
cryopreservation of biological specimens which in the past had
resisted efforts of cryopreservation to result in a useful
percentage of viable preserved specimens. Notably, pig embryos and
horse embryos can now be vitrified according to the present
invention, and wherein at least 25, 35, 40, or 45 percent, and more
preferably, 50, 55, 60, 65, 70 or 75 percent, of the vitrified
embryos will be viable after being thawed and cultured.
[0040] According to the present invention, one or more biological
specimens are collected by any means well known in the art, and are
preferably transferred to a base medium. The base medium may
contain one or more optional ingredients, such as a cryoprotectant
to protect the biological specimen from cold and/or a viscosity
increasing compound, to assist in maintaining the material within a
transfer instrument, preferably a loop. The viscosity increasing
compound can be any such compound known in the art, including, but
not limited to, Ficoll, Percoll, hyaluronic acid, albumin,
polyvinyl pyrrolidone (PVP), and glycerol.
[0041] According to the present invention, the biological specimen
is preferably placed on a transfer instrument. The specimen may be
placed in a base medium, and the transfer instrument, such as a
loop or paddle, used to scoop the biological specimen from the base
medium. In a preferred embodiment, the transfer instrument is a
loop, and the loop is preferably dipped in base medium to form a
thin film of the base material on the loop, and the biological
material is deposited via pipette directly into the loop. If
development cells such as embryos, sperm or oocytes are utilized as
the biological specimen, one or more may be placed within each
loop.
[0042] The transfer instrument containing the biological specimen
is then quickly placed in a freezing material, such that the
biological specimen is directly exposed to the freezing material,
allowing vitrification of the biological specimen. Preferably, the
time between pipetting the biological specimen onto the transfer
instrument and the placement of the biological specimen into the
freezing material is 45 sec. or less, more preferably 30 sec. or
less. The freezing material may be liquid nitrogen, ethane slush,
or any other freezing material well known in the art. Preferably,
the biological specimen is held within the freezing material during
all manipulations subsequent to vitrification, until the specimen
is to be thawed.
[0043] The vitrified biological specimen is then transferred into a
storage container. In a preferred embodiment, the transfer
instrument is a loop which is attached to the inside of a vial cap.
The vial is filled with the freezing material, and resides in the
same reservoir as the freezing material used for vitrification of
the biological specimen. After vitrification, the biological
specimen, still contained within the loop, may be sealed in the
vial without having been removed from the freezing material. The
sealed vial, which contains the vitrified biological specimen and
the loop within the freezing material, can then be cryopreserved
indefinitely.
[0044] Thereafter, the biological specimen may be thawed, and the
viable biological specimen may be further developed. Thawing is
accomplished by removing the vial containing the vitrified
biological specimen from any storage tank in which it resides, and
quickly removing the transfer instrument containing the biological
specimen from the vial, and plunging the transfer instrument and
specimen into a thaw solution. In a preferred embodiment, the
storage vial is placed in a reservoir containing a freezing
material, preferably the same freezing material as contained within
the vial. While within the freezing material, the vial is opened
and the transfer instrument, containing the biological specimen, is
removed and quickly plunged into the thaw solution. The thaw
solution may be any solution or material that is sufficient to
allow the biological specimen to thaw while preserving its
viability, including but not limited to, media known in the art
that is appropriate as a base medium for the particular biological
specimen. After thawing, the biological specimen can be further
manipulated in any appropriate manner known for the species and
process for which the specimen is being utilized.
[0045] A preferred method of the present invention is further
illustrated in accordance with FIG. 1. As shown in I, a biological
sample in an appropriate base medium is applied directly to a loop
or scooped directly into the loop. As shown in I, the loop is
attached to a magnetic vial cap. Immediately thereafter, the loop
is plunged directly into the freezing material contained within a
reservoir, which as illustrated may be a insulated box filled with
liquid nitrogen. Alternatively, the freezing material may be placed
directly into the vial, and the biological specimen may be
vitrified by being directly exposed to the freezing material within
the vial itself, thereby eliminating the need for a separate
reservoir. While under the liquid nitrogen, the loop is secured
into the storage vial, with the vitrified biological specimen
remaining within the loop. Multiple vials can be filled by keeping
them upright in vial-sized holes within the reservoir, or,
alternatively, single vials could be held under the nitrogen with a
forceps or other tool. Multiple vials can then be cryopreserved
indefinitely in any suitable container, such as a standard dewar,
as illustrated in III. At any time thereafter, the loop may be
removed from the vial while under a freezing material such as
liquid nitrogen, as shown in IV, in the exact reversal of the
vitrification procedure as described above. It is convenient, but
not necessary, to use a reservoir of freezing material to surround
the vial, before thawing, but the freezing material contained
within the vial itself should be sufficient to keep the biological
specimen cryopreserved during manipulation prior to being thawed.
The biological specimen is then plunged directly into a thaw
solution. The thaw solution may be contained in any manner which is
convenient, including an open culture dish as shown in V, or in a
straw for direct loading into a transfer gun. The biological
specimen is instantly diluted into the thaw solution, and floats
away from the loop. The biological specimen can then be cultured in
any appropriate fashion known in the art.
[0046] Vitrification of sensitive biological specimens such as
sperm, oocytes and embryos using the method of the present
invention has advantages over conventional cryopreservation
procedures in that the present method lacks any insulating layer
between the biological specimen and the freezing material. This
factor, coupled with the very small volume of less than 1-5 .mu.l
for the typical biological specimen used, or media, solution or
other material containing the biological specimen used, results in
both very rapid and uniform heat exchange during cooling. High
rates of cooling prevent chilling injury to sensitive cells such as
developmental cells. The extremely rapid cooling rate obtained with
the present invention also substantially reduces the exposure time
to any optional cryoprotectants used and thereby reduces their
cytotoxicity to the specimen.
[0047] Other major benefits of the methods of the present invention
include: an open system enabling ready visualization of the sample
during manipulation; rapid freezing of a large number of samples
with no need for expensive or complicated equipment; very
straightforward labeling and storage; and trivial and instantaneous
sample warming and recovery. For applications that require a closed
system such as human clinical applications, the use of standard
cryovials enable them to be closed in standard plastic sheets, or
alternatively, the release hole within the cryovial cap may be
sealed shut, preventing any possible viral cross-transmission.
[0048] The ultimate test of viability of embryos following
cryopreservation is the ability to establish and maintain a
pregnancy resulting in normal fertile young. The hamster is a good
model for this for two reasons. Firstly their sensitivity to the in
vitro environment makes them a very sensitive model as evidenced by
the fact that the inventors of the present invention are unaware of
any report in the literature that has successfully produced hamster
pups following cryopreservation using any method. Secondly, the
hamster has a gestation period of only 16 days and sexual maturity
is reached after 3-4 months. The following Examples demonstrate the
success of the present methodology for cryopreservation of viable
embryos which can then be thawed and used to produce normal young,
resulting in a success rate of at least 90%.
[0049] Bovine embryos and in particular bovine oocytes are reported
to be very sensitive to chilling injury. Furthermore, the high
lipid content in the embryo has been linked to the increased
sensitivity of bovine embryos to cryopreservation procedures. As
shown in the following Examples, vitrification using the method of
the present invention with oocytes and cleavage stage embryos of
bovines allowed subsequent development to the morula/blastocyst
stage in culture, with high percentage rates of successful
hatching.
[0050] The present invention also relates to a kit for the
vitrification of a biological specimen. The kit will generally
contain instructions describing the vitrification of a biological
specimen wherein the specimen is directly exposed to a freezing
material. The kit will also include one or more optional
ingredients, including, but not limited to, a transfer instrument,
most preferably a loop, a vial which is of the proper size and
shape to hold the loop and the vitrified specimen it contains, a
base medium, a transfer solution, and a cryoprotectant.
[0051] This invention is illustrated further by the following
nonlimiting Examples. All of the references listed in the
application are hereby incorporated by reference.
EXAMPLE 1
Methodologies and Materials for Vitrification of Bovine and Hamster
Oocytes and Embryos
A. Media
[0052] The medium used in the following Examples was Hamster Embryo
Culture Medium-10 (HECM-10), prepared as described by Lane et al.,
Mol. Reprod. Dev. 50:443-450 (1998). For embryo collection and
cryopreservation, a Hepes-buffered modification of HECM-10 where 20
mM NaHCO.sub.3 was replaced with 20 mM Hepes (pH 7.35) was used.
Cryoprotectant solutions were added to the medium immediately prior
to use. Media for bovine embryo culture were G1.2 and G2.2, as
taught by Gardner et al., Hum. Reprod. 13:3434-3440 (1998). All
salts, carbohydrates, amino acids, dimethylsulfoxide (DMSO),
ethylene glycol and sucrose were purchased from Sigma Chemical
Company (St. Louis, Mo.). Bovine Serum Albumin was purchased from
Bayer Diagnostics.
B. Hamster Embryo Collection and Culture
[0053] Hamster embryos were collected from super-ovulated females
as previously described by Lane et al., Mol. Reprod. Dev.
50:443-450 (1998). Hamster embryos were cryopreserved at either the
pronuclear 1-cell or 2-cell stages. Hamster embryos were cultured
in HECM-10 as taught by Lane et al., Mol. Reprod. Dev. 50:443-450
(1998). Cell number of resultant blastocysts was assessed by
propidium iodide staining following triton treatment.
C. In Vitro Maturation/In Vitro Fertilization/In Vitro Culture
(IVM/IVF/IVC) of Bovine Embryos
[0054] Immature bovine oocytes were isolated from ovaries and
matured as described by Krisher et al., Biol. Reprod. 60:1345-1352
(1999). Mature oocytes were either vitrified and thawed, or not
subject to vitrification and thawing when used as controls, and
fertilized in vitro, by the methods taught by Krisher et al., Biol.
Reprod. 60:1345-1352 (1999). Following fertilization, putative
zygotes were isolated and cultured in sequential media G1.2 and
G2.2 for 72 hr. in each medium. After a total of 144 hr.
development to the morula/blastocyst and blastocyst stages was
assessed.
D. Vitrification Using Loop
[0055] Loops used for vitrification consisted of a nylon loop (20
.mu.m width; 0.5-0.7 mm diameter) mounted on a stainless steel pipe
held by epoxy to the lid of a cryovial (Hampton Research, Laguna
Niguel, CA). Oocytes and embryos were vitrified using a 2-step
loading with cryoprotectants. Initially oocytes and embryos were
placed in cryoprotectant solution I which contained 10% DMSO and
10% ethylene glycol for 1-3 min. before being transferred to
solution II, which contained 20% DMSO and 20% ethylene glycol, 10
mg/ml Ficoll (MW 400,000) and 0.65 M sucrose for approximately 20
sec. Cells are then transferred to the loop that had previously
been dipped into solution II to create a thin-film. For hamster
embryos, 10-12 embryos were placed on the loop, and for bovine
embryos 3-6 embryos were placed on each loop. The embryos suspended
in the nylon loop were then plunged directly into liquid nitrogen.
By previously submerging the cryovial under liquid nitrogen, the
loop containing the embryos was plunged into the cryovial
containing liquid nitrogen and sealed under liquid nitrogen in one
motion.
[0056] Oocytes and embryos were thawed using a 2-step dilution with
sucrose. With the cryovial submerged under liquid nitrogen, the
vial was opened and the loop containing cells was removed from the
liquid nitrogen, and was then inserted directly into a well of the
base medium containing 0.25 M sucrose. The oocytes/embryos
immediately fell from the loop into the thaw solution. Oocytes were
moved from this solution after 2 min. and transferred to base
medium containing 0.125 M sucrose for a further 5 min.
Subsequently, oocytes/embryos were washed twice in the base medium
for 5 min. and were then returned to culture.
E. Vitrification Using Open Pulled Straw Technique
[0057] For comparison purposes, hamster embryos were vitrified
using the open-pulled straw (OPS) technique described by Vajta et
al., Cryo-Letters 18:191-195 (1997). Ten-twelve embryos were
exposed to a 2-step loading of cryoprotectants consisting of
ethylene glycol and DMSO at the same concentrations as above.
Embryos were pipetted into a 1 .mu.l drop of the second
cryopreservation solution and then loaded into a pulled straw using
capillary action and the straw containing the embryos was plunged
directly into liquid nitrogen. For thawing, embryos were expelled
from the straw by pressure build-up during warming and thawed as
above.
F. Embryo Transfer
[0058] Hamster morulae/blastocysts were transferred to day 3 (-1
day asynchronous) pseudo-pregnant recipients. Eight embryos were
transferred to each uterine horn. On day 14 of pregnancy some
animals were euthanized and implantation and fetal development
rates determined. The remaining females were allowed to litter on
day 16 of pregnancy and the number of pups was recorded soon after
birth.
G. Statistical Analyses
[0059] Differences in development among treatments were assessed
using linear-logistic regression where the distribution was
binomial (Glim 4.0, Numerical Algorithms Group, Oxford, UK). Day of
experiment was fitted as a factor. Differences in cell numbers were
assessed using Analysis of Variance as both Gaussian normality and
equal variances were confirmed. Multiple comparisons between
treatments were assessed by Bonferroni's procedure for multiple
comparisons.
EXAMPLE 2
Vitrification and Subsequent Development of Hamster Embryos
[0060] Hamster 2-cell embryos were vitrified using a loop according
to the method of the present invention, and compared with results
of control embryos exposed to cryoprotectant or embryos vitrified
using the OPS method, as described in Example 1. Hamster embryos
were collected from the oviduct, and allocated to either the
control, loop or OPS vitrification. Significantly more embryos
developed to the morula/blastocyst and blastocyst stage when
vitrified within the loop compared to those vitrified using OPS, as
shown below in Table 1.
[0061] Significantly fewer 2-cell embryos were able to continue
development to the morula/blastocyst or blastocyst stages in
culture following vitrification by either technique compared to
control embryos, as shown in Table 1. However, the cell numbers of
the blastocysts (an indicator of cleavage rates) resulting from
vitrified 2-cell embryos were statistically equivalent to 2-cell
embryos that were not vitrified, as shown in Table 1. Rat 2-cell
embryos were also successfully vitrified using the loop and could
develop normally after thawing with cleavage rates of 75%, similar
to control embryos (n=10).
[0062] To further assess the ability to vitrify sensitive embryos,
the experiment was repeated with 1-cell embryos, although the
length of time that the 1-cell embryos were exposed to the initial
dilution of cryoprotectant was reduced from 2 min. to 1 min.
Preliminary studies demonstrated that a 2 min. exposure (without
vitrification) of 1 cell embryos to the cryoprotectant solutions
severely reduced development. Again embryos were collected from the
oviduct and allocated to either the control, loop or OPS
vitrification.
[0063] Hamster 1-cell embryos were able to cleave and continue
development in culture to the morula/blastocyst stage following
vitrification with the loop, as shown in Table 1. Developmental
rates after vitrification were significantly better for embryos
vitrified using the loop compared to those vitrified using OPS
(Table 1). Hamster oocytes were also able to be successfully
vitrified using the loop (n=20) and subsequently fertilized and
developed to the morula/blastocyst stage at rates of around 10%,
comparable to control non-cryopreserved oocytes. TABLE-US-00001
TABLE 1 Development of Hamster Embryos in Culture Following
Vitrification Blastocyst Stage of M/B B Cell Number Development
Treatment (%) (%) (mean .+-. sem) 1-cell Control 79.5 30.1 18.9
.+-. 3.1 Loop 39.8.sup.a 15.5.sup.a 11.9 .+-. 1.1.sup.a OPS
22.0.sup.b 5.0.sup.b 9.2 .+-. 1.2.sup.b 2-cell Control 98.5 94.2
24.1 .+-. 2.8 Loop 64.2.sup.a 43.1.sup.a 19.7 .+-. 1.8.sup.a OPS
50.5.sup.a 29.6.sup.b 19.6 .+-. 1.4.sup.a M/B morula/blastocyst
development B blastocyst development N = at least 100 embryos
cultured per treatment for 1-cell embryos (4 replicates) and at
least 400 embryos per treatment for 2-cell embryos (8 replicates)
.sup.asignificantly different from control (P < 0.05)
.sup.bsignificantly different from control and from loop
vitrification (P < 0.05).
EXAMPLE 3
Viability of Hamster 1-Cell and 2-Cell Embryos Following
Vitrification
[0064] Hamster embryos were vitrified using either the loop method
or by OPS. Following warming, embryos were cultured to the
morula/blastocyst stage (both vitrified and control embryos) before
transfer to pseudo-pregnant recipients. There was no difference in
the viability of morula/blastocyst stage embryos that had been
previously vitrified at the 2-cell stage to implant and develop to
a viable fetus compared to control embryos that were not
cryopreserved, as shown below in Table 2. However, significantly
fewer embryos were able to implant and develop to a viable fetus
when vitrified using OPS, as shown in Table 2. Two additional
females that received morulae/blastocysts which were vitrified at
the 2-cell stage using the loop were allowed to litter and 5 normal
pups were born. These pups developed into morphologically sound and
fertile adults.
[0065] Similarly, for hamster 1-cell embryos, implantation and
fetal development were not affected by vitrification using the
loop, as shown in Table 2. No embryos vitrified using OPS were
transferred due to the low survival rates in culture observed in
the previous experiment. Again two females which received
morulae/blastocysts vitrified at the 2-cell stage using the loop
were allowed to litter and a total of 9 pups were born. One pup was
eaten by the mother 6 to 9 days after birth. The remaining pups
developed into morphologically sound and fertile adults.
TABLE-US-00002 TABLE 2 Development of Hamster Embryos in utero
Following Vitrification Stage of Development Implantation Fetuses
for Vitrification n Method N(%) N(%) 1-cell 20 Control 8(40) 6(30)
17 Loop 7(41) 5(29) 2-cell 40 Control 34(85) 26(65) 72 Loop
39(54).sup.a 36(50) 112 OPS 48(43).sup.a 40(36).sup.a
.sup.asignificantly different from control embryos (P <
0.05)
EXAMPLE 4
Vitrification of Bovine Oocytes, Cleavage Stage Embryos and
Blastocysts
[0066] To determine the ability of the vitrification method of the
present invention to successfully vitrify embryos with different
cellular properties, bovine oocytes and embryos were vitrified as
taught in Example 1, and their survival and subsequent development
assessed, and the results shown in Table 3. Oocytes and embryos
were allocated to either the control group or to loop vitrification
using the methodology of the present invention. In vitro produced
bovine blastocysts were successfully vitrified using the loop with
more than 80% of expanded blastocysts being able to both re-expand
and hatch following vitrification, as shown in Table 3. Culture of
control blastocysts resulted in 100% hatching after 48 hr. of
culture. Furthermore, 75% of completely hatched blastocysts could
also be successfully vitrified using loop vitrification. Eight-cell
bovine embryos vitrified using the vitrification method of the
present invention could be vitrified and warmed with subsequent
survival rates (assessed by development to the morula/blastocyst
and blastocyst stages) equivalent to those obtained for fresh
embryos that had not been cryopreserved, as shown in Table 3.
Vitrification of embryos at the 4-cell stage resulted in slightly
reduced survival rates compared to the fresh embryos, however many
were able to complete normal development to the morula/blastocyst
stage, as shown in Table 3. Bovine oocytes are extremely sensitive
to chilling damage and few reports have demonstrated any success
following cryopreservation. In vitro matured bovine MII oocytes
were successfully vitrified using the loop. Vitrified and warmed
oocytes were subsequently fertilized and of these 33% continued
development to the morula/blastocyst stage (n=42). TABLE-US-00003
TABLE 3 Development of Bovine Embryos in Culture Following
Vitrification B at HB at Stage of 8-Cell at M/B at 144 Hr. 168 Hr.
Development Treatment 72 Hr. (%) 144 Hr. (%) (%) (%) 4-cell Control
0 46 15 n/d Loop 4 24 16 n/d 8-cell Control n/a 59 50 n/d Loop n/a
52 41 n/d Blastocyst Control n/a n/a n/a 100 Loop n/a n/a n/a 80.5
M/B, morula/blastocyst development B blastocyst development HB
hatched blastocyst development n/a not applicable n/d not
determined
EXAMPLE 5
Methodologies and Materials for Vitrification of Human and Mouse
Blastocysts
A. Culture Media
[0067] Media for embryo culture was G1.2 and G2.2 (IVF Sciences
Scandinavian, Gothenburg, Sweden). Media for embryo collection was
a HEPES-modification of G1.2 (H-G1.2) and the base medium for
cryopreservation and thawing was a HEPES-buffered modification of
G2.2 without amino acids and vitamins (H-G2.2). In both cases the
media were modified by replacing 20 mM NaHCO.sub.3 with 20 mM HEPES
and adjusted to pH 7.35.
B. Mice
[0068] Embryos were collected from 4-6 week old F1 (C57BL6xCBa)
females. Females were stimulated with 5 iu of pregnant mare's
gonadotrophin (Sigma Chemical Co., St. Louis, Mo.) and 48 hr. later
with 5 iu of human chorionic gonadotrophin (hCG; Sigma Chemical
Co.). Following the hCG injection females were placed with males of
the same strain and the following morning the presence of a vaginal
plug indicated that mating had taken place. Zygotes were collected
at 22 hr. post-hCG and denuded from surrounding cumulus by
incubation in H-G1.2 with 0.5 mg/ml hyalronidase for less than 1
min. Zygotes were washed twice in H-G1.2 and placed in culture.
C. Mouse Embryo Culture
[0069] Mouse zygotes were cultured in groups of 10 in 20 .mu.l
drops of medium G1.2 at 37.degree. C. in an humidified atmosphere
of 5% CO.sub.2 in air. After 48 hr. of culture, 8-cell embryos were
washed 3 times in medium G2.2 and cultured for a further 48 hr. in
20 .mu.l drops of medium G2.2. Blastocyst development was assessed
after 96 hr. of culture.
D. Human Embryo Culture
[0070] The culture system for blastocyst growth was done according
to Gardner et al., Hum. Reprod. 13:3434-40 (1998). Following oocyte
retrieval, cumulus enclosed oocytes were incubated in Ham's F-10
supplemented with fetal cord serum (FCS) for insemination. Semen
was prepared with a 50-70-95 discontinuous gradient or
mini-gradient method (Pure Spemm, Nidacon, Gothenburg), depending
on the initial semen parameters. The resulting pellet was washed in
Ham's F-10. For normal insemination, to each oocyte, 50-100,000
sperm/mL were added. If intracytoplasmic injection (ICSI) was
performed, oocytes were denuded using hyaluronidase and drawn
pipettes. Each mature oocyte was placed in a 6 .mu.l droplet of
phosphate buffered saline supplemented with 15% FCS. The partner's
sperm was placed in a 6 .mu.l droplet of PVP (IVF Sciences
Scandinavian). All droplets were overlaid with Ovoil (IVF Sciences
Scandinavian). ICSI was performed on a Nikon inverted microscope
with Narishige micromanipulators. Injected oocytes were then rinsed
and placed in tubes of G1.2 until fertilization was assessed.
Fertilization was assessed 15-18 hr. post insemination or ICSI.
Cumulus and corona cells were removed by dissection with 27-gauge
disposable needles in an organ culture dish. Resulting 2 pronuclear
embryos were washed well and subsequently cultured in groups of 2-3
in G1.2 medium in 1-mL Falcon culture tubes in 5% CO.sub.2 in
air.
[0071] After 48 hr. of culture, embryos were rinsed 3 times and
cultured for a further 72 hr. Blastocysts on day 6 that were not
considered of good enough quality to cryopreserve by previous
methods known in the art, i.e. not fully expanded or with poor
inner cell mass development, were donated for vitrification by the
method of the present invention.
E. Vitrification Using a Loop
[0072] Loops used for vitrification consisted of a nylon loop (20
.mu.m width; 0.5-0.7 mm diameter) mounted on a stainless steel pipe
inserted into the lid of a cryovial. The loops were purchased
mounted (Hampton Research, Laguna Niguel, CA) and then epoxied into
vials. A metal insert on the lid enables the use of a handle with a
small magnet for manipulation of the loop if desired.
[0073] Blastocysts were vitrified using a 2-step loading with
cryoprotectants. Initially blastocysts were placed in
cryoprotectant solution I which contained 10% DMSO and 10% ethylene
glycol for 2 min. before being transferred to solution II, which
contained 20% DMSO and 20% ethylene glycol, 10 mg/ml Ficoll (MW
400,000) and 0.65 M sucrose for around 20 sec. These concentrations
of cryoprotectants and length of exposure have previously been
demonstrated to be optimal for the vitrification of both rodent and
domestic animal embryos using the loop procedure. While blastocysts
are in cryoprotectant solution I, the loop is dipped into
cryoprotectant solution II to create a thin film on the loop. The
blastocysts were then transferred from solution II onto the film of
cryoprotectant on the loop. The loop containing the blastocyst was
then plunged into the cryovial which is submerged and filled with
liquid nitrogen. By previously submerging the cryovial under liquid
nitrogen, the loop containing the blastocysts could be plunged into
the cryovial containing liquid nitrogen and sealed under liquid
nitrogen in one motion. The vials were stored in standard
canes.
[0074] Blastocysts were thawed using a 2-step dilution with
sucrose. With the cryovial submerged under liquid nitrogen the vial
was opened and the loop containing blastocysts removed from the
liquid nitrogen and placed directly into a well of the base medium
containing 0.25 M sucrose. The blastocysts immediately fell from
the loop into the thaw solution. Blastocysts were moved from this
solution after 2 min. and transferred to base medium containing
0.125 M sucrose for a further 3 min. Subsequently, blastocysts were
washed twice in the base medium for 5 min. and were then returned
to culture.
[0075] Following-vitrification, mouse and human blastocysts were
cultured in medium G2.2 for 6 hr. to assess re-expansion before
assessment of blastocyst outgrowth. A 6 hr. incubation was chosen
as this is the normal time period used for the assessment of thawed
blastocysts prior to transfer.
F. Assessment of Blastocyst Outgrowth
[0076] Both mouse and human blastocysts were assessed for outgrowth
as a marker of subsequent viability. Blastocysts were transferred
to medium G2.2 supplemented with 10% fetal cord serum to assess
blastocyst attachment and outgrowth. Blastocysts were cultured in 4
well plates (Nunclon, Denmark) previously coated with 0.1% gelatin
in 500 .mu.l drops at 37.degree. C. in 5% CO.sub.2 in air for 48
hr. Blastocyst hatching and attachment were assessed after 24 hr.
and outgrowth assessed after a further 24 hr. of culture. Outgrowth
of inner cell mass (ICM) and trophectoderm was given a score
between 0 and 3 based on the amount of outgrowth, where 0 was no
growth and 3 was extensive growth as described by Spindle and
Pederson, J. Exp. Zool., 186:305-318 (1972).
G. Assessment of Blastocyst Viability in Mice
[0077] Viability of mouse blastocysts following vitrification was
assessed by transfer to pseudo-pregnant recipients. Following
warming, blastocysts were cultured for 6 hr. in medium G2.2 prior
to transfer. All blastocysts that re-expanded after the 6 hr.
period were pooled and blastocysts for transfer randomly selected.
Six blastocysts were transferred to each uterine horn. On day 15 of
pregnancy, implantation, fetal development and fetal weights were
assessed. Non-cryopreserved blastocysts served as the control.
H. Statistical Analyses
[0078] Differences in hatching, attachment and viability following
vitrification were assessed by Chi-square analysis with Yates
Correction. Data for outgrowth of both the ICM and trophectoderm
were initially subjected to a Kolmogorov-Smirnov test to determine
the normality of the data. An F-test was then used to assess that
the two groups of data had equal variances. Once the normality and
equal variances were established differences in outgrowth were
assessed by Student's t-test.
EXAMPLE 6
Vitrification of Mouse Blastocysts
[0079] A total of 160 mouse blastocysts were vitrified using a loop
according to the present invention. Following vitrification, 100%
of these blastocysts were able to re-expand in culture. There was
no difference in the ability of vitrified blastocysts to hatch and
attach in culture compared to control embryos, as shown in Table 4.
Similarly, there was no difference in the ability of either the ICM
or trophectoderm to outgrow in culture between the control and
vitrified blastocysts, as demonstrated in Table 4.
[0080] Following vitrification and thawing, 60 blastocysts were
transferred to pseudo-pregnant recipients and their viability
compared to sibling control blastocysts that were not
cryopreserved. There was no difference in the ability of vitrified
blastocysts to implant and develop to a fetus compared to control
blastocysts. Resultant fetal weights were also similar for
blastocysts that were vitrified (0.245.+-.0.021 g) compared to
control blastocysts (0.250.+-.0.017 g). All fetuses resulting from
both vitrified and control blastocysts were morphologically normal.
Additionally a recipient female that received 8 vitrified
blastocysts (4 per uterine horn) was allowed to litter. Three
morphologically normal pups were born. TABLE-US-00004 TABLE 4
Effect of Loop Vitrification of Mouse Blastocysts on Re-Expansion
and Outgrowth Study group Vitrified Treatment Control
blastocysts.sup.1 Re-expansion (%) -- 100 Hatching (%) 87.5 95.5
Attachment (%) 78.1 85.9 ICM outgrowth 2.21 .+-. 0.10 2.17 .+-.
0.09 (means .+-. SEM) trophectoderm outgrowth 2.00 .+-. 0.09 2.14
.+-. 0.09 (means .+-. SEM) .sup.1there was no difference between
control and vitrified blastocysts for any parameter measured. n
.gtoreq. 100 for both control and vitrified blastocysts
EXAMPLE 7
Human Blastocyst Vitrification
[0081] Eighteen human blastocysts between minimally to
semi-expanded were vitrified using a loop according to the
methodology of the present invention. Of these 11 (83.3%)
re-expanded in culture. Ability to hatch in culture and outgrowth
of the ICM and trophectoderm were similar for blastocysts that were
vitrified and control blastocysts that were not cryopreserved, as
shown in Table 5. TABLE-US-00005 TABLE 5 Effect of Loop
Vitrification of Human Blastocysts on Re-Expansion and Outgrowth
Study group Control Vitrified blastocysts.sup.1 Treatment n = 12 n
= 18 Re-expansion (%) -- 83.3 Hatching (%) 63.6 73.3 Attachment (%)
36.0 60.0 ICM outgrowth 2.0 .+-. 0.2 1.7 .+-. 0.2 (means .+-. SEM)
trophectoderm outgrowth 1.7 .+-. 0.2 2.0 .+-. 0.2 (means .+-. SEM)
.sup.1there was no difference between control and vitrified
blastocysts for any parameter measured.
EXAMPLE 8
Vitrification of 8 Day Bovine Blastocyst
[0082] The following solutions were used in this Example:
Freeze Solutions:
[0083] Solution 1: Base medium (as taught in Example 1) containing
10% ethylene glycol and 10% DMSO
[0084] Solution 2: Base medium containing 0.65 M sucrose and 20%
ethylene glycol and 20% DMSO and 10 mg/ml Ficoll
Thaw Solutions:
[0085] Solution 1: Base medium with 0.25 M sucrose
[0086] Solution 2: Base medium with 0.125M sucrose
[0087] Solution 3: Base medium
[0088] Solution 4: Base medium
[0089] Bovine blastocysts were produced according to the methods
described in Example 1. The blastocysts were all expanded to some
degree prior to vitrification. The blastocysts were pipetted using
standard equipment onto a loop that had been dipped into Freeze
Solution 2. Each loop contained a single blastocyst. The loops
containing the blastocysts was then treated for 2 min. in Freeze
Solution 1, followed by 30 sec. of treatment in Freeze Solution 2
which included a viscosity solution of Ficoll and then immediately
plunged directly into liquid nitrogen and vitrified. The
blastocysts were kept frozen in liquid nitrogen for 30-90 min. and
then thawed.
[0090] To thaw the blastocysts, the blastocysts and the loop in
which they were vitrified were placed for five min. each in Thaw
Solutions 1-4 sequentially.
[0091] Of the 13 blastocysts that were vitrified and thawed
according to the present invention, 9 of the blastocysts hatched
after being cultured after 48 hr.
[0092] A second set of 4 blastocysts were vitrified, thawed and
cultured as described above, and all 4 blastocysts successfully
hatched after being cultured for 48 hr.
[0093] A third set of 8-day bovine blastocysts were produced by the
methods described in Example 1. A total of 12 blastocysts were
used, and 2 expanded blastocysts were placed in each loop. The
blastocysts were subject to vitrification, thawing and culturing as
described, except the thawing process was conducted according to
the following regime. Two min. in Solution 1; 5 min. in Solution 2;
5 min. in Solution 3 and 5 min. in Solution 4. After being cultured
for 48 hr., 11 of the blastocysts had hatched.
EXAMPLE 9
Vitrification of Nine Day Bovine Blastocysts
[0094] Bovine blastocysts were produced according to methods
described in Example 1 and vitrified as described in Example 8. The
blastocysts were then thawed and cultured as described in Example
8. After 48 hr., 80% of the blastocysts had hatched.
EXAMPLE 10
Vitrification of Seven Day Bovine Blastocysts
[0095] Seven day bovine blastocysts were produced as described in
Example 1 and cultured using G1.2/G2.2 as described in Example 1. A
total of 20 blastocysts were vitrified and thawed according to the
method described in Example 8 and frozen for 2 hr. with 2 to 3
blastocysts per loop. The blastocysts were then thawed and cultured
as described in Example 8. After the thawed blastocysts were
cultured for 48 hr., 15 of the 20 blastocysts had-hatched and 2 had
re-expanded.
[0096] A second experiment was conducted with seven day blastocysts
according to the procedures described above, with 1 to 2
blastocysts per loop. After 48 hr. of culture, 29 of the 33
vitrified blastocysts had hatched.
EXAMPLE 11
Vitrification of Bovine Oocytes
[0097] In this Example, the vitrification of bovine oocytes using
methodology of the present invention was compared to oocytes which
were vitrified using the known open pulled straw (OPS) methodology
and also to control oocytes which had not been frozen. The oocytes
which were to be vitrified were treated for 35 sec. in Solution 1
and for 30 sec. in either Solution 2 alone or Solution 2 plus a
viscosity solution, using the Solutions described in Example 8. The
oocytes were then frozen either 1-3 per straw or 1 to 3 per loop by
plunging them into liquid nitrogen. After vitrification, all
oocytes were thawed according to the following regime: 1 min. in
Solution 1; 5 min. in Solution 2; 5 min. in Solution 3; and 5 min.
in Solution 4, using the Solutions of Example 8. The bovine oocytes
were then returned to maturation media for 2 hr. and then
fertilized in the normal fashion. After one day, the oocytes were
moved to medium G1.2 as described in Example 1. After 4 days, the
cleaved embryos were moved to fresh medium G1.2, as described in
Example 1. The control oocytes showed 37.5% cleavage, the oocytes
which had undergone vitrification according to the current
methodology showed 19% cleavage, and those which were vitrified
using the OPS methodology showed 14% cleavage. The oocytes were
then moved to fresh G2.2 medium and cultured for an additional 4
days. At the end of eight days, only 2 of the 24 original oocyte
denuded controls had reached the morula stage of development. In
comparison, 3 of the 16 oocytes which had been vitrified according
to the present invention had reached the morula stage of
development. In comparison, of the 28 oocytes which had been
subjected to vitrification using the OPS methodology, none of them
had reached the morula stage of development, with only 4 such
oocytes surviving, with the most successful single example of the
OPS method having progressed to the 16-32 cell stage.
EXAMPLE 12
Methodologies and Materials for the Vitrification of Mouse and
Human Oocytes
A. Media
[0098] Media for embryo culture was G1.2 and G2.2 supplemented with
5 mg/ml human serum albumin (Gardner et al., Hum. Reprod.
13:3434-40 (1998)). Media for embryo collection and vitrification
was a HEPES-buffered modification of G1.2 without EDTA, modified by
replacing 20 mM NaHCO.sub.3 with 20 mM HEPES and adjusted to pH
7.35.
B. Mice
[0099] Oocytes were collected from 4-5 week old F1 (C57BL6xCBa)
females. Females were stimulated with 5 iu of pregnant mare's
gonadotrophin (Sigma Chemical Co., St. Louis, Mo.) and 53 hr. later
with 5 iu of human chorionic gonadotrophin (hCG; Sigma Chemical
Co.). Oocytes were collected at 14 hr. post-hCG and denuded from
surrounding cumulus by incubation in H-G1.2 with 0.5 mg/ml
hyularonidase for less than 1 min. Oocytes were washed twice in
H-G1.2 and either inseminated or cryopreserved.
C. Human Oocyte Collection
[0100] Patients were stimulated to produce multiple oocytes
according to Gardner et al., Hum. Reprod. 13:3434-40 (1998).
Oocytes were flushed from the follicles and placed into culture at
G1.2 for 4 hr. Oocytes were denuded from surrounding cumulus by
incubation in G1.2 with hyularonidase. Immature oocytes were then
placed into culture in G2.2 with fetal cord serum for 24 hr. Mature
MII oocytes were then either allocated to control or were vitrified
using the loop.
D. Loop Vitrification of Mouse and Human Oocytes
[0101] Oocytes were vitrified using a 2-step loading with
cryoprotectants. Initially oocytes were placed in cryoprotectant
solution I which contained 10% DMSO and 10% ethylene glycol for 1
min before being transferred to solution II, which contained 20%
DMSO and 20% ethylene glycol, 10 mg/ml Ficoll (MW 400,000) and 0.65
M sucrose for around 20 sec. Oocytes were then transferred to the
loop that had previously been dipped into solution II to create a
thin-film, and plunged directly into liquid nitrogen. By previously
submerging the cryovial under liquid nitrogen, the loop containing
the oocytes could be plunged into the cryovial containing liquid
nitrogen and sealed under liquid nitrogen in one motion. The vials
were stored in standard canes.
[0102] Oocytes were thawed using a 2-step dilution with sucrose.
With the cryovial submerged under liquid nitrogen the vial was
opened and the loop containing cells removed from the liquid
nitrogen and placed directly into a well of the base medium of
H-G1.2 containing 0.25 M sucrose. The oocytes immediately fall from
the loop into the thaw solution. Oocytes were moved from this
solution after 2 min and transferred to base medium H-G1.2
containing 0.125 M sucrose for a further 3 min. Subsequently,
oocytes were washed twice in H-G1.2 for 5 min. and were then
returned to culture.
E. In Vitro-Fertilization and Embryo Culture of Mouse Oocytes
[0103] Spermatozoa were aspirated from the epididymis of 12-16 week
old F1 (C57BL6xCBa) male mice into medium FG1 (as described by
Gardner and Lane, 1997, Hum. Reprod. Update 3:367-382) supplemented
with 1 mg/ml glutathione and 5 mg/ml HSA (Scandinavian IVF
Sciences, Gothenburg, Sweden). Spermatozoa were capacitated for 1.5
hr. before insemination. Before oocytes were inseminated, a small
hole (5 .mu.M) was made in the zona of the oocytes using a
Fertilase 670 nm laser aiming beam and a collimated 1.48 .mu.M
laser beam (MTM. Medical Technologies, Montreux, Switzerland).
Oocytes were placed in 100 .mu.l drops of FG1 and co-incubated with
approximately 1.times.10.sup.4 sperm for 4 hr. Oocytes were washed
twice and cultured in 20 .mu.l drops of G1.2 at 37.degree. C. in 6%
CO.sub.2, 5% O.sub.2 and 89% N.sub.2. Fertilization was assessed by
the presence of 2-cell embryos the following morning. All 2-cells
were moved to fresh drops of G1.2. After 48 hr. culture embryos
were washed well in G2.2 and cultured for a further 48 hr. in
medium G2.2 to the blastocyst stage.
F. Viability Assessment of Human Oocytes
[0104] Viability of human oocytes was assessed by dye-exclusion.
Oocytes were placed in H-G1.2 containing 25 .mu.g/ml of propidium
iodide for 10 min., then washed in H-G1.2 for 5 min. Oocytes that
had not survived the vitrification procedure had positive staining
of nuclei material, while surviving oocytes demonstrate no
staining.
G. Statistical Analysis
[0105] Differences in development among treatments were assessed
using linear-logistic regression where the distribution was
binomial (Glim 4.0, Numerical Algorithms Group, Oxford, UK). Day of
experiment was fitted as a factor. Differences in cell numbers were
assessed using Analysis of Variance as both Gaussian normality and
equal variances were confirmed. Multiple comparisons between
treatments were assessed by Bonferroni's procedure for multiple
comparisons.
EXAMPLE 13
Development in Culture of Mouse Oocytes Following
Cryopreservation
[0106] Oocytes vitrified using the method of the present invention
had significantly higher rates of survival compared to oocytes
cryopreserved using the slow-freezing procedure, as shown in Table
6. Similarly, fertilization rates were significantly higher in
oocytes that were vitrified using the method of the present
invention compared to the slow-freezing procedure as shown in Table
6. Oocytes vitrified using the present method had equivalent
fertilization rates to control fresh oocytes which were
inseminated, as shown in Table 6. Rates of fertilization were
significantly lower in oocytes cryopreserved using the
slow-freezing procedure as shown in Table 6. Control embryos that
were not frozen developed to the blastocyst stage at rates of 70.0%
from total oocyte or 95.4% from 2-cell embryos. There was no
difference in blastocyst development rates from oocytes that were
vitrified using the loop compared to the control oocytes as shown
in Table 6. In contrast, rates of blastocyst development from total
oocytes or from 2-cell embryos was significantly reduced in oocytes
that were cryopreserved by slow-freezing as shown in Table 6.
TABLE-US-00006 TABLE 6 Effect of Cryopreservation of Mouse Oocytes
on Mouse Fertilization and Embryo Development Survival
Fertilization Blastocyst/Total Blastocyst/2- Treatment (%) (%) (%)
Cell (%) Control 100 73.4 70.0 95.4 Loop 99.2 69.8 67.4 96.5
Slow-freeze 80.9* 39.5** 25.7** 65.1** N = at least 300 embryos per
treatment *significantly different from all other treatments (P
< 0.05) **significantly different from all other treatments (P
< 0.01)
EXAMPLE 14
Subsequent Viability of Mouse Oocytes Following
Cryopreservation
[0107] Blastocysts derived from either fresh or cryopreserved
oocytes (either by vitrification with the present methodology or
slow freezing) were transferred to pseudo-pregnant recipients and
implantation and fetal development assessed and the results given
in Table 7. Blastocysts that were derived from oocytes that were
vitrified using the method of the present invention had similar
implantation rates to fresh oocytes, however fetal development was
slightly lower. In contrast, oocytes that were frozen using the
slow-freeze procedure had significantly reduced implantation and
fetal development rates compared to either control oocytes or
oocytes vitrified using the method of the present invention.
Additionally, 2 females which each had 8 blastocysts transferred
that resulted from oocytes that were vitrified were allowed to
litter. Eleven pups were born from these females (7 and 4) and all
developed into morphologically normal and fertile adult mice. Of
the eleven pups born, 8 were females and 3 males. TABLE-US-00007
TABLE 7 Effect of Cryopreservation on Mouse Viability Following
Transfer Implantation Fetal Development Fetal Development/
Treatment (%) (%) Implantation (%) Control 86.sup.a 68.0.sup.a
79.1.sup.a Loop 88.0.sup.a 56.5.sup.b 64.2.sup.b Slow-freeze
52.4.sup.b 26.2.sup.c 50.0.sup.b N = at least 50 blastocysts
transferred per treatment group .sup.a-cdifferent letters are
significantly different (P < 0.05)
EXAMPLE 15
Survival Rates of Human Oocytes Following Cryopreservation
[0108] Viability of human oocytes was assessed following
vitrification by the present methodology. High rates of survival
were observed, as shown in Table 8. TABLE-US-00008 TABLE 8 Survival
Rates of Human Oocytes Following Cryopreservation Treatment Number
of Embryos Survival (%) Fresh oocytes 12 100 Vitrified oocytes 21
81.9
EXAMPLE 16
Vitrification of Mouse Cleavage Stage Embryos
[0109] Embryos were collected according to the methods described in
Example 1. One-cell embryos were vitrified using the loop
immediately following collection while 2-cell embryos were obtained
following 24 hr. culture according to the methods described in
Example 1.
[0110] One-cell and 2-cell embryos were vitrified using the loop
according to the methods described in Example 12. There was no
difference in the ability of mouse 1-cell or 2-cell embryos to
develop to the blastocyst stage in culture compared to fresh
embryos that were not cryopreserved. The results are shown below in
Table 9. TABLE-US-00009 TABLE 9 Development of Mouse 1-cell and
2-cell Embryos Following Vitrification According to the Present
Invention Treatment.sup.# Number of Embryos Blastocyst (%) Fresh
1-cells 20 95.0 Vitrified 1-cells 20 90.0 Fresh 2-cells 30 96.7
Vitrified 2-cells 30 93.3 .sup.#There was no difference in the
ability of loop vitrified 1-cell or 2-cell embryos to develop in
culture compared to fresh embryos.
EXAMPLE 17
Vitrification of Mouse Spermatozoa Using the Present Invention
[0111] Mature mouse spermatozoa were collected according to the
methods described in Example 12.
[0112] A 1 .mu.l drop of cryoprotectant solution I which contained
10% DMSO and 10% ethylene glycol was placed on the lid of a petri
dish. A 1 .mu.l of sperm solution was added to the drop of solution
I. After 20 sec, 1 .mu.l of solution II, which contained 20% DMSO
and 20% ethylene glycol, 10 mg/ml Ficoll (MW 400,000) and 0.65 M
sucrose was added and the whole drop placed on a loop which was
plunged into liquid nitrogen. For thawing, the loop was placed into
a 20 .mu.l of base medium containing 0.25M sucrose for 30 sec.,
when 20 .mu.l of base medium was added to the drop for a further 1
min, finally 2 ml of base medium was added. Following this
procedure, viable sperm could be obtained as determined by
motility.
[0113] It is understood that the invention is not confined to the
particular embodiments set forth herein as illustrative, but
embraces all such modified forms thereof as come within the scope
of the following claims.
* * * * *