WO2012054892A1 - Cryopreservation solutions and uses therefor - Google Patents
Cryopreservation solutions and uses therefor Download PDFInfo
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- WO2012054892A1 WO2012054892A1 PCT/US2011/057379 US2011057379W WO2012054892A1 WO 2012054892 A1 WO2012054892 A1 WO 2012054892A1 US 2011057379 W US2011057379 W US 2011057379W WO 2012054892 A1 WO2012054892 A1 WO 2012054892A1
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- vessel
- membrane
- cells
- cryopreservation
- solutes
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Definitions
- cryopreservation systems including aqueous solutions and associated devices, that allow the viability of mammalian and non-mammalian gametes, gonadal tissue, pre-implantation embryos, and the like to be preserved for long periods of time.
- the combination of all of the elements of this system will result in improvements in end use.
- aqueous cryopreservation media comprising:
- membrane-non-diffusible solutes refers to solutes selected from the group consisting of sucrose, trehalose, mannitol, glucose, galactose, fructose, and the like.
- each R b is independently hydrogen, hydroxy, Ci to C 6 alkoxy, or
- reproductive material is cooled fall in the range of about -180 up to about -120 °C.
- cryopreserved reproductive material is warmed to a temperature in the range of about -20 up to about +20 °C at a rate in the range of about 10 up to about 5000 °C/minute.
- Presently preferred warming rates fall in the range of about 100 up to about 2500 °C/minute.
- warming is carried out in two stages, wherein the cryopreserved reproductive material is warmed at a rate in the range of about 10 up to about 100 °C/minute for about 10-30 seconds, and thereafter, warmed at a rate in the range of about 1000 up to about 2500 °C/minute.
- This two-stage warming protocol is especially beneficial if the cryopreserved material has been previously subjected to slow cooling (as described herein).
- aqueous cryopreservation medium and the cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison. See, for
- aqueous pre-cryopreservation medium comprising:
- the above-described pre- cryopreservation media can be employed in a stepwise method for introducing membrane- diffusible solutes into cells, said method comprising incorporating one or more pre- cryopreservation media as described in the preceding paragraph, and thereafter incorporating into said cells an aqueous cryopreservation medium comprising:
- pre-exposure solutions that are designed to incorporate permeating cryoprotectants into the cell at a concentration lower that that contained in the full-strength cryopreservation solution
- the pre-exposure solutions should contain a reduced concentration of non- permeating agents, such as those commonly contained in cell culture media, such that the total concentration is between 25 and 75% of that found in an isotonic media,
- a total combination of solutes should be present that precludes ice formation when a cylindrical plastic holding container containing the solution is cooled and warmed using a combination of cooling and warming rates as described herein, and viewed under a dissecting microscope during warming to observe the incidence of ice formation
- the solutes in the full-strength cryopreservation media should include a combination of solutes, some of which can diffuse across a biological membrane and some of which cannot diffuse across a biological membrane, Regarding the solutes that can diffuse across the biological membrane, these should include ethylene glycol and/or dimethyl sulfoxide, with or without one or more of the following amides (urea, hydroxyurea, formamide, acetamide, N-methyl formamide, and the like).
- the total concentration of these compounds should range between 20% and 50% (w/w),
- sucrose trehalose, and the like
- concentration of these compounds should range between 10 and 40%> (w/w)
- Z-1000 it should be included at a concentration ranging between 0.1% to 10% (w/w), and
- polyvinylpyrrilodone it should be included at a concentration ranging between 0.1 % to 10%> (w/w).
- aqueous media useful for removing membrane-diffusible solutes from cells by diluting the cryoprotectant from said cells employing a medium comprising:
- the use of specially-formulated media to dilute the cryoprotectant from the cryopreserved cells include the following features: o
- the use of non-permeating solutes such as those found in standard cell culture media at concentrations close to those found in standard cell culture media, and
- non-permeating solutes including compounds such as sucrose or trehalose and at total concentrations ranging from 1 to 30% (w/w).
- vessels in which gametes, gonadal tissue, pre -implantation embryos, and the like can be contained and cryopreservation medium can be introduced into and removed therefrom. See, for example, Figures 1 and 2.
- invention vessels comprise a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnel-like devices designed to facilitate placing a label into one end of the hollow cylindrical structure, and aqueous cryopreservation medium and cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison.
- funnel-shaped end caps provides:
- invention vessels further comprise a structure for holding and storing gametes, gonadal tissue, pre-implantation embryos, and the like to be
- cryopreserved wherein said vessel is designed with any geometry allowing cells to be placed into the vessel before preservation and removed from the vessel after preservation and also allowing media including said cryopreservation media to be moved through said vessel under positive or negative pressure.
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Abstract
Cryopreservation systems, including aqueous solutions and associated devices, that allow the viability of mammalian and non- mammalian gametes and pre-implantation embryos to be preserved for long periods of time. The aqueouscryopreservation media comprises membrane-diffusible solutes, membrane-non-diffusible solutes, and at least one polymeric material. The vessel employed is a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnel-like devices which facilitate placing a label into one end of the hollow cylindrical structure, and the aqueous cryopreservation medium and the cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison.
Description
CRYO PRESERVATION SOLUTIONS AND USES THEREFOR
FIELD OF THE INVENTION
[0001] The present invention relates to cryopreservation solutions and uses therefore.
Invention solutions are particularly useful for the cryopreservation of gametes, gonadal tissue, pre-implantation embryos, and the like.
SUMMARY OF THE INVENTION
[0002] In accordance with the present invention, there are provided cryopreservation systems, including aqueous solutions and associated devices, that allow the viability of mammalian and non-mammalian gametes, gonadal tissue, pre-implantation embryos, and the like to be preserved for long periods of time. The combination of all of the elements of this system will result in improvements in end use.
BRIEF DESCRIPTION OF THE FIGURES
[0003] Figure 1 is an illustration of an exemplary vessel useful in the practice of the present invention.
[0004] Figure 2 is an exploded view of the vessel illustrated in Figure 1.
[0005] Figure 3 presents an image of material which has been cryopreserved using invention solutions, and slow rates of cooling and warming. Note the clean appearance of the solution.
[0006] Figure 4 presents an image of material which has been cryopreserved using rapid rates of cooling and warming. Note the messy appearance of the solution, which contains many bubbles formed as a result of significant thermal gradients being built up in the solution during cooling, resulting in cavitation of the solution upon warming. The thermal stress associated with the rapid cooling and warming resulting in the cavitation
bubbles could also be harmful to biomaterial being cryopreserved in the same manner, arguing for the use of relatively slow rates of cooling and warming during cryopreservation.
DETAILED DESCRIPTION OF THE INVENTION
[0007] In accordance with the present invention, there are provided aqueous cryopreservation media comprising:
20-50% (w/w) membrane-diffusible solutes,
10-40% (w/w) of membrane-non-diffusible solutes,
0.1-10% (w/w) of at least one polymeric material.
[0008] As employed herein, the term "membrane-diffusible solutes" refers to compositions comprising at least ethylene glycol. Optional additional components in membrane-soluble solutes include one or more components selected from the group consisting of dimethyl sulfoxide, urea, hydroxyurea, formamide, acetamide, N-methyl formamide, 1 ,2-propanediol, glycerol, 1,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, 3- methoxy-l,2-propanediol, and the like.
[0009] As employed herein, the term "membrane-non-diffusible solutes" refers to solutes selected from the group consisting of sucrose, trehalose, mannitol, glucose, galactose, fructose, and the like.
[0010] As employed herein, the term "polymeric material" refers to such polymers as:
(i) a compound of the formula:
[-CR2CR'(OH)-]n,
wherein:
each R is independently hydrogen or an alkyl group,
each R' is independently hydrogen or an alkyl group,
n is at least 3 up to about 500,
up to 25 mol % of the hydroxyl groups are replaced with acetyl groups, said compound has a mean molecular weight of less than 10,000
daltons, and
said compound is present at a concentration in the range of 1 part per million up to about 10% by weight;
(ii) a compound of the formula:
Ra[-OCR2CR(OH)CR2-]nRb,
wherein:
each R is independently hydrogen, hydroxy, Ci to C6 alkoxy, or amino, each Ra is independently hydrogen, hydroxy, Ci to C6 alkoxy, or
amino,
each Rb is independently hydrogen, hydroxy, Ci to C6 alkoxy, or
amino,
n is 2 up to about 1000, and
said compound is covalently bound to a hydrophobic group which renders the compound soluble in a hydrophobic solvent; and
(iii) polyvinylpyrrolidone having a molecular weight in the range of about 1000 up to about 25,000.
[0011] Exemplary materials set forth as option (i) above are described in U.S. Pat.
No. 6,391 ,224, the entire contents of which are hereby incorporated by reference herein.
[0012] Exemplary materials set forth as option (ii) above are described in U.S. Pat.
No. 6,616,858, the entire contents of which are hereby incorporated by reference herein.
[0013] In accordance with another embodiment of the present invention, there are provided methods for the cryopreservation of reproductive material, said methods comprising cooling a combination of said reproductive material and cryopreservation medium according to the invention to a temperature in the range of about -210 up to about -1 10 °C at a rate in the range of about 10 up to about 2500 °C/minute. Presently preferred cooling rates fall in the range of about 50 up to about 500 °C/minute, with cooling rates in the range of about 50 up to about 100 °C/minute being especially preferred.
[0014] The use of cryopreservation solutions containing a specific combination of solutes, including "ice blocker" polymers (see, for example, U.S. Pat. Nos. 6,391 ,224 and 6,616,858) provides:
o Ice-free cryopreservation (i.e., vitrification) when using a cylindrical plastic holding container combined with specific cooling and warming rates,
o The use of cryopreservation agents that penetrate an intact membrane bilayer (often referred to as permeating cryoprotectants) at concentrations similar to those that have been shown to be non-toxic to mammalian oocytes and pre- implantation embryos and still retain all of the benefits described above, o The ability to vitrify a sample using cooling rates that are far slower than those currently in use, and the like.
[0015] The use of cooling rates between 10 and 2500 °C/minute provides:
o Ease of use by allowing the end user more flexibility in the step associated with cooling the sample,
o Improved biosecurity by allowing the use of a cylindrical plastic holding
container which can be securely sealed,
o Significant reduction in mechanical stress build-up in the sample during
cooling, virtually eliminating the incidence of stress fracturing and solution cavitation during cooling, and the like.
[0016] As used herein, "reproductive material" refers to gametes, gonadal tissue, pre- implantation embryos, and the like.
[0017] Presently preferred temperatures to which reproductive material is cooled fall in the range of about -180 up to about -120 °C.
[0018] In accordance with another embodiment of the present invention,
cryopreserved reproductive material is warmed to a temperature in the range of about -20 up to about +20 °C at a rate in the range of about 10 up to about 5000 °C/minute. Presently preferred warming rates fall in the range of about 100 up to about 2500 °C/minute. In a presently preferred embodiment, warming is carried out in two stages, wherein the cryopreserved reproductive material is warmed at a rate in the range of about 10 up to about 100 °C/minute for about 10-30 seconds, and thereafter, warmed at a rate in the range of about 1000 up to about 2500 °C/minute. This two-stage warming protocol is especially beneficial
if the cryopreserved material has been previously subjected to slow cooling (as described herein).
The use of warming rates between 100 and 5000 degrees C per minute
Prevention of ice formation upon warming when the sample has been cooled using the rates described herein, in combination with appropriate cryopreservation solutions containing the solutes in concentrations as described herein,
Ease of use by allowing the end user more flexibility in the step associated with warming the sample,
Improved biosecurity by allowing the use of a cylindrical plastic holding container which can be securely sealed,
Significant reduction in mechanical stress build-up in the sample during warming, virtually eliminating the incidence of stress fracturing and solution cavitation during warming, and the like.
[0020] In certain embodiments of the present invention, reproductive material is contained in a vessel configured such that invention cryopreservation medium can be introduced into and removed from said vessel.
[0021] In certain embodiments of the present invention, the vessel employed is a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnel-like devices which facilitate placing:
a label into one end of the hollow cylindrical structure, and
the aqueous cryopreservation medium and the cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison. See, for
Example, Figures 1 and 2.
[0022] In certain embodiments of the invention, the vessel employed to facilitate carrying out the invention method comprises a structure for holding and storing cells to be cryopreserved, and is designed with any geometry allowing cells to be placed into the vessel before preservation and removed from the vessel after preservation and also allow media
including said cryopreservation media to be transported through said vessel under positive or negative pressure.
[0023] In accordance with yet another embodiment of the present invention, there are provided aqueous pre-cryopreservation medium comprising:
5-30% (w/w) membrane-diffusible solutes,
2-5% (w/w) of membrane-non-diffusible solutes,
0-1% (w/w) of at least one polymeric material.
[0024] In certain embodiments of the present invention, the above-described pre- cryopreservation media can be employed in a stepwise method for introducing membrane- diffusible solutes into cells, said method comprising incorporating one or more pre- cryopreservation media as described in the preceding paragraph, and thereafter incorporating into said cells an aqueous cryopreservation medium comprising:
20-50%) (w/w) membrane-diffusible solutes,
10-40%) (w/w) of membrane-non-diffusible solutes,
0.1-10% (w/w) of at least one polymeric material.
The use of specifically formulated solutions to cryopreserve cells include the
A series of two or more pre-exposure solutions that are designed to incorporate permeating cryoprotectants into the cell at a concentration lower that that contained in the full-strength cryopreservation solution, The pre-exposure solutions should contain a reduced concentration of non- permeating agents, such as those commonly contained in cell culture media, such that the total concentration is between 25 and 75% of that found in an isotonic media,
For the full-strength cryopreservation media, a total combination of solutes should be present that precludes ice formation when a cylindrical plastic holding container containing the solution is cooled and warmed using a combination of cooling and warming rates as described herein, and viewed under a dissecting microscope during warming to observe the incidence of ice formation,
The solutes in the full-strength cryopreservation media should include a combination of solutes, some of which can diffuse across a biological membrane and some of which cannot diffuse across a biological membrane, Regarding the solutes that can diffuse across the biological membrane, these should include ethylene glycol and/or dimethyl sulfoxide, with or without one or more of the following amides (urea, hydroxyurea, formamide, acetamide, N-methyl formamide, and the like). The total concentration of these compounds should range between 20% and 50% (w/w),
Regarding the solutes that cannot diffuse across the biological membrane, these should include sucrose, trehalose, and the like, and the concentration of these compounds should range between 10 and 40%> (w/w),
Regarding the solutes that cannot diffuse across the biological membrane, these should include polymeric material known commercially as X-1000 (see U.S. Pat. No. 6,391,224 Bl) and Z-1000 (see U.S. Pat. No. 6,616,858 B2) and polyvinylpyrrilodone, with a molecular weight ranging from 1000 and 25,000, Regarding the use of X-1000, it should be included at a concentration ranging between 0.1% to 10% (w/w),
Regarding the use of Z-1000, it should be included at a concentration ranging between 0.1% to 10% (w/w), and
Regarding the use of polyvinylpyrrilodone, it should be included at a concentration ranging between 0.1 % to 10%> (w/w).
[0026] In accordance with still another embodiment of the present invention, there are provided aqueous media useful for removing membrane-diffusible solutes from cells by diluting the cryoprotectant from said cells employing a medium comprising:
0-30%) (w/w) membrane-diffusible solutes,
5-30%) (w/w) of membrane-non-diffusible solutes,
0-1% (w/w) of at least one polymeric material.
[0027] The use of specially-formulated media to dilute the cryoprotectant from the cryopreserved cells include the following features:
o The use of non-permeating solutes such as those found in standard cell culture media at concentrations close to those found in standard cell culture media, and
o The use of additional non-permeating solutes including compounds such as sucrose or trehalose and at total concentrations ranging from 1 to 30% (w/w).
[0028] In accordance with yet another embodiment of the present invention, there are provided methods for removing membrane-diffusible solutes from cells by diluting the cryoprotectant from said cells employing a medium as described in the preceding paragraph.
[0029] In accordance with still another embodiment of the present invention, there are provided vessels in which gametes, gonadal tissue, pre -implantation embryos, and the like can be contained and cryopreservation medium can be introduced into and removed therefrom. See, for example, Figures 1 and 2.
[0030] In certain aspects, invention vessels comprise a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnel-like devices designed to facilitate placing a label into one end of the hollow cylindrical structure, and aqueous cryopreservation medium and cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison.
[0031] The use of a cylindrical plastic holding container divided into two sections with a barrier having an outer diameter slightly larger than the inner diameter of the hollow cylindrical structure provides numerous advantages, including:
o Ease of handling,
o Familiarity,
o Ease of labeling, including the insertion of internally-secure labels, o Improved biosecurity through ease of sealing,
o Greater thermal mass, reducing the likelihood of accidentally warming to dangerous temperatures,
o Maintenance of sterility,
o Ease of incorporation into currently-used storage schemes,
o Ease of sample expulsion by allowing the plug to be pushed through the cylindrical plastic holding container, forcing the fluid containing the biomaterial out of the cylindrical plastic holding container, and the like.
The use of funnel-shaped end caps provides:
Ease of use by facilitating the insertion of very thin pipettes containing the biological samples,
Ease of use by facilitating the insertion of very thin pipettes containing the solution to be used to vitrify the biomaterial,
Ease of use by facilitating the insertion of labels, and the like.
[0033] The use of a solid barrier device having an outer diameter slightly larger than the inner diameter of the cylindrical plastic holding container provides for ease of use by facilitating the expulsion of the solution and biomaterial from the cylindrical plastic holding container after preservation.
[0034] The use of thin pipettes having an outer diameter smaller than the inner diameter of the cylindrical plastic holding container provides for ease of use by facilitating:
o Filling the cylindrical plastic holding container with a cryopreservation
solution,
o The insertion of biological samples into the cylindrical plastic holding
container, and the like.
[0035] In certain aspects, invention vessels further comprise a structure for holding and storing gametes, gonadal tissue, pre-implantation embryos, and the like to be
cryopreserved, wherein said vessel is designed with any geometry allowing cells to be placed into the vessel before preservation and removed from the vessel after preservation and also allowing media including said cryopreservation media to be moved through said vessel under positive or negative pressure.
[0036] While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
Claims
1. An aqueous cryopreservation medium comprising:
20-50% (w/w) membrane-diffusible solutes,
10-40% (w/w) of membrane-non-diffusible solutes,
0.1-10% (w/w) of at least one polymeric material.
2. The cryopreservation medium of claim 1 wherein said membrane-diffusible solutes comprise ethylene glycol and/or dimethyl sulfoxide.
3. The cryopreservation medium of claim 2, further comprising one or more components selected from the group consisting of urea, hydroxyurea, formamide, acetamide, N-methyl formamide, 1,2-propanediol, glycerol, 1,3-butanediol, 1 ,4-butanediol, 2,3- butanediol, and 3-methoxy- 1,2-propanediol.
4. The cryopreservation medium of claim 1 wherein said membrane-non- diffusible solutes are selected from the group consisting of sucrose, trehalose, mannitol, glucose, galactose, and fructose.
5. The cryopreservation medium of claim 1 wherein said polymeric material is selected from the group consisting of:
a compound of the formula:
[-CR2CR'(OH)-]n,
wherein:
each R is independently hydrogen or an alkyl group,
each R' is independently hydrogen or an alkyl group,
n is at least 3 up to about 500,
up to 25 mol %> of the hydroxyl groups are replaced with acetyl groups, said compound has a mean molecular weight of less than 10,000
daltons, and
said compound is present at a concentration in the range of 1 part per million up to about 10%> by weight;
a compound of the formula: Ra[-OCR2CR(OH)CR2-]„Rb,
wherein:
each R is independently hydrogen, hydroxy, Ci to C6 alkoxy, or amino, each Ra is independently hydrogen, hydroxy, Ci to C6 alkoxy, or
amino,
each Rb is independently hydrogen, hydroxy, Ci to C6 alkoxy, or
amino,
n is 2 up to about 1000, and
said compound is covalently bound to a hydrophobic group which renders the compound soluble in a hydrophobic solvent; and polyvinylpyrrolidone having a molecular weight in the range of about 1000 up to about 25,000.
6. An aqueous pre-cryopreservation medium comprising:
5-30% (w/w) membrane-diffusible solutes,
2-5% (w/w) of membrane-non-diffusible solutes,
0-1% (w/w) of at least one polymeric material.
7. A method for introducing membrane-diffusible solutes into cells, said method comprising incorporating one or more pre-cryopreservation media according to claim 6 into said cells, and thereafter incorporating into said cells an aqueous cryopreservation medium comprising:
20-50%) (w/w) membrane-diffusible solutes,
10-40%) (w/w) of membrane-non-diffusible solutes,
0.1-10% (w/w) of at least one polymeric material.
8. The method of claim 7, further comprising removing the membrane-diffusible solutes from the cells by diluting the cryoprotectant from said cells employing a medium comprising:
0-30%) (w/w) membrane-diffusible solutes,
5-30%o (w/w) of membrane-non-diffusible solutes,
0-1% (w/w) of at least one polymeric material.
9. A method for the cryopreservation of reproductive material, said method comprising cooling a combination of said reproductive material and cryopreservation medium according to claim 1 to a temperature in the range of about -210 up to about -110 °C at a rate in the range of about 10 up to about 2500 °C/minute.
10. The method of claim 9 wherein said reproductive material is selected from the group consisting of gametes, gonadal tissue, and pre-implantation embryos.
11. The method of claim 9 wherein said combination is cooled to a temperature in the range of about -180 up to about -120 °C.
12. The method of claim 9 further comprising warming the cryopreserved reproductive material to a temperature in the range of about -20 up to about +20 °C at a rate in the range of about 100 up to about 5000 °C/minute.
13. The method of Claim 9 wherein said cells are contained in a vessel and said cryopreservation medium can be introduced into and removed from said vessel.
14. The method of Claim 13 wherein said vessel is a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnel-like devices which facilitate placing:
a label into one end of the hollow cylindrical structure, and
the aqueous cryopreservation medium and the cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison.
15. The method of Claim 13 wherein said vessel comprises a structure for holding and storing cells to be cryopreserved, and is designed with any geometry allowing cells to be placed into the vessel before preservation and removed from the vessel after preservation and also allow media including said cryopreservation media to be transported through said vessel under positive or negative pressure.
16. A vessel in which gametes and pre-implantation embryos can be contained and cryopreservation medium can be introduced into and removed therefrom.
17. The vessel Claim 16, wherein said vessel comprises a hollow cylindrical structure separated into two regions by a barrier, and having placed upon the ends two funnellike devices designed to facilitate placing a label into one end of the hollow cylindrical structure, and aqueous cryopreservation medium and cells to be cryopreserved into the opposite end of the hollow cylindrical structure independently or in unison.
18. The vessel of Claim 16 further comprising a structure for holding and storing gametes and pre-implantation embryos to be cryopreserved, wherein said vessel is designed with any geometry allowing cells to be placed into the vessel before preservation and removed from the vessel after preservation and also allowing media including said cryopreservation media to be moved through said vessel under positive or negative pressure.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40607910P | 2010-10-22 | 2010-10-22 | |
| US61/406,079 | 2010-10-22 |
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| Publication Number | Publication Date |
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| WO2012054892A1 true WO2012054892A1 (en) | 2012-04-26 |
Family
ID=45975646
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| PCT/US2011/057379 WO2012054892A1 (en) | 2010-10-22 | 2011-10-21 | Cryopreservation solutions and uses therefor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115399311A (en) * | 2021-05-26 | 2022-11-29 | 北京大学深圳医院 | Vitrification refrigerating fluid set and preparation method and application thereof |
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|---|---|---|---|---|
| US5856081A (en) * | 1991-07-08 | 1999-01-05 | The American National Red Cross | Computer controlled cryoprotectant perfusion apparatus |
| US5952168A (en) * | 1996-11-07 | 1999-09-14 | 21St Century Medicine, Inc. | Method for vitrification of biological materials using alkoxylated compounds |
| US5985538A (en) * | 1997-08-01 | 1999-11-16 | Saint Barnabas Medical Center | Cryopreservation and cell culture medium comprising less than 50 mM sodium ions and greater than 100 mM choline salt |
| US20080268492A1 (en) * | 2007-04-12 | 2008-10-30 | The Curators Of The University Of Missouri | Methods for Determining Optimal Techniques for Vitrification of Isolated Cells |
| WO2010056755A2 (en) * | 2008-11-11 | 2010-05-20 | Craig H Randall | Microfluidic embryo and gamete culture systems |
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2011
- 2011-10-21 WO PCT/US2011/057379 patent/WO2012054892A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5856081A (en) * | 1991-07-08 | 1999-01-05 | The American National Red Cross | Computer controlled cryoprotectant perfusion apparatus |
| US5952168A (en) * | 1996-11-07 | 1999-09-14 | 21St Century Medicine, Inc. | Method for vitrification of biological materials using alkoxylated compounds |
| US5985538A (en) * | 1997-08-01 | 1999-11-16 | Saint Barnabas Medical Center | Cryopreservation and cell culture medium comprising less than 50 mM sodium ions and greater than 100 mM choline salt |
| US20080268492A1 (en) * | 2007-04-12 | 2008-10-30 | The Curators Of The University Of Missouri | Methods for Determining Optimal Techniques for Vitrification of Isolated Cells |
| WO2010056755A2 (en) * | 2008-11-11 | 2010-05-20 | Craig H Randall | Microfluidic embryo and gamete culture systems |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115399311A (en) * | 2021-05-26 | 2022-11-29 | 北京大学深圳医院 | Vitrification refrigerating fluid set and preparation method and application thereof |
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