Claims
- 1. A method for preserving a suspension of biological material comprising:
- (a) preparing a cryosolution of a suspension of biological material, said cryosolution comprising a buffer, a cryoprotectant and a biological material;
- (b) nebulizing said cryosolution with a low frequency, ultrasonic nebulizer to form microdroplets;
- (c) cooling the microdroplets at a rate which results in the formation of amorphous phase ice and/or cubic phase ice with or without hexagonal phase ice in said cryosolution by spraying the microdroplets on a solid cryogenic surface;
- (d) continuously removing said cryosolution from said solid cryogenic surface after cooling to replenish said surface for further cooling of additional microdroplets; and
- (e) drying the cooled cryosolution to form a dried cryosolution.
- 2. The method of claim 1 wherein said dried cryosolution has a final residual moisture content of less than about 5%.
- 3. The method of claim 1 wherein said cryosolution further comprises a dry protectant.
- 4. The method of claim 2 further comprising the step of sealing said dried cryosolution in a container without increasing the residual water content of said dried cryosolution above about 5%.
- 5. The method of claim 4 wherein said sealing is under low pressure or in the presence of a dry inert gas.
- 6. The method of claim 1 wherein said buffer is selected from the group consisting of 2-amino-2-hydroxymethyl-1,3-propanediol; 2-ethanesulfonic acid; 2-amino-2-methyl-1,3-propanediol; 3-propanesulfonic acid; piperazine-N-N'-bis; 1,4-piperazinediethanesulfonic acid and combinations thereof.
- 7. The method of claim 1 wherein said buffer is a low salt buffer mixture selected from the group consisting of (1) 8 gms glycine, 20 mls of 0.15M phosphate buffer pH 6.7-8.4 and 1.8 gm sodium chloride made up to 1000 ml., pH 6.7; (2) 16 gms glycine, 20 mls of 0.15M phosphate buffer pH 6.7-8.4, 1.8 gm sodium chloride, 5.0 gms glucose, 0.06 gm calcium chloride and 0.1 gms adenine made up t 1000 ml., pH 6.7; (3) a solution containing 0.9% NaCl, 0.75% mannitol, 2.2% dextrose and 0.027% adenine; (4) a solution containing 0.875% dextrose, 0.9% NaCl, 0.214% mannitol, 0.008% adenine, 0.2555% sodium citrate, 0.029% citric acid and 0.022% monobasic sodium phosphate; (5) 16 mM phosphate buffer, pH 7.4, 5 mM KCl, 4 g/dl BSA and 6.9 g/lit. lactose; and (6) 16 mM phosphate buffer, pH 7.4, 5 mM KCl and 15 g/lit. lactose.
- 8. The method of claim 1 wherein said cryoprotectant is selected from the group consisting of dimethyl sulfoxide, 2-3 butane diol, polyvinylpyrrolidone, propylene glycol, 1,2 propanediol, glycerol, fructose, trehalose, raffinose, hydroxyethyl starch, dextran, sucrose, sorbitol, proline, human serum albumin and combinations thereof.
- 9. The method of claim 8 wherein said cryoprotectant is selected from the group consisting of
- (1) a vitrification solution comprising a mixture of 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol, 1.0M proline, 2.5% raffinose, 15% PVP with an average molecular weight of about 40,000 and 15% dextran with an average molecular weight from about 40,000 to about 70,000;
- (2) a vitrification solution comprising a mixture of 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol, 10% raffinose, 6% trehalose, 6% sucrose, 12% PVP with an average molecular weight of about 40,000 and 12% dextran with an average molecular weight from about 40,000 to about 70,000; and
- (3) a vitrification solution comprising a mixture 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol. 2.5% raffinose, 12% sucrose, 15% PVP with an average molecular weight of about 40,000 and 15% dextran with an average molecular weight from about 40,000 to about 70,000.
- 10. The method of claim 3 wherein said dry protectant is selected from the group consisting of: sucrose, raffinose, trehalose, zinc, proline, myristic acid, spermine and combinations thereof.
- 11. The method of claim 1 wherein said drying comprises freeze drying.
- 12. The method of claim 1 wherein said drying comprises molecular distillation drying.
- 13. The method of claim 1 wherein said biological material comprises red blood cells.
- 14. The method of claim 1 wherein said biological material comprises mammalian cultured cells.
- 15. The method of claim 1 wherein said biological material comprises platelets.
- 16. The method of claim 1 wherein said biological material comprises leukocytes.
- 17. The method of claim 1 wherein said biological material comprises Factor VIII.
- 18. The method of claim 1 wherein said biological material comprises sperm.
- 19. The method of claim 1 wherein said biological material comprises pancreatic islets.
- 20. The method of claim 1 wherein said biological material comprises bone marrow cells.
- 21. The method of claim 1 wherein said biological material comprises a virus.
- 22. The method of claim 1 wherein said biological material comprises a vaccine.
- 23. The method of claim 1 further comprising rehydrating the dried cryosolution.
- 24. A method for preserving a suspension of a microscopic biological material comprising:
- (a) preparing a cryosolution of a microscopic biological material, said cryosolution comprising a buffer, a cryoprotectant, and a biological material;
- (b) nebulizing said cryosolution With a low frequency, ultrasonic nebulizer to form discrete droplets, said droplets being less than about 200 .mu.m in diameter;
- (c) rapidly cooling said droplets resulting in the formation of amorphous phase ice, cubic phase ice and hexagonal phase ice by spraying the droplets on a solid cryogenic surface;
- (d) continuously removing the rapidly cooled cryosolution from said solid cryogenic surface after said ice has been formed to replenish said surface for cooling of additional droplets; and
- (e) drying said rapidly cooled cryosolution to remove said amorphous phase ice, cubic phase ice and hexagonal ice to form a dried cryosolution, said drying being completed to a final residual moisture content of less than about 5%.
- 25. The method of claim 24 further comprising the step of sealing said dried cryosolution in a container without increasing the residual water content of said dried cryosolution above about 5%.
- 26. The method of claim 25 wherein said sealing is performed in a dry inert gas atmosphere at a low pressure of from about 10.sup.-6 mbar to 10.sup.-1 mbar.
- 27. The method of claim 24 further comprising rehydrating the dried cryosolution.
RELATED APPLICATIONS
This application is a continuation of application Ser. No. 07/709,504, filed on Jun. 3, 1991, now abandoned, which is a continuation-in-part of application Ser. No. 07/581,584, filed on Sep. 12, 1990, now abandoned.
US Referenced Citations (18)
Foreign Referenced Citations (1)
Number |
Date |
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0267026 |
Nov 1988 |
EPX |
Continuations (1)
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Number |
Date |
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Parent |
709504 |
Jun 1991 |
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Continuation in Parts (1)
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Number |
Date |
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581584 |
Sep 1990 |
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