Methods for the Cryopreservation of Mammalian Cells

• US 20090305224A1
• Filed: 04/12/2007
• Published: 12/10/2009
• Est. Priority Date: 04/14/2006
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Toth, Thomas L., He, Xiaoming, Toner, Mehmet, Edd, Jon
• Granted Patent: US 9,538,745 B2
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 1/0268 Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
• CPC Class Codes: A01N 1/0278 Physical preservation processes

Abstract

The present invention features novel methods for the cryopreservation of mammalian cell that combine the advantages of the slow-freezing and vitrification approaches while avoiding their shortcomings. Generally, the methods include the use of a capillary tube made of a thermally conductive wall material and a thin wall such that the ratio of the thermal conductivity of the wall material to the wall thickness is at least 1,000-500,000. The solution is then exposed to temperatures equal to or less than −80° C. and the vitrification solution containing the mammalian cells is cooled at a rate equal to or greater than 30,000-100,000,000° C./minute. The exposure of the capillary tube with a thermally conductive and thin wall allows for vitrification of the solution in the absence of ice formation. Cryoprotectants can also be added to the vitrification solution to further prevent ice formation.

Claims

1. A method for cryopreserving a cell comprising:

(a) suspending said cell in a vitrification solution;

(b) placing said cell in said vitrification solution in a capillary tube having a wall made of a material that is thermally conductive, wherein said wall material of said capillary tube has a ratio of thermal conductivity to wall thickness of at least 1,000; and

(c) exposing at least a portion of said capillary tube of step (b) to a temperature less than or equal to −

80°

C.,

wherein said cell in said vitrification solution is cooled at a rate equal to or greater than 30,000°

C./min, wherein said cooling causes vitrification of said cell in said vitrification solution in the absence of ice formation.

2. The method of claim 1, wherein said cell is a mammalian cell.

5. The method of claim 1, wherein said cell is an oocyte, a sperm, a stem cell, an embryo, or a zygote.

6. The method of claim 1, wherein said capillary tube is a microcapillary tube.

7. The method of claim 1, wherein said capillary tube comprises a wall having a thickness of 1 to 100 μ

m.

8. The method of claim 7, wherein said capillary tube comprises a wall having a thickness of 7 to 14 μ

m.

9. The method of claim 8, wherein said capillary tube comprises a wall having a thickness of 10 μ

m.

10. The method of claim 1, wherein said capillary tube comprises an inner diameter of about 1 to 400 μ

m.

11. The method of claim 1, wherein said material has a thermal conductivity of at least 0.2 W m¹ K⁻

1.

12. The method of claim 1, wherein said material is selected from the group consisting of plastic, glass, quartz, stainless steel, sapphire, silver, copper, diamond, gold, titanium, palladium, platinum.

13. The method of claim 12, wherein said material is quartz.

14. The method of claim 1, wherein said cell in said vitrification solution is cooled at a rate equal to or greater than 50,000°

C./min.

15. The method of claim 14, wherein said cell in said vitrification solution is cooled at a rate equal to or greater than 100,000°

C./min.

16. The method of claim 1, wherein said wall of said capillary tube has a ratio of thermal conductivity to wall thickness of at least 500,000.

17. The method of claim 1, wherein said temperature in step (c) is less than or equal to −

100°

C.

18. The method of claim 17, wherein said temperature in step (c) is less than or equal to −

196°

C.

19. The method of claim 1, wherein said vitrification solution comprises at least one cryoprotective agent.

20. The method of claim 19, wherein said at least one cryoprotective agent is selected from the group consisting of a sugar, glycerol, ethylene glycol, 1,2-propanediol, and DMSO.

22. The method of claim 19, wherein said at least one cryoprotective agent is present at a concentration less than or equal to 4M.

23. The method of claim 22, wherein said at least one cryoprotective agent is present at a concentration less than or equal to 2M.

27. The method of claim 1, wherein said vitrification solution comprises at least one nanoparticle or microparticle.

28. The method of claim 27, wherein said nanoparticle or microparticle comprises carbon or a noble metal.

29. The method of claim 27, wherein said nanoparticle or microparticle is selected from the group consisting of gold, silver, titanium, palladium, platinum, and copper.

30. The method of claim 1, wherein said vitrification solution comprises polymers or peptides that inhibit ice nucleation in said vitrification solution.

31. The method of claim 30, wherein said polymer or peptide is selected from the group consisting of polyvinyl alcohol, polyglycerol, and antifreeze proteins.

32. The method of claim 1, wherein said cell in said vitrification fluid is emulsified into a droplet of vitrification solution surrounded by an immiscible biocompatible fluid.

33. The method of claim 32, wherein immiscible biocompatible fluid is oil.

34. The method of claim 1, further comprising shaking the housing at a frequency of at least 1.0-100 Hz.

3-4. -4. (canceled)

21. (canceled)

24-26. -26. (canceled)