Compositions and methods for cryopreservation of cells

Abstract

To provide a composition and a method for cryopreserving a cell.SOLUTION: A composition for cryopreservation contains a sugar component having a total concentration of sugar components in a composition of 300 mM or less; a sugar alcohol component having a total concentration of sugar alcohol components in a composition of 2M or less; and at least one of polymeric component and albumin, with the proviso that the composition for cryopreservation contains less than a cryopreservative amount of dimethyl sulfoxide (DMSO). The method for cryopreserving a cell includes: adding a cell to the composition for cryopreservation; freezing the composition for cryopreservation; preserving the frozen composition for cryopreservation; thawing the composition for cryopreservation; removing the cell from the thawed composition for cryopreservation; and culturing the cell under conditions effective for the cell to remain viable . The freezing can include cooling at the speed of 0.1°C / min. to 5°C / min. The method can be executed without a cleaning step after thawing.SELECTED DRAWING: None

Claims

1. A composition for cryopreservation, wherein the cryopreservation composition is A sugar component, wherein the total concentration of sugar components in the freeze-preservation composition is 300 mM or less, A sugar alcohol component wherein the total concentration of sugar alcohol components in the cryopreservation composition is 1.2 M or less, Polymer components containing poloxamer at concentrations of 1% to 5%, An amino acid component in a concentration of 0.71 mM to 50 mM, comprising glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, Arbitrarily, albumin at concentrations of 0.5% to 10%, A cryopreservation composition comprising, however, less than 140 mM of dimethyl sulfoxide (DMSO) in the cryopreservation composition.

2. The cryopreservation composition according to claim 1, wherein the sugar component is provided at a concentration of 1 mM to 250 mM, 10 mM to 200 mM, 20 mM to 120 mM, 25 mM to 100 mM, or 30 mM to 80 mM.

3. The cryopreservation composition according to claim 1 or 2, wherein the sugar component comprises at least one sugar selected from the group consisting of trehalose, maltose, lactose, fructose, sucrose, glucose, dextran, melegitose, raffinose, nigerotriose, maltotriose, maltotriulose, kestose, cellobiose, chitobiose, and lactulose.

4. The cryopreservation composition according to claim 3, wherein the sugar component comprises at least one sugar selected from the group consisting of trehalose, maltose, and lactose.

5. The cryopreservation composition according to claim 4, wherein the sugar component comprises trehalose.

6. The cryopreservation composition according to any one of claims 1 to 5, wherein the sugar component comprises trehalose, maltose, lactose, or a combination thereof in an amount of 10 mM to 200 mM, 20 mM to 120 mM, or 30 mM to 80 mM.

7. The cryopreservation composition according to claim 6, wherein the sugar component comprises trehalose in an amount of 10 mM to 200 mM, 20 mM to 120 mM, or 30 mM to 80 mM.

8. The cryopreservation composition according to claim 7, comprising 30 mM to 80 mM trehalose.

9. The cryopreservation composition according to any one of claims 1 to 8, wherein the sugar alcohol component is provided at a concentration of 0.2 M to 1.2 M, 0.2 M to 1 M, or 0.3 M to 0.8 M.

10. The cryopreservation composition according to any one of claims 1 to 9, wherein the sugar alcohol component comprises at least one sugar alcohol selected from the group consisting of glycerol, sorbitol, ethylene glycol, propylene glycol, inositol, xylitol, mannitol, arabitol, ribitol, erythritol, sreitol, galactitol, and pinitol.

11. The cryopreservation composition according to claim 10, wherein the sugar alcohol component comprises at least one sugar alcohol selected from the group consisting of glycerol, sorbitol, ethylene glycol, propylene glycol, inositol, xylitol, and mannitol.

12. The cryopreservation composition according to claim 10, wherein the sugar alcohol component comprises glycerol.

13. A cryopreservation composition according to any one of claims 1 to 12, comprising 0.4 M to 1 M glycerol.

14. A cryopreservation composition according to any one of claims 1 to 13, comprising albumin at a concentration of 0.5% to 8%.

15. The cryopreservation composition according to claim 14, wherein the concentration of albumin is 1% to 5%.

16. The solution further contains ionic components at concentrations of at least 0.05% (w / v), at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, or at least 0.7%, and 2.5% (w / v) or less, 2% or less, 1.5% or less, 1.3% or less, 1.2% or less, 1.1% or less, or 1.0% or less, wherein the ionic components include salts, acids, bases, or combinations thereof, and optionally, the salt is CaCl 2 MgCl 2 MgSO 4 , KCl, KH 2 PO 4 NaHCO 3 , NaCl, and Na 2 HPO 4 A cryopreservation composition according to any one of claims 1 to 15, selected from the above.

17. The cryopreservation composition according to claim 16, wherein the concentration of the ionic component is 0.2% to 2%.

18. The cryopreservation composition according to claim 17, wherein the concentration of the ionic component is 0.3% to 1.6%.

19. The cryopreservation composition according to any one of claims 1 to 18, wherein the amino acid component further comprises at least one of isoleucine and creatine.

20. A cryopreservation composition according to any one of claims 1 to 19, further comprising a second amino acid component comprising one or more amino acids, amino acid derivatives, peptides, or combinations thereof.

21. The cryopreservation composition according to claim 20, wherein the second amino acid component comprises one or more of valine, histidine, cysteine, tryptophan, tyrosine, arginine, glutamine, taurine, betaine, ectoin, dimethylglycine, ethylmethylglycine, RGD peptide, or a combination thereof.

22. A cryopreservation composition according to any one of claims 1 to 21, further comprising cells.

23. The cryopreservation composition according to claim 22, wherein the cells are iPS cells, embryonic stem cells, cardiac progenitor cells, cardiomyocytes, neural progenitor cells, neurons, glial cells, beta cells, endothelial cells, epithelial cells, smooth muscle cells, tendinocytes, osteocytes, chondrocytes, adipocytes, corneal cells, retinal cells, trabecular reticular cells, intestinal cells, renal cells, hematopoietic cells, gametes, or a combination thereof.

24. The cryopreservation composition according to claim 23, wherein the cells are iPS cells, embryonic stem cells, cardiac progenitor cells, cardiomyocytes, neural progenitor cells, neurons, glial cells, epithelial cells, endothelial cells, retinal cells, or a combination thereof.

25. The cryopreservation composition according to claim 24, wherein the cells are iPS cells.

26. The cryopreservation composition according to any one of claims 22 to 25, wherein the cells are viable, recovered, cryopreserved cells.

27. A method for cryopreserving cells, (a) Adding cells to the cryopreservation composition according to any one of claims 1 to 21, (b) Initiating the crystallization of molecular components in the cryopreservation composition at the ice nucleation temperature, and (c) Cooling the cryopreservation composition at a certain rate. A method that includes this.

28. The method according to claim 27, wherein the ice nucleation temperature is 0°C to -3°C, -1°C to -20°C, -1°C to -12°C, -12°C to -20°C, -6°C to -12°C, -1°C to -8°C, -1.5°C to -7°C, -2°C to -6°C, -3°C to -6°C, or -3°C to -5°C.

29. The method according to claim 28, wherein the ice nucleus formation temperature is -4°C.

30. The method according to any one of claims 27 to 29, wherein the cooling rate of the cryopreservation composition is 0.1°C / min to 5°C / min, 0.3°C / min to 3°C / min, or 0.8°C / min to 1.2°C / min.

31. The method according to any one of claims 27 to 30, comprising thawing and using the cryopreservation composition without a washing step.

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