CRYOPRESERVATION MEDIUM AND METHOD TO PREVENT RECRYSTALLIZATION

Abstract

The present invention is directed to a medium for preserving cells at non-cryogenic freezing temperatures. The medium comprises a hydrophilic and nontoxic polymer or other macromolecule, an aqueous liquid, and a cryoprotectant. The molecules of the macromolecule form compact three-dimensional structures that are spherical in shape when dissolved in the aqueous liquid. The medium of the present invention can be used for long-term storage of cells at non-cryogenic temperatures with outcomes similar to those seen with storage at cryogenic temperatures.

Claims

1. A medium for preserving cells at a non-cryogenic freezing temperature comprising: a hydrophilic and nontoxic macromolecule; an aqueous liquid; and a cryoprotectant; wherein the macromolecule is at a concentration in the medium equal to or greater than about 20% (w/v), and wherein molecules of the macromolecule form compact three-dimensional structures that are spherical in shape when dissolved in the aqueous liquid; wherein when the medium is in use and is at the non-cryogenic freezing temperature, the compact and spherical structures are concentrated in an unfrozen portion of the medium with the cells being preserved; and wherein a crowding effect prevents ice recrystallization during storage at the non-cryogenic temperatures.

2. (canceled)

3. (canceled)

4. The medium of claim 1, wherein the concentration of the macromolecule in the medium is about 25% (w/v) or greater.

5. The medium of claim 4, wherein the concentration of the macromolecule in the medium is about 35% (w/v) or greater.

6. The medium of claim 5, wherein the concentration of the macromolecule in the medium is about 50% (w/v) or greater.

7. The medium of claim 1, wherein the cryoprotectant is at a concentration equal to or greater than about 20% of the concentration of the macromolecule in the medium.

8. The medium of claim 7, wherein the concentration of the cryoprotectant in the medium is equal to or greater than about 75% of the concentration of the macromolecule in the medium.

9. The medium of claim 8, wherein the concentration of the cryoprotectant in the medium is equal to or greater than about 100% of the concentration of the macromolecule in the medium.

10. The medium of claim 1, wherein the macromolecule is a polymer.

11. The medium of claim 10, wherein the polymer comprises molecules that form the compact three-dimensional structures that are approximately spherical in shape when dissolved in the aqueous liquid.

12. The medium of claim 11, wherein the polymer is selected from the group consisting of spherical hydrophilic polysaccharides, polymerized cyclodextrin or saccharides, globular proteins or spheroproteins, spherical glycoproteins formed by attaching oligosaccharide chains to those globular proteins, other derivatives of those globular proteins and combinations thereof.

13. The medium of claim 12, wherein the polymer is a hydrophilic polysaccharide.

14. The medium of claim 13, wherein the polymer is a polymer formed by the copolymerization of sucrose and epichlorohydrin.

15. (canceled)

16. (canceled)

17. (canceled)

18. The medium of claim 1, wherein the cells are eukaryotic cells.

19. The medium of claim 18, wherein the suspended cells are mammalian cells and the mammalian cells are selected from the group consisting of murine cells, porcine cells, human cells, and combinations thereof.

20. The medium of claim 18, wherein the mammalian cells are selected from the group consisting of stem cells, somatic cells, reproduction cells and combinations thereof.

21. The medium of claim 1, wherein the cells are prokaryotic cells.

22. The medium of claim 1, wherein the compact approximately spherical structures are about 100 nm (nanometer) or less in their widest dimension.

23. The medium of claim 17, wherein the compact approximately spherical structures comprise structures ranging from about 1 to 50 nm in their widest dimension.

24. (canceled)

25. The medium of claim 1, wherein the medium is substantially free of serum, animal proteins or human proteins.

26. A method for preserving cells at a non-cryogenic freezing temperature comprising: providing a cryopreservation medium comprising a hydrophilic and nontoxic macromolecule, a cryoprotectant, and an aqueous liquid, wherein the macromolecule is at a concentration in the medium greater than about 10% (w/v), and wherein the macromolecule forms a highly compact approximately spherical structure when dissolved in the aqueous liquid; using the medium to suspend the cells to form a medium-cellular suspension; cooling the medium-cellular suspension to the non-cryogenic freezing temperature, wherein the non-cryogenic freezing temperature is about 85 C. or higher; and maintaining the medium-cellular suspension at or near the non-cryogenic freezing temperature or a different non-cryogenic freezing temperature, without losing water due to sublimation or without any need for lyophilization or freeze drying processing, for a time period longer than three weeks while maintaining post-thaw cell survival rates of the cells equal to or about the same as would be obtained for storage of the cells in liquid nitrogen for the period of time.

27.-44. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] FIG. 1A. depicts the molecular dynamic demonstration of the macromolecular crowding behavior of a Ficoll-DMSO-water system at 80 C., and their interaction with a growing ice nucleus.

[0094] FIG. 1B depicts the molecular dynamic demonstration of an evenly distributed Sucrose-DMSO-water system at 80 C., and their interaction with a growing ice nucleus.

[0095] FIG. 1C depicts the molecular dynamic demonstration of an evenly distributed DMSO-water system at 80 C., and their interaction with a growing ice nucleus.

[0096] FIG. 2 depicts the molecular dynamic simulation results the values of the root-mean-square (RMS) distance of water atomic positions of three systems with the growing ice nucleus shown in FIG. 1. The top curve for DMSO-water; middle curve for sucrose-DMSO-water; and bottom curve for Ficoll-DMSO-water.

[0097] FIG. 3A depicts the SEM observation of fractured samples of the normal frozen cryopreservation solution.

[0098] FIG. 3B depicts the SEM observation of fractured samples of a medium of the present invention.

[0099] FIG. 4 depicts the optical observation of samples of (A) a frozen normal cryopreservation solution and (B) a medium of the present invention after 5 week storage at 80 C.

[0100] FIG. 5 depicts an assessment of cell recovery of nave type O2K porcine iPSC cryopreserved with different post-mixture concentrations in the medium-cellular suspension (i.e. the concentration values are calculated after mixing embodiments of the media of the present invention with cell suspensions) of Ficoll 70 in (A) FBS based or (B) serum-free DMEM/F12 based media.

[0101] FIG. 6 depicts post-thaw recovery of colonies from the (A) nave type O2K porcine iPSC, (B) epiblast type ID6 porcine iPSC, (C) epiblast type human iPSC and (D) epiblast type H1 hESC, over extended storage periods. For each storage period: Left bars: cells stored in liquid nitrogen. Center bars: cells stored in 80 C. freezer without using the medium of the present invention. Right bars: cells stored in 80 C. freezers using the medium of the present invention.

[0102] FIG. 7 depicts dissociation of epiblast type stem cells by different methods.

[0103] FIG. 8 depicts a comparison of efficacy of four different cryopreservation protocols performed on H1 hESC after single cell dissociation by trypsin with the aid of ROCKi. The left bars are cells stored in liquid nitrogen. For each storage period: The center left bars are for cryopreservation using the medium of the present invention in liquid nitrogen, showing that it is suitable for both 80 C. and liquid nitrogen storage or at any temperature in-between. The right center bars are cells stored in 80 C. freezer without using the medium of the present invention. The right bars are cells stored in 80 C. freezers using the medium of the present invention.

[0104] FIG. 9 depicts the expression of biomarkers characteristic of pluripotency of all above four stem cell types (in the same order as in FIG. 6) after recovery from cryopreservation using the medium of the present invention at 80 C.

[0105] FIG. 10 depicts the morphologies of ID6 porcine iPSC colonies, which were broken into large clumps (100 cells), following 2 weeks of cryopreservation.

REFERENCES

[0106] Mazur, P. Cryobiology: the freezing of biological systems. Science 168, 939-949 (1970). [0107] Mazur, P. Freezing of living cells: mechanisms and implications. Am J Physiol. 247, 125-142 (1984). [0108] Valeri, C. R. & Pivacek, L. E. Effects of the temperature, the duration of frozen storage, and the freezing container on in vitro measurements in human peripheral blood mononuclear cells. Transfusion 36, 303-308 (1996). [0109] Cohen, R. I., Thompson, M. L., Schryver, B., Ehrhardt, R. O. Standardized cryopreservation of pluripotent stem cells. Curr Protoc Stem Cell Biol. Unit 1C.14 (2014). [0110] Massie, I., Selden, C., Hodgson, H., Fuller, B. Storage temperatures for cold-chain delivery in cell therapy: a study of alginate-encapsulated liver cell spheroids stored at 80 C. or 170 C. for up to 1 year. Tissue Eng Part C Methods. 19:189-95 (2013) [0111] Fahy, G. M. & Wowk, B. Principles of cryopreservation by vitrification. Methods Mol Biol. 1257, 21-82 (2015). [0112] Jin, B., Mochida, K., Ogura, A., Koshimoto, C., Matsukawa, K., Kasai, M., Edashige, K. Equilibrium vitrification of mouse embryos at various developmental stages. Mol Reprod 79, 785-94 (2012). [0113] Baudot, A. & Odagescu, V. Thermal properties of ethylene glycol aqueous solutions. Cryobiology 48, 283-94 (2004). [0114] Forsyth, M. & MacFarlane, D. R. Recrystallization revisited. Cryo-Letters 7, 367-378 (1986). [0115] Gao, D. & Critser, J. K. Mechanisms of cryoinjury in living cells. ILAR J 41, 187-196 (2000). [0116] Venketesh, S. & Dayananda, C. Properties, potentials, and prospects of antifreeze proteins. Crit Rev Biotechnol. 28, 57-82 (2008). [0117] Chantelle, J. et al. Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations. Sci Rep 5, 9692 (2005). [0118] Dauty, E., & Verkman, A. S. Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy. J. Mol. Recognit 17, 441-447 (2004). [0119] Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. 26, 597-604 (2001). [0120] Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental biology 227, 271-278 (2000). [0121] Ezashi, T., Das, P. & Roberts, R. M. Low O2 tensions and the prevention of differentiation of hES cells. Proceedings of the National Academy of Sciences of the United States of America 102, 4783-4788 (2005). [0122] Roberts, R. M., Yuan, Y., Genovese, N. & Ezashi, T. Livestock Models for Exploiting the Promise of Pluripotent Stem Cells. ILAR Journal 00 (2015). [0123] Ezashi, T., Yuan, Y. & Roberts, R. M. Pluripotent Stem Cells from Domesticated Mammals. Annual review of animal biosciences (2015). [0124] Telugu, B. P. et al. Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos. The Journal of biological chemistry 286, 28948-28953 (2011) [0125] Lee, J. E. & Lee, D. R. Human Embryonic Stem Cells: Derivation, Maintenance and Cryopreservation. International journal of stem cells 4, 9-17 (2011). [0126] Fujioka, T., Yasuchika, K., Nakamura, Y., Nakatsuji, N. & Suemori, H. A simple and efficient cryopreservation method for primate embryonic stem cells. The International journal of developmental biology 48, 1149-1154 (2004). [0127] Reubinoff, B. E., Pera, M. F., Vajta, G. & Trounson, A. O. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Human reproduction 16, 2187-2194 (2001). [0128] Richards, M., Fong, C. Y., Tan, S., Chan, W. K. & Bongso, A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem cells 22, 779-789 (2004). [0129] Ezashi, T. et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America 106, 10993-10998 (2009). [0130] McElroy, S. L. & Reijo Pera, R. A. Culturing human embryonic stem cells in feeder-free conditions. CSH protocols 2008, pdb prot5044 (2008). [0131] Martin-Ibanez, R. et al. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Human reproduction 23, 2744-2754 (2008). [0132] Kurosawa, H. Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells. Journal of bioscience and bioengineering 114, 577-581 (2012). [0133] Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature biotechnology 25, 681-686 (2007). [0134] Baharvand, H., Salekdeh, G. H., Taei, A. & Mollamohammadi, S. An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells. Nature protocols 5, 588-594 (2010). [0135] Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nature methods 8, 409-412 (2011). [0136] Lee, K. et al. Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proceedings of the National Academy of Sciences of the United States of America 111, 7260-7265 (2014). [0137] Girish, V. & Vijayalakshmi, A. Affordable image analysis using NIH Image/ImageJ. Indian journal of cancer 41, 47 (2004). [0138] Johnson, L. A., Weitze, K. F., Fiser, P., Maxwell, W. M. C., Storage of Boar Semen, Animal Reproduction Science 62, 143-172 (2000). [0139] Yang, Jun, et al. The effects of storage temperature on PBMC gene expression, BMC Immunology 17, 6-21 (2016). [0140] Cody, William L., et al. Skim Milk Enhances the Preservation of Thawed 80 C. Bacterial Stocks, J Microbiol Methods 75, 135-138 (2008).