Cryopreservation and Thawing of Mammalian Cells

Abstract

Cryopreservation is the process by which intact living cells are preserved at cryogenic temperatures in liquid nitrogen. Freezing cells allow them to be stored, often for years, in a state where their normal metabolic activity is suspended in order to protect them from damage due to chemical reactivity and time. The cells can then be thawed and resuscitated as needed. Cells are cryopreserved in order to guard against genetic drift in continuous cell lines, or against transformation or differentiation in noncontinuous cell lines, such as stem cells and primary cells. In the case of primary cells that are isolated directly from the tissue of interest, they have a finite ability to divide; therefore, cryopreservation is necessary to preserve their unique characteristics for future experiments. Optimal cryopreservation of cells relies on proper freezing and thawing methods. To be protected from structural damage during the freeze‐thaw process, mammalian cells are frozen in the presence of cryoprotectant. Post‐thaw viability assays are then conducted to measure the success of the cryopreservation techniques by calculating the percentage of frozen cells that are alive and able to recover normal function once thawed.

Key Concepts:

  • Cryopreservation is used to enable long‐term storage, preserve current genetic state, prevent transformation or differentiation during subculture and provide a back‐up stock in case of infection/contamination during culture.

  • A controlled rate of freezing and rapid thawing is necessary for optimal cryopreservation and recovery.

  • Care must be taken to minimize transient warming events during transfer and storage, as it impacts viability and recovery.

  • Active thawing results in higher cell viability and recovery than passive thawing.

  • Timing is critical to all stages of the cryopreservation process. All materials should be ready before beginning the procedure, and steps should be taken to ensure each sample is handled with minimal delay.

  • Method standardisation and quality control are necessary to maintain sample integrity and reproducibility.

Keywords: cryopreservation; controlled‐rate freezing, cryoprotectant; viability; freezing; cell thawing

Figure 1.

CoolCell® cell freezing container for twelve 1–2 mL cryovials. Radially symmetric thermal‐exchange design ensures uniform cooling profiles to all vial positions. Composed of high‐density materials used in aerospace industry for lightweight and insulate properties as well as a highly thermo‐conductive alloy core. CoolCell containers offer equal or better results than programmable freezers and alcohol‐based freezing units. © BioCision LLC.

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References

Armitage WJ and Mazur P (1984) Toxic and osmotic effects of glycerol on human granulocytes. American Journal of Physiology 247(5 Pt 1): C382–C389.

Berz D, McCormack EM, Winer ES, Colvin GA and Quesenberry PJ (2007) Cryopreservation of hematopoietic stem cells. American Journal of Hematology 82: 463–472.

Bissoyi A, Nayak B, Pramanik K and Sarangi SK (2014) Targeting cryopreservation‐induced cell death: a review. Biopreservation and Biobanking 12(1): 23–34.

Carrell DT, Wilcox AL and Urry RL (1996) Effect of fluctuations in temperature encountered during handling and shipment of human cryopreserved semen. Andrologia 28(6): 315–319.

Chen X and Thibeault S (2013) Effect of DMSO concentration, cell density, and needle gauge on the viability of cryopreserved cells in three‐dimensional hyaluronan hydrogel. Conference Proceedings IEEE Engineering in Medicine and Biology Society 2013: 6228–6231.

Choi J and Bischof JC (2011) Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells. Cryobiology 63: 285–291.

Chua KJ and Chou SK (2009) On the study of the freeze‐thaw thermal process of a biological system. Applied Thermal Engineering 29: 3696–3709.

Cosentino LM, Corwin W, Baust JM et al. (2007) Preliminary report: evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation. Cell Preservation Technology 5: 189–204.

Cox MA, Kastrup J and Hrubisko M (2012) Historical perspectives and the future of adverse reactions associated with haemopoietic stem cells cryopreserved with dimethyl sulfoxide. Cell Tissue Bank 13(2): 203–215.

Foussat A, Rietze R, Pottier E et al. (2014) Effective cryopreservation and recovery of human regulatory T‐cells. BioProcess International 12(3): 34–38.

Gao D and Critser JK (2000) Mechanisms of cryoinjury in living cells. ILAR Journal 41(4): 187–196.

Grein TA, Freimark D, Weber C et al. (2010) Alternatives to dimethylsulfoxide for serum‐free cryopreservation of human mesenchymal stem cells. International Journal of Artificial Organs 33(6): 370–380.

Gurtovenko AA and Anwar J (2007) Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide. Journal of Chemistry 111: 10453–10460.

Hunt CJ, Armitage SE and Pegg DE (2003) Cryopreservation of umbilical cord blood: 2. Tolerance of CD34+ cells to multimolar dimethyl sulphoxide and the effect of cooling rate on the recovery after freezing and thawing. Cryobiology 46: 76–87.

Klossner DP, Robilotto AT and Clarke DM (2007) Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems. Cryobiology 55: 189–199.

Mazur P, Leibo SP, Farrant J et al. (1970) Interactions of cooling rate, warming rate, and protective additive on the survival of frozen mammalian cells. Ciba Foundation Symposium on the Frozen Cell. London: J&A Churchill.

Morris C, de Wreede L, Scholten M et al. (2014) Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide. Transfusion 54: 2514–2522.

Naaldijk Y, Staude M, Fedorova V and Stolzing A (2012) Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide. BMC Biotechnology 12: 49.

Norkus M, Kilmartin L, Fay D et al. (2013) The effect of temperature elevation on cryopreserved mesenchymal stem cells. Cryoletters 34: 349–359.

Yokoyama WM, Thompson ML and Ehrhardt RO (2012) Cryopreservation and thawing of cells. Current Protocols in Immunology Appendix 3(3G): doi:10.1002/0471142735.ima03gs99.

Further Reading

Baust JM (2005) Advances in media for cryopreservation and hypothermic storage. BioProcess International 4: S46–S54.

Li Y and Ma T (2012) Bioprocessing of cryopreservation for large‐scale banking of human pluripotent stem cells. BioResearch Open Access 5: 205–214.

Li Y and Wang H (2013) Intracellular ice formation (IIF) during freeze–thaw repetitions. International Journal of Heat and Mass Transfer 64: 436–443.

Mullen SF and Critser JK (2007) The science of cryobiology. Cancer Treatment and Research 138: 83–109.

Pegg DE (2007) Principles of cryopreservation. Methods in Molecular Biology 368: 39–57.

Sigma‐Aldrich (2010) Cryopreservation and storage of cells. Fundamental Techniques in Cell Culture Laboratory Handbook‐2nd Edition, vol. 12. St. Louis, MO: Sigma‐Aldrich Corp.

Thirumala S, Goebel WS and Woods EJ (2009) Clinical grade adult stem cell banking. Organogenesis 5(3): 143–154.

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How to Cite close
Thompson, Maria L, Kunkel, Eric J, and Ehrhardt, Rolf O(Dec 2014) Cryopreservation and Thawing of Mammalian Cells. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002561.pub2]