Cryopreservation and Thawing of Mammalian Cells


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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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. [doi: 10.1002/9780470015902.a0002561.pub2]