Cryopreservation of Cells

• Cell culture is not static. Cells in culture acquire changes that can be either genetically programmed (e.g., senescence in primary culture) or due to the accumulation of genetic abnormalities (mutations, gain or loss of whole chromosomes or part of chromosomes). In addition to this, changes in gene expression patterns and epigenetic modifications due to several reasons including fluctuations in culture condition, contamination, mishandling, and stressful conditions to culture, can also lead to permanent changes in cell behavior (e.g., stem cell culture can differentiate, or lose its ability to differentiate). Therefore, we need a method to preserve cell culture that stops or slows down these processes.
• Cryopreservation is an efficient way to preserve cells at ultra-low temperatures (below -135°C) which stops all physiological processes and biological aging. It is a routinely used technique in all cell culture laboratories.
• During preservation at ultra-low temperatures, cells die due to many reasons including cell lysis due to ice crystal formation, pH change, dehydration, and alterations in the concentration of electrolytes. Four distinct phases of cell preservation and revival process can cause damage to cells…………
  • when the temperature is reduced to freezing point (hypothermia)
  • when the temperature reduced to below the freezing point
  • during frozen state
  • during revival
• Cryopreservation methods ensure that cells are alive at ultra-low temperatures and maintain their features when revived after a long-term frozen state.
• Most cryopreservation methods rely on
  • cryoprotectants
  • slow cooling
  • rapid revival
• To cryopreserve, cells are suspended in a freezing medium, followed by slow cooling and subsequently stored in liquid nitrogen.
• A freezing medium is nothing but a growth medium supplemented with a cryoprotectant. A high amount of serum (up to 90%) can also be present in the freezing medium if the cells are maintained in a serum-supplemented growth medium.
• Cryoprotectants, the most important component of the freezing medium, function by preventing the formation of ice crystals, thus protecting cells from lysis.
• DMSO and polyalcohols (e.g., glycerol, ethylene glycol, 2,3 butanediol) can be used as cryoprotectants, often a concentration varies from 5 – 20%. Most cryoprotectants have the ability to penetrate the cell membrane and function by replacing part of the water in the cell.
• DMSO is the most frequently used cryoprotectant. However, some cell lines are sensitive to DMSO. In such situations, glycerol can be used. Glycerol is less toxic than DMSO, however, osmotic problems associated with glycerol at the time of thawing restrict its uses.
• A high concentration of serum can also be added to the freezing medium. High serum concentration correlates with better survival upon thawing.
• Serum-free chemically defined freezing mediums are also available which can be prepared by adding cryoprotectant to the serum-free chemically defined growth medium.
• Serum-containing freezing mediums are used for cell lines growing in serum-supplemented growth medium whereas serum-free freezing medium is used for those cell lines which are maintained in serum-free chemically defined medium.

RELATED POSTS:

• Protocol – Cryopreservation of Adherent Cell Culture
• Protocol – Cryopreservation of Adherent Cells Growing in Serum-free Medium 
• Protocol – Thawing and Revival of Cryopreserved Cells

FURTHER READING

• Pegg, 2007. Principles of cryopreservation. Methods Mol Biol. 368:39-57. PMID-18080461; Full-Text Link: Springer 
• Fujisawa et al., 2019. Cryopreservation in 95% serum with 5% DMSO maintains colony formation and chondrogenic abilities in human synovial mesenchymal stem cells. BMC Musculoskelet Disord. 2019, 20(1):316. PMID-31279341; Full-Text Links: Biomedcentral, PMC
• Baust et al. 2009. Cryopreservation: An emerging paradigm change. Organogenesis. 5(3), 90-6. PMID-20046670; Full-Text Links: Tandfonline, PMC2781087