Cryopreservation of Cells

• Cell culture is not static; cells undergo changes that can be genetically programmed, such as senescence in primary cultures, or result from genetic abnormalities like mutations or chromosomal alterations. Additionally, fluctuations in culture conditions, contamination, mishandling, and stress can lead to changes in gene expression and epigenetic modifications, affecting cell behavior—such as stem cells losing their differentiation potential. Therefore, it is essential to implement preservation methods that halt or slow these processes. (read more: Dynamic Nature of Cells in Culture)
• Cryopreservation is an effective method for preserving cells at ultra-low temperatures (below -135°C). This process halts all physiological activity and biological aging. As a result, cryopreservation is a standard technique in all cell culture labs. 
• During preservation at ultra-low temperatures, cells can die for several reasons, including cell lysis due to ice crystal formation, pH changes, dehydration, and alterations in electrolyte concentration. The cell preservation and revival process involves four distinct phases that can affect cells in various ways, inducing damage through different mechanisms:
  • Hypothermia: When the temperature is reduced to the freezing point. (read more: Impact of Hypothermia on Cell Preservation)
    
  • Below Freezing: When the temperature drops below the freezing point.
  • Frozen State: While the cells remain frozen.
  • Revival: During the process of reviving the cells.
• 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 storage in liquid nitrogen.
• The freezing medium typically consists of a growth medium supplemented with a cryoprotectant. If the cells are maintained in a serum-supplemented growth medium, the freezing medium can contain serum concentrations between 10% and 20%, but it can be increased upto 90%. A high concentration of serum (upto 90%) can enhance cell survival upon thawing.
  
• Cryoprotectants are the most critical components of the freezing medium, as they prevent the formation of ice crystals, thereby protecting cells from lysis.
• Common cryoprotectants include DMSO and polyalcohols such as glycerol, ethylene glycol, and 2,3-butanediol, typically used at concentrations ranging from 5% to 20%. These cryoprotectants penetrate the cell membrane and partially replace the water inside the cells.
• DMSO is the most frequently used cryoprotectant; however, some cell lines are sensitive to it. In such cases, glycerol can be used as an alternative. Glycerol is less toxic than DMSO, but osmotic issues during thawing can limit its application.
• 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 suitable for cell lines grown in serum-supplemented environments, while serum-free freezing mediums are appropriate for those maintained in serum-free, chemically defined conditions.
  

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