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 (Cryo: icy cold or frost; Preservation: storage) is an effective method for long-term preservation of cell cultures at ultra-low temperatures below -135°C. Preserved cells can be revived whenever needed.
• Cryopreservation completely halts physiological activity and biological aging, preserving cells in a suspended state. Additionally, it safeguards cell cultures from accidental loss due to mishandling, contamination, or equipment failure. As a result, cryopreservation is an essential technique in all cell culture laboratories. 
• Without proper cryopreservation, cells stored at ultra-low temperatures are susceptible to damage and death due to factors such as ice crystal formation, cell lysis, pH fluctuations, dehydration, and electrolyte imbalances. The cryopreservation process mitigates these risks by protecting cells from freezing-induced stress, ensuring their viability and structural integrity during long-term storage.
• 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