Cryopreservation of cell culture

  • Cell culture is not static. Cells in culture acquire changes which can be either genetically programmed (e.g., senescence in primary culture) or due to accumulation of genetic abnormalities (mutations, gain or loss of whole chromosomes or part of chromosomes). In addition to this, changes in gene expression pattern and epigenetic modifications due to several reasons including fluctuations in culture condition, contamination, mishandling and stressful condition 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 which stop or slow down these processes.
  • Cryopreservation is an efficient way to preserve cells at ultra-low temperature (below -135°C) which stop all physiological processes and biological aging. It is a routinely used technique in all cell culture laboratories.
  • During preservation at ultra-low temperature, cells die due to many reason including 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 to damage to cells…………
    • when temperature reduced to above freezing point (hypothermia)
    • when temperature reduced to below freezing point
    • during frozen state
    • during revival
  • Cryopreservation methods ensure that cells are alive at ultra-low temperature and maintain their features when revived after long term frozen state.
  • Most cryopreservation methods rely on
    • cryoprotectants
    • slow cooling
    • rapid revival
  • To cryopreserve cells, cells are suspended in freezing medium, followed by slow cooling and subsequently storage in liquid nitrogen.
  • Freezing medium is nothing but growth medium supplemented with cryoprotectant. Serum containing growth medium contains high amount of serum (upto 90%).
  • Cryoprotectants, the most important component of freezing medium, function by preventing the formation of ice crystals, thus protect cells from lysis.
  • Polyalcohols (e.g., glycerol, ethylene glycol, 2,3 butanediol) and DMSO can be used as cryoprotectants, often a concentration varies from 5 – 20%. Most cryoprotectants have ability to penetrate the cell membrane and function by replacing part of the water in the cell.
  • DMSO is most frequently used cryoprotectant. However, some cells lines are sensitive to DMSO. In such situation, glycerol can be  used. Glycerol is less toxic than DMSO, however, osmotic problem associated with glycerol at the time of thawing restrict its uses.
  • High concentration of serum can also be added in freezing medium. High serum concentration correlate with better survival upon thawing.
  • Serum-free chemically defined freezing medium are also available which are prepared by adding cryoprotectant to serum-free chemically defined medium growth.
  • 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.