Cryopreservation and cryopreservatents of animal cell culture
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Cryopreservation of Cell Lines
OPS Diagnostics has several products used for cryogenically storing cell lines, such as Cryogenic Vials and Cryogenic Storage Boxes. Please visit our website to see these and other cryogenic products.
Introduction
The preservation of cells is an extremely important aspect of cell culture. The only effective means of preservation of animal cells is by freezing, which can be accomplished with either liquid nitrogen or by employing cryogenic freezers. The freezing process involves slowly reducing the temperature of prepared cells to -30 to -60°C followed by a transfer to temperatures less than -130°C. Once at ultralow temperatures, the cells are biologically inert and can be preserved for years.
Preparation
Cryopreserving cultured cells differs from preserving bacteria and fungi in that higher viability is required. Where a 1% survival rate of a microbial culture can be practical, such low viability is unacceptable with cultured cells. High survival rates are clearly important for cell lines due to the expense and difficulty in preparation , slow relative rate of growth, and tendency to change with repeated passage in culture. Consequently, methods used for cell culture cryopreservation must ensure high viability (i.e., >90%).
Factors that can affect the viability of cryopreserved cells include growth conditions prior to harvesting, the physiological state of the cells, the cell density, choice of cyroprotectant, and handling techniques. Actively growing cells harvested from late-logarithmic to early-stationary phase cells usually yield the highest number of viable cells following freezing. Once harvested, the desirable final concentration of cells should be between 106 to 107 cells/ml. Higher densities are often useful with adherent cells since thawed cells can be diluted and plated at a desired density. Cryoprotectants such as DMSO and glycerol are valuable to prevent cell dehydration during the freezing process. The cell suspension is generally prepared at a concentration twice that desired for freezing so that an equal volume of 2X cyroprotectant can be added. Gentle handling techniques during harvest and concentration will improve viability of the recovered cells. Excessive enzymatic treatment, vigorous pipetting, and high-speed centrifugation should be avoided.
Cryoprotectants
The diffusion of cryoprotective agents such as glycerol or dimethylsulfoxide (DMSO) into a cell will result in a partial replacement of intracellular water and help to prevent dehydration (from ice formation) during freezing. Glycerol is also known to stabilize proteins in their native states and to assist in the maintenance of critical macromolecular interactions at subzero temperatures. The cryoprotectant should be prepared separately by combining the cryoprotective agent and the growth medium for the cells. Cryoprotective agents are usually used individually in concentrations ranging from 5-15% (v/v) with the optimum varying with the cell type. It is important that the cryoprotective agents be of highest possible quality and sterilized prior to use. Glycerol may be sterilized by autoclaving for 15 minutes and should be stored in small aliquots to prevent introduction of contaminants. DMSO should be sterilized by filtration with a 0.2 µm nylon syringe filter and stored at -20°C in small, single-use sealed aliquots. Air oxidation of DMSO is relatively rapid and these products are toxic to cells. DMSO should not be allowed to come into contact with the skin as it is rapidly absorbed and is a reported neurotoxin. Preformulated cryoprotective media can also be purchased.
Equilibration
Cells mixed with the cryoprotectant require an equilibration time at room temperature prior to the onset of the cooling process. This time generally ranges from 15 to 45 minutes and allows penetration of the cell by the cryoprotectant for maximum protective effect.
OPS Diagnostics has several products used for cryogenically storing cell lines, such as Cryogenic Vials and Cryogenic Storage Boxes. Please visit our website to see these and other cryogenic products.
Introduction
The preservation of cells is an extremely important aspect of cell culture. The only effective means of preservation of animal cells is by freezing, which can be accomplished with either liquid nitrogen or by employing cryogenic freezers. The freezing process involves slowly reducing the temperature of prepared cells to -30 to -60°C followed by a transfer to temperatures less than -130°C. Once at ultralow temperatures, the cells are biologically inert and can be preserved for years.
Preparation
Cryopreserving cultured cells differs from preserving bacteria and fungi in that higher viability is required. Where a 1% survival rate of a microbial culture can be practical, such low viability is unacceptable with cultured cells. High survival rates are clearly important for cell lines due to the expense and difficulty in preparation , slow relative rate of growth, and tendency to change with repeated passage in culture. Consequently, methods used for cell culture cryopreservation must ensure high viability (i.e., >90%).
Factors that can affect the viability of cryopreserved cells include growth conditions prior to harvesting, the physiological state of the cells, the cell density, choice of cyroprotectant, and handling techniques. Actively growing cells harvested from late-logarithmic to early-stationary phase cells usually yield the highest number of viable cells following freezing. Once harvested, the desirable final concentration of cells should be between 106 to 107 cells/ml. Higher densities are often useful with adherent cells since thawed cells can be diluted and plated at a desired density. Cryoprotectants such as DMSO and glycerol are valuable to prevent cell dehydration during the freezing process. The cell suspension is generally prepared at a concentration twice that desired for freezing so that an equal volume of 2X cyroprotectant can be added. Gentle handling techniques during harvest and concentration will improve viability of the recovered cells. Excessive enzymatic treatment, vigorous pipetting, and high-speed centrifugation should be avoided.
Cryoprotectants
The diffusion of cryoprotective agents such as glycerol or dimethylsulfoxide (DMSO) into a cell will result in a partial replacement of intracellular water and help to prevent dehydration (from ice formation) during freezing. Glycerol is also known to stabilize proteins in their native states and to assist in the maintenance of critical macromolecular interactions at subzero temperatures. The cryoprotectant should be prepared separately by combining the cryoprotective agent and the growth medium for the cells. Cryoprotective agents are usually used individually in concentrations ranging from 5-15% (v/v) with the optimum varying with the cell type. It is important that the cryoprotective agents be of highest possible quality and sterilized prior to use. Glycerol may be sterilized by autoclaving for 15 minutes and should be stored in small aliquots to prevent introduction of contaminants. DMSO should be sterilized by filtration with a 0.2 µm nylon syringe filter and stored at -20°C in small, single-use sealed aliquots. Air oxidation of DMSO is relatively rapid and these products are toxic to cells. DMSO should not be allowed to come into contact with the skin as it is rapidly absorbed and is a reported neurotoxin. Preformulated cryoprotective media can also be purchased.
Equilibration
Cells mixed with the cryoprotectant require an equilibration time at room temperature prior to the onset of the cooling process. This time generally ranges from 15 to 45 minutes and allows penetration of the cell by the cryoprotectant for maximum protective effect.
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