Licia Miller Product Manager
Cryopreservation is a method of preserving cells at low temperatures in order to maintain cell activity and function for a long period of time. This method is of great significance for the long-term preservation of cell lines, experimental repeatability, the establishment of gene banks, and the storage of cell therapy. Cryopreservation can reduce genetic variation in cells during passage and avoid cell loss due to contamination, environmental changes, or experimental errors. The best time for cryopreservation is when the cells are at their optimal growth rate.
Cryoprotectants are key components in cell cryopreservation. Commonly used cryoprotectants include dimethyl sulfoxide (DMSO) and glycerol. The main function of these cryoprotectants is to lower the freezing point of the solution and reduce the formation of ice crystals, thereby protecting cells from mechanical damage by ice crystals. DMSO is a commonly used cryoprotectant that is suitable for most cell types, but some cells are sensitive to DMSO, in which case glycerol can be used as a substitute.
Phase 1 Cryopreservation
Experimental Steps
1. Label the cryovials with the date, researcher’s name, cell number, passage number, and cell type (as well as any other useful information, such as genetic modification).
2. If the cells are adherent, remove the cell culture medium first, wash with PBS, add an appropriate amount of trypsin to cover the cells, and incubate in a 37°C incubator for about 2 minutes (the specific incubation time should be determined according to the actual cell type being cultured) .
Add an equal amount of culture medium to terminate the digestion, gently pipette the cells to wash off the adherent cells, and transfer them to a 50 mL Falcon tube.
3. For suspension cultured cells, transfer the required volume of cells directly into a 50 mL Falcon tube.
4. Count the cells using a hemocytometer to determine their viability. The viability of the cryopreserved cells should be at least 75%.
5. At room temperature, centrifuge at 300 × g for 5 min.
6. Prepare freezing medium (see Table 1) .
Table 1. Cryopreservation medium formulas for different mammalian cell lines :
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Mix well and warm to 37°C before use.
Freezing media contains essential cryoprotectants, such as dimethyl sulfoxide (DMSO) , to prevent the formation of intracellular and extracellular ice crystals that could damage cell membranes and components.
Note that DMSO is not suitable for all cell types and in some cases glycerol can be used as an alternative.
7. Remove the supernatant.
8. Add freezing medium to the desired cell density.
For mammalian cells, this concentration is usually 1×106/mL cryopreservation medium. Cells should not be left in cryopreservation medium at room temperature for more than 10 minutes.
9. Aliquot 1 mL of sample into cryovials and securely cap.
10. Transfer the cryovials to a freezer box at room temperature and place in a -80°C freezer. Add an appropriate amount of isopropyl alcohol to the periphery of the freezer box to ensure that the temperature drops steadily at a rate of 1°C per minute.
A slow freezing process (-1ºC per minute) helps prevent the formation of ice crystals inside the cells, since more water will be expelled before freezing.
11. After approximately 24 hours, remove the cryovials from the cryobox and transfer to liquid nitrogen for long-term storage.
Generally, frozen cells can be stored in liquid nitrogen for several years. Ideally, frozen cells should not be kept at -80ºC for a long time (up to a week) and should be transferred to liquid nitrogen as quickly as possible. Long-term storage of cells at -80ºC may compromise cell viability.
Phase 2 Thawing frozen cell lines
Experimental Steps
1. Remove the cryovials from the liquid nitrogen storage. Quickly place in a 37°C water bath until approximately 80% thawed (do not thaw at room temperature). This process should not take more than 1 minute.
Because a slow thawing process can damage cells, cells should generally be thawed as quickly as possible.
At the same time, the cryoprotectant DMSO has certain cytotoxicity, so cells should be thawed faster than at room temperature to quickly dilute and remove DMSO.
Another purpose of thawing cells quickly is to prevent any crystals that form during the freezing process from damaging the cell membranes or components.
2. Use a pipette to transfer the contents of the cryovial to a 15 mL Falcon tube containing approximately 10 mL of pre-warmed culture medium.
3. Centrifuge at 300 × g for 5 min under 4°C, discard the supernatant, and resuspend the cell pellet in an appropriate amount of culture medium.
4. Transfer the cell suspension to a culture container and then transfer to a 37°C incubator for culture.
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