Cryopreservation is the process of preserving cells by freezing them at ultra-low temperatures, typically -80°C or in liquid nitrogen (-196°C). This process allows researchers to store and maintain cell lines, such as HEK293, for long periods, ensuring the availability of viable cells for future experiments and reducing the need for continuous passaging. Cryopreservation is especially useful for maintaining early passages of cell lines, which can help minimize the effects of genetic drift and phenotypic changes that may occur over time.
Here is a general protocol for cryopreserving HEK293 cells:
- Harvest the cells: When the HEK293 cells reach around 80-90% confluency, harvest them by detaching the cells from the culture flask using trypsin-EDTA. After incubation, neutralize the trypsin by adding complete growth medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin).
- Centrifuge the cells: Transfer the cell suspension to a sterile centrifuge tube and centrifuge the cells at approximately 200-300 x g for 5 minutes to pellet the cells.
- Prepare freezing medium: Prepare a cryoprotective medium containing 90% complete growth medium (without antibiotics) and 10% dimethyl sulfoxide (DMSO). DMSO is a commonly used cryoprotectant that helps prevent the formation of ice crystals within cells during the freezing process.
- Resuspend the cells: After centrifugation, aspirate the supernatant and gently resuspend the cell pellet in the prepared freezing medium. The cell concentration should typically be between 1-5 million cells/mL, depending on the desired cell density upon thawing.
- Aliquot and freeze the cells: Transfer 1-2 mL of the cell suspension to cryovials, label the vials with relevant information (e.g., cell line, passage number, date), and immediately place the vials in a freezing container. The freezing container should provide a controlled cooling rate of approximately -1°C per minute, which can be achieved using a commercially available freezing container or a homemade device, such as a foam container filled with isopropanol.
- Store the cells: After a minimum of 4 hours in the -80°C freezer, transfer the cryovials to liquid nitrogen for long-term storage. It is recommended to store the cells in the vapor phase rather than directly in liquid nitrogen to prevent potential contamination.
When needed, HEK293 cells can be thawed and cultured according to standard protocols. It is important to monitor the viability and characteristics of the cells after thawing to ensure consistency and reproducibility in experimental results.