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TOPIC: Cryo Vial Cell Revival: 5 Step Guide to Revive Cells

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Date: October 10th

Cryo Vial Cell Revival: 5 Step Guide to Revive Cells	Permalink

Cryo vial of cryo storage is important in cell revival procedure. Cell revival refers to the process of thawing cells stored in liquid nitrogen or a -70°C freezer and re-culturing them to allow the cells to resume growth. When returned to normal temperatures, the cell’s structure remains intact, and its biochemical reactions immediately resume. The warming process should be fast, following the principle of slow freezing and quick thawing, to prevent water from entering the cells during thawing and forming ice crystals, which could affect cell viability. Steps for Cell Revival 1. Remove the cryo vial from the liquid nitrogen tank and place it in a -80°C freezer for a few minutes before quickly immersing it in a 37°C water bath. Shake occasionally, ensuring the vial thaws within 2 minutes. Be careful not to submerge the vial cap in the water to avoid contamination. During the revival process, the typical cell death rate is around 25%. 2. After complete thawing, wipe the cryovial with 75% alcohol to sterilize it, then open the vial and transfer the cell suspension into a T25 or T75 culture flask with culture medium. Incubate at 37°C with 5% CO2. If the cryoprotectant needs to be removed, transfer the suspension into a sterile centrifuge tube, add 5 mL of culture medium, and centrifuge at an appropriate speed based on the cell type. Discard the supernatant, resuspend the cells in pre-warmed medium, and ensure they are fully suspended. 3. Dilute the suspension as needed, then seed the resuspended cells into culture dishes or flasks. After 24 hours, replace the medium to remove dead cells, and change the medium every three days. Once the cells reach 80-90% confluence, perform sub-culturing. Precautions for Cell Revival 1. Always use protective gear when handling frozen cells, including cryogenic gloves and goggles, as improperly sealed cryovials can absorb liquid nitrogen and may explode upon thawing. 2. While thawing the cells in the water bath, use tweezers to hold the cryovial and shake it occasionally for even heating. The faster the thawing, the better the cell viability. 3. Disinfect the cryovial with an alcohol swab before opening it. Ensure aseptic conditions throughout the procedure, and keep the cells on ice during post-thaw handling to minimize the toxicity of the cryoprotectant. 4. Complete the thawing process within 2 minutes and avoid excessive pipetting or bubble formation, which can harm cell growth post-revival. 5. Prevent contamination by ensuring the cryovial cap stays dry, as contact with water may introduce mycoplasma. Regularly fumigate the lab with hydrogen peroxide, and use mycoplasma detection kits when necessary. __________________

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