About Thawing of Frozen Cell

Cells that are cryopreserved can be used again whenever required.

The procedure of bringing frozen cells to room temperature from a metabolically inactive state to an active state without causing much damage is called as thawing.

Principle of Thawing or Cell Revival

When cryopreserved cells are needed for study, they should be thawed rapidly and plated at high density to optimize recovery.

The ampoule should be thawed as rapidly as possible, to minimize intracellular ice crystal growth during the warming process.

This can be done in warm water, in a bucket or water bath.

The cell suspension should be diluted slowly after thawing as rapid dilution reduces viability.

Some mammalian cells (often suspension-growing cells) are more sensitive to cryoprotectants, particularly DMSO, and must be centrifuged after thawing but still need to be diluted slowly in medium first.

Thawing Requirements

Sterile:

1. Culture flask

2. Centrifuge tube (if centrifugation is required)

3. Growth medium

4. Pipettes, 1 ml, 10 ml

Non-sterile:

5. Protective gloves and face mask

6. Sterile water at 37°C, 10 cm deep in a clean, alcohol-swabbed bucket with lid

7. 70% IPA, cotton swabs.

Thawing Procedure

1. Bring the cryovial out of freezer in ice. Thaw.

2. Incubate in water bath at 37o C for 1-2 minutes.

3. Double-check the label to confirm the identity of the cells; then swab the vial thoroughly with 70% alcohol, and open it in a laminar-flow hood.

4. Transfer the contents of the vial to a 2ml microfuge tube and seal with parafilm.

5. Centrifuge at 1300 rpm for 10 minutes at room temperature.

6. Resuspend the pellet in 2 ml medium.

7. Recentrifuge at 1300 rpm for 10 minutes at room temperature to remove traces of DMSO. Pellet should be resuspended in 1 ml of medium.

8. Transfer the suspension to 25 cm2 flask. Add medium slowly to the cell suspension (5 ml). For cells in suspension culture also require centrifugation to remove the cryoprotectant.

9. Incubate cells in a humidified incubator with 5% CO2, at 37oC.

10. Check after 24 h.

Post Thawing Observations

For attached monolayer cells, confirm attachment and try to estimate percentage survival based on photographs of cells at the expected density (cells/cm2 ) with full survival.

For cells growing in suspension culture, check appearance (clear cytoplasm, lack of granularity and dilute to regular seeding concentration.

This can be made more precise if the cells are counted and an estimate of viability is made, in which case the cells can be diluted to the regular seeding concentration of viable cells.

Thawing Precaution

Dilution should be slow in case of DMSO because sudden dilutions can cause severe osmotic change and reduce the viability.

On removal from storage, extreme caution must be exercised to prevent explosion of the cryo vial because of sudden expansion of the trapped nitrogen.

To retain maximum viability during cryopreservation, cells must be cooled at a constant slow rate, -1 to -5 C/min.

To minimize contamination risk, all components should be pretested.

Resuspending cells for freezing in precooled freeze medium, 0-4 C, may improve their survival prior to freezing.

When this approach is used, it is important to maintain the cells at a constant temperature during all subsequent handling by placing ampoules in ice until they are frozen.

After thawing cells it is necessary to slowly dilute the cryoprotectant to prevent osmotic shock.

If the cryovial is stored in the liquid N2 it may expand and on thawing it may crack.