About Cryopreservation

Serial sub-culturing is a time consuming and can lead to contamination or genetic drift as smaller and smaller portions of a population are selected.

However, a cell populations can be stabilized by subjecting them to cryogenic temperatures. Stabilizition of cells at cryogenic temperatures is called cryopreservation.

What is Cryopreservation?

Ice forms at different rates during the cooling process.

As ice forms, water is removed from the extracellular environment and an osmotic imbalance occurs across the cell membrane leading to loss of water from the cell.

The increase in solute concentration can be detrimental to cell survival.

Too much water retention inside the cell cause damage to the cells and thus reduce the cell viability due to ice crystal formation and recrystallization during warming can occur.

The rate of cooling has a dramatic effect on over all cryopreservation process.

Rapid cooling minimizes the solute concentration and thus affects the uniform ice formion, but leads to more intracellular ice.

On the other hand, slow cooling, results in a greater loss of water from the cell and less internal ice, but increases the solution effects.

A cooling rate of 1°C per minute is generally preferred. Cryoprotective additives or chemicals, for example dimethylsulfoxide (DMSO) and glycerol can protect the cells during freezing and minimize the detrimental effects of increased solute concentration and ice crystal formation.

Cryopreservation media generally consists of a base medium, cryopreservative and a protein source.

The cryopreservative and protein protect the cells from the stress of the freeze-thaw process.

Additionally, it maintains frozen cells at the proper storage temperature and using an appropriate warming rate will minimize damage to frozen cells.

Adopted from BioRender

Cryopreservation Requirements

Sterile:

1. Culture to be frozen

2. If monolayer: PBS and 0.25% trypsin (for adherent cells)

3. Growth medium with serum (If serum is being used with serum-free cultures, it should be washed off after thawing)

4. Cryoprotectant DMSO or glycerol, (free of impurities)

5. Pipette

6. Cryovials (1.0 ml), prelabeled with the cell line designation and the date of freezing

Non sterile:

7. Hemocytometer

8. Insulated container for freezing (Ice box)

9. Protective gloves, nitrile

Cryopreservation Procedure

1. Before cryopreservation, cells should be characterized and checked for contamination.

2. Check visually and on microscope for healthy appearance, morphological characteristics.

3. Grow the culture up to the late log phase and if cells are as a monolayer, trypsinize and count the cells.

If suspension is used count, centrifuge the cells and resuspend at 2 × 10^6–2 × 10^7 cells/ml.

4. Constitute the freezing media by adding 90% serum and 10% v/v DMSO.

5. Dilute the cell suspension in ratio of 1:1 with freezing medium to give approximately 1 × 10^6 –1 × 10^7 cells/ml

6. Dispense the cell suspensions into pre-labeled cryovials.

7. Allow slow freezing by keeping vials at 4 °C followed by -20 °C and finally stored at – 80 °C (storage in liquid nitrogen is preferable).

8. When the vials are safely located in the freezer, complete the appropriate entries in the freezer index.

Cryopreservation Precaution

Cells harvested for cryopreservation should be at their optimum viability to ensure maximum survival during freezing and after thawing.

This is especially relevant when the method of cryopreservation reduces the number of viable cells and increases the chances of selecting freeze-tolerant populations that may have different characteristics from the original population.

DMSO is toxic if left in contact with cells for more than a short period of time. Once the cells have been prepared for freezing, they should be ampuled and frozen as soon as possible.

DMSO can penetrate many synthetic and natural membranes, including skin.

Hence, DMSO should always be handled with caution, particularly in the presence of any toxic substances.