Cell cryopreservation considerations - Database & Sql Blog Articles

Principles of Cryopreservation and Resuscitation: Slow Freezing and Rapid Thawing

When cells are cooled below zero degrees Celsius, several critical changes occur within the cellular structure. These include dehydration of organelles, an increase in the concentration of intracellular solutes, and the formation of ice crystals inside the cells.

If the freezing process is done slowly, the cells can gradually lose water, preventing the formation of large, damaging ice crystals. On the other hand, rapid freezing leads to the formation of small ice crystals that can cause mechanical damage to cell membranes and organelles. To avoid this, the resuscitation (thawing) process must be fast, as slow thawing can lead to recrystallization—where small ice crystals grow into larger ones, further harming the cells.

Slow Freezing Protocols

1. Standard Procedure: Using a controlled-rate freezer or a cell cryostat.

- When the temperature is above -25°C, cool at a rate of 1–2°C per minute.

- Once the temperature drops below -25°C, increase the cooling rate to 5–10°C per minute.

- When the temperature reaches -100°C, transfer the sample directly into liquid nitrogen for long-term storage.

2. Simple Procedure:

- Place the cryotube (with the nozzle facing up) into a gauze bag, secure it with a rope, and attach it to the liquid nitrogen tank using a wire.

- Allow the temperature to decrease at a rate of 1–2°C per minute until it reaches the surface of the liquid nitrogen overnight (about 40 minutes).

- The next morning, transfer the tube into the liquid nitrogen for long-term storage.

3. Traditional Procedure:

- Pre-cool the cryotube at 4°C for 10 minutes.

- Then move it to -20°C for 30 minutes.

- Next, place it at -80°C for 16–18 hours (or overnight).

- Finally, store it in a liquid nitrogen tank for long-term preservation.

Cryopreservation Method

1. Pre-formulated Cryopreservation Solution:

- Mix 10% DMSO with cell growth medium (e.g., 20% serum + basal medium).

2. Cell Preparation:

- Harvest cells during the logarithmic growth phase.

- Treat them with trypsin to detach from the culture dish.

- Add an appropriate volume of the cryopreservation solution and gently pipette to create a uniform cell suspension (concentration: 1×10⁶ to 5×10⁶ cells/mL).

3. Freezing:

- Transfer 1 mL of the cell suspension into a cryotube.

- Seal the tube, label it with the cell type and freezing date.

- Store the tube in liquid nitrogen for long-term preservation.

Resuscitation Protocol

1. Quick Thawing:

- Remove the frozen cell sample from liquid nitrogen.

- Immediately place it in a 37°C water bath.

- Gently shake the tube until the contents are fully melted (within 1 minute, not exceeding 3 minutes).

2. Post-Thaw Handling:

- Directly inoculate the thawed cells into a pre-warmed culture flask containing complete growth medium.

- Replace the old culture medium with fresh complete medium after 24 hours to remove residual DMSO.

3. Special Cases:

- If the cells are particularly sensitive to cryoprotectants (like DMSO), centrifuge the thawed cells to remove the cryoprotectant before reseeding into fresh medium.