Cell cryopreservation considerations - Database & Sql Blog Articles

Principles of Cryopreservation and Resuscitation: Slow Freezing and Rapid Thawing

Cryopreservation is a technique used to preserve cells, tissues, or organs by cooling them to very low temperatures. When cells are cooled below zero degrees Celsius, several critical changes can occur, including dehydration of cellular organelles, an increase in the concentration of solutes inside the cell, and the formation of ice crystals within the cell structure.

If the freezing process is too rapid, large ice crystals can form inside the cells, which may damage or rupture the cell membrane and organelles. To avoid this, slow freezing is recommended. During slow freezing, the cells gradually lose water, preventing the formation of large ice crystals. This helps maintain cell viability and function after thawing.

On the other hand, the resuscitation process should be fast. Rapid thawing prevents the formation of new, larger ice crystals from smaller ones—a process known as recrystallization—which can also harm the cell structure. Therefore, both the freezing and thawing steps must be carefully controlled to ensure successful cryopreservation.

Slow Freezing Protocol

1. Standard Procedure: Using a Cell Cryostat

• When the temperature is above -25°C, cool at a rate of 1–2°C/min.
• Once the temperature drops below -25°C, increase the cooling rate to 5–10°C/min.
• At around -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 and secure it with a rope.
• Suspend the gauze bag in a liquid nitrogen tank using a wire rope.
• Allow the temperature to decrease at a rate of 1–2°C per minute, which typically takes about 40 minutes to reach the surface of the liquid nitrogen overnight.
• The next morning, place the sample into the liquid nitrogen tank for long-term storage.

3. Traditional Procedure:

• Store the cryotube at 4°C for 10 minutes.
• Move to -20°C for 30 minutes.
• Then store at -80°C for 16–18 hours (or overnight).
• Finally, transfer to a liquid nitrogen tank for long-term preservation.

Cryopreservation Method

1. Pre-formulated Cryopreservation Solution:

• Use a solution containing 10% DMSO mixed with cell growth medium (which includes 20% serum and basal medium).

2. Prepare the Cells:

• Harvest cells during the logarithmic growth phase.
• After trypsinization, add an appropriate volume of the cryopreservation solution.
• Pipette the mixture to create a uniform cell suspension, ideally between 1×10⁶ and 5×10⁶ cells/mL.

3. Freeze the Cells:

• Add 1 mL of the cell suspension to a cryotube.
• Seal the tube, label it with the cell name and freezing date.
• Store the sample in liquid nitrogen for long-term preservation.

Resuscitation Method for Cryopreserved Cells

1. Quick Thawing:

• Remove the frozen cell sample from the liquid nitrogen.
• Immediately place the cryotube in a 37°C water bath.
• Gently shake the tube until the contents are fully melted—this should take approximately 1 minute (no longer than 3 minutes).

2. Reintroduce the Cells:

• Directly inoculate the thawed cells into a culture flask containing pre-warmed 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 them into a fresh culture medium.