Properly Thawing Cryopreserved PBMCs: A Step-by-Step Guide

Peripheral Blood Mononuclear Cells (PBMCs) are a crucial component in immunological research and clinical studies. Cryopreservation is a common technique used to store PBMCs for extended periods while maintaining their viability and functionality. However, the process of thawing these cells is equally important to ensure they remain viable and effective for subsequent experiments. Here’s a comprehensive guide on how to properly thaw cryopreserved PBMCs.

1. Preparation Before Thawing

a. Warm the Thawing Medium:

• Prepare a water bath at 37°C. This is the optimal temperature for quickly and evenly thawing cryopreserved cells.
• Pre-warm the thawing medium, such as RPMI-1640 supplemented with 10% FBS, to 37°C. Having a warm medium ready helps minimize thermal shock to the cells.

b. Gather Necessary Equipment:

• Personal protective equipment (PPE): gloves, lab coat, and safety goggles.
• Sterile 15 mL and 50 mL centrifuge tubes.
• Pipettes and sterile pipette tips.
• Cryovials containing PBMCs.

2. Thawing the PBMCs

a. Retrieve the Cryovials:

• Remove the cryovials containing PBMCs from liquid nitrogen storage using appropriate cryogenic handling tools to avoid any thermal injury.

b. Quick Thawing:

• Immediately place the cryovials into the 37°C water bath. Submerge the vial just below the cap to avoid contamination.
• Gently swirl the vial in the water bath to ensure even thawing. This should take approximately 1-2 minutes, or until only a small ice crystal remains.

c. Rapid Transfer:

• As soon as the cells are almost completely thawed, promptly remove the vial from the water bath.
• Wipe the outside of the vial with 70% ethanol to disinfect it before opening.

3. Dilution and Washing

a. Dilution:

• Transfer the thawed cells slowly into a pre-warmed 15 mL centrifuge tube containing 10 mL of the thawing medium. Add the cells dropwise while gently swirling the tube to mix. This gradual dilution helps reduce osmotic shock.

b. Washing:

• Centrifuge the cells at 300 x g for 10 minutes at room temperature to pellet the PBMCs.
• Carefully aspirate the supernatant without disturbing the cell pellet.
• Resuspend the cell pellet in 10 mL of fresh, pre-warmed culture medium.

4. Assessing Cell Viability

a. Count the Cells:

• Use a hemocytometer or an automated cell counter to determine the total number of cells and assess viability.
• Typically, trypan blue exclusion is used to differentiate viable (unstained) from non-viable (blue-stained) cells.

b. Adjust Cell Concentration:

• Based on the cell count, adjust the concentration of the PBMC suspension to the desired level for your specific application.

5. Culture and Recovery

a. Initiate Culture:

• Plate the PBMCs in the appropriate culture vessels with the recommended density and culture medium.

b. Monitor and Maintain:

• Incubate the cells at 37°C in a humidified incubator with 5% CO2.
• Monitor the cells for recovery and viability over the next 24-48 hours before proceeding with downstream applications.

Tips for Optimal Thawing

1. Minimize Time in the Water Bath:

   • Prolonged exposure to temperatures above 37°C can harm the cells. Thaw them quickly and proceed to dilution immediately.

2. Use Pre-warmed Media:

   • Cold media can shock the cells during thawing. Always use media that is at the correct temperature to ensure smooth recovery.

3. Gentle Handling:

   • PBMCs are delicate after thawing. Handle them gently to avoid mechanical stress that can affect their viability.

4. Immediate Washing:

   • Washing away the DMSO (cryoprotectant) as quickly as possible is crucial since it can be toxic to cells at higher temperatures.

Properly thawing cryopreserved PBMCs is critical for maintaining their viability and functionality for research applications. By following these steps and tips, researchers can ensure that their PBMCs are in optimal condition for their studies, leading to more reliable and reproducible results.

For more information on best practices in biospecimen handling and other related topics, visit BioSample Connect.