Cryopreserved PBMCs are ideal for many research applications as they enable a greater degree of flexibility in the experimental design. Researchers using cryopreserved PBMCs can control the timing of experiments and access to multiple donor samples simultaneously, whereas fresh PBMCs are logistically challenging to obtain and fresh cells have a short shelf life.
Although cryopreserved cells offer key advantages, they also require careful handling and special techniques for the PBMC thawing protocol. Achieving high PBMC recovery and viability is dependent upon laboratory personnel carefully following a validated thawing procedure.
The protocol described below is what our team at Cytologics and many of our scientific partners routinely use to thaw PBMCs.
What Happens to PBMCs During Cryopreservation?
The PBMC cryopreservation process involves reducing the mobility of water and the activity of degradative enzymes.
Cryoprotectants containing 10% dimethyl sulfoxide (DMSO) are a reliable, general purpose freezing medium to reduce the formation of intracellular ice crystals and resulting cell damage. PBMCs cryopreserved in 10% DMSO should be stored at temperatures below -150°C to ensure the cells remain stable.
Proper training and protocols can help reduce cell death during freezing and thawing of samples during retrieval from a repository.
PBMC Thawing Protocol
The range of temperatures traversed during freezing is also traversed during thawing (but in reverse). As with cooling, the manner in which this process is performed is critically important to post-thaw recovery.
The thawing rate must be one order of magnitude greater than the cooling rate for most conventionally cryopreserved cells. Typically, thawing rates of >60°C/min are desired.
You can monitor PBMC warming rate using a stopwatch and thermometer or thermocouple device. The average warming rate is the change in temperature (melting temperature of the solution minus the storage temperature) divided by the time to thaw.
This protocol describes a method for thawing PBMCs cryopreserved in CryoStor® CS-10, which is a serum-free, animal component-free DMSO solution widely used to freeze PBMCs.
Figure 1. PBMC Thawing Protocol: Key Steps.
Equipment and Materials
- 37°C water bath
- Transfer pipettes
- 25 mL serological pipettes
- 50 mL conical tubes
- Human primary cells (cryopreserved)
- Appropriate culture media. Examples include:
- Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% fetal bovine serum (FBS)
- DMEM with 4500 mg/L D-Glucose with 10% FBS
- RPMI 1640 Medium with 10% FBS
- Warm appropriate culture medium in a 37°C water bath.
- When removing frozen cells from storage, it is important to minimize exposure to room temperature (15 – 25°C). If not proceeding directly to thawing, place the cells on dry ice or in a liquid nitrogen container.
- Quickly thaw cells in a 37°C water bath by gently swirling the vial. Remove the vial when a small amount of ice remains. This should take approximately 1 – 2 minutes. Do not vortex the cells.
- Transfer vial to a biosafety hood and wipe the outside of the vial with 70% ethanol or isopropanol.
- Transfer the cell suspension to a 50 mL conical tube using a transfer pipette.
- Using a transfer pipet, rinse the vial with 1 mL of medium and add it dropwise to the cells, while gently swirling the 50 mL tube.
- Wash cells by adding 15 – 20 mL of medium dropwise, while gently swirling the tube.
- Centrifuge the cell suspension at 300 x g for 10 minutes at room temperature (15 – 25°C).
- Carefully remove the supernatant with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed. Resuspend the cell pellet by gently flicking the tube. DNase can be added to prevent cell clumping.
- Gently add 15 – 20 mL of medium to the tube.
- Centrifuge the cell suspension at 300 x g for 10 minutes at room temperature.
- Carefully remove the supernatant with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed. Resuspend the cell pellet by gently flicking the tube and adding appropriate medium.
- Cells are now ready for use in downstream applications.
Proper techniques and handling are critical for ensuring optimal viability and recovery of the cells for downstream applications. By following this PBMC thawing protocol, you’ll find that cells have viability of at least 90% and maximum cell recovery.