Cryopreserved PBMCs are ideal for many research applications as they enable a greater degree of flexibility (timing, number of samples, etc.) in the experimental design. A validated PBMC cryopreservation protocol is perhaps the most critical factor in achieving high post-thaw cell viability and functionality.
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. CryoStor® CS-10 is serum-free, animal component-free DMSO solution that is widely used to freeze PBMCs.
Cryoprotectants containing fetal bovine serum (FBS) and human serum albumin (HSA) are good alternative freezing mediums, but these solutions contain human and animal proteins which may negatively impact certain downstream experiments.[1]
The protocol below describes the PBMC cryopreservation using CryoStor® CS-10.
Why Cryopreserve PBMCs?
The goal of the cryopreservation process is to retain the critical biological properties and functionality of the cells post-thaw. On a molecular level, freezing stabilizes the cells while minimizing ice crystal formation which can cause cell death.
All cells contain degradative enzymes and freezing is effective at reducing the activity of those enzymes. As the temperature is reduced, the activity of the enzymes decreases and there is a threshold at which the protein is no longer active and therefore cannot participate in degradation of the cell.[2]
How Does Cryopreservation Media Work?
Cryopreservation media are typically hypertonic, meaning they have a higher osmotic pressure than intracellular fluid. Exposing cells to the cryopreservation media can result in cell death from osmotic stresses unless a careful process is followed.[3]
When cells are placed in cryopreservation media, the initial response of the cells is to dehydrate in order to reduce the chemical potential difference between the intracellular and extracellular fluids. This is followed by a gradual increase in cell volume resulting from penetration of the cryoprotectant into the cell. The cell may lyse during this initial transfer phase unless the temperature, volume and cell concentration conform to the media manufacturer’s recommendations.[4]
Certain cryoprotective agents are toxic to the cells and result in cell losses with time of exposure to the media in liquid state (pre-freeze and post-thaw).[5] Laboratories should develop and optimize a process for rapid introduction and removal of the cryopreservation media to minimize cell losses from toxic exposure.
PBMC Cryopreservation Protocol
Materials
> CryoStor® CS10
> Cryogenic vials and labels
> Isopropanol freezing container or controlled-rate freezer
> Pipettor
> Serological pipettes
Protocol
1. Wipe down all non-sterilized materials with 70% ethanol.
2. Label cryogenic vials
Use Brady B-492 labels or similar.
3. Suspend freshly isolated PBMCs in cold (2 – 8°C) CryoStor® CS-10
It is recommended to suspend PMBCs pellet in a concentration between 5 x 106 and 10 x 106 cells per mL of CryoStor®.
4. Mix the suspension thoroughly
5. Incubate the PBMCs at 2 – 8°C for 10 minutes
6. Aliquot the PBMC suspension in cryogenic vials
7. Freeze the PBMCs at a rate of -1°C using a controlled-rate freezer
If using an isopropanol freezing container (e.g., Nalgene® Mr. Frosty), place the vials into the container and store in a -80°C freezer overnight to allow gradual and even cooling.
8. For long-term storage, transfer the vials from the freezer to vapor phase liquid nitrogen
Recommend storage temperature below -135°C.[6]
Interested in learning more on this topic? Watch our video on isolating PBMCs from whole blood to achieve maximum purity and viability.
References
[1] Best BP. Cryoprotectant Toxicity: Facts, Issues, and Questions. Rejuvenation Research, Oct 2015. 422-436. http://doi.org/10.1089/rej.2014.1656.
[2] Hubel A, Spindler R, Skubitz AP. Storage of human biospecimens: selection of the optimal storage temperature. Biopreserv. Biobank. 2014; 12: 165–75.
[3] Levin RL, Cravalho EG, Huggins CE. A membrane model describing the effect of temperature on the water conductivity of erythrocyte membranes at subzero temperatures. Cryobiology 1976; 13: 415–29.
[4] Ibid.
[5] Fahy GM. Cryoprotectant toxicity neutralization. Cryobiology 2010; 60, S45–53.
[6] Hunt CJ. Technical Considerations in the Freezing, Low-Temperature Storage and Thawing of Stem Cells for Cellular Therapies. Transfus Med Hemother. 2019; 46(3):134-150. doi:10.1159/000497289.
