Peripheral blood mononuclear cells, or PBMCs, are the major cells of the human body that are involved in immunity. By definition, PBMCs are the subset of cells in the blood that have one round nucleus, including lymphocytes (T cells, B cells, and natural killer [NK] cells), monocytes, and macrophages. Erythrocytes and platelets have no nucleus, while granulocytes (basophils, neutrophils, and eosinophils) have multi-lobed nuclei; therefore, none of these are considered to be PBMCs. Researchers and clinicians alike are interested in PBMCs, as they are incredibly useful in such areas as regenerative therapy, cancer immunotherapy, autoimmune disease, and biomarker identification.
Once PBMCs have been isolated from whole blood, either they can be used fresh or they can be cryopreserved for future use. However, freezing and thawing can affect the quality of the cells. For this reason, experts from Precision for Medicine have shared with us a list of 10 steps to follow in order to thaw cryopreserved PBMCs for maximized cell recovery and viability.
Step 1. Keep the cells on dry ice
When transferring the vials of cells from the freezer to your workstation, even if it’s a short distance, keep the vials on dry ice so that the cells stay cold. Additionally, don’t try to thaw more than four vials at a time. Focusing on more vials may divide your attention and prevent you from treating each vial with optimum care.
Step 2. Warm the cell medium
Place the container of medium that you will be using to suspend the cells into a water bath that is warmed to 37°C ± 3°.
Step 3. Warm the cells in a water bath
Hold your vials in a water bath that is warmed to 37°C ± 3°. Do not immerse the vial below the cap level, as this can allow water to leak inside and contaminate your samples. Keep a close eye on your vials; when there is still one ice crystal left on the external surface of the vial, remove the vial from the water bath. Wipe it down using a sterile alcohol wipe, especially around the cap, to prevent your cells from becoming contaminated.
Vial warmed at 37°C ± 3°
Step 4. Transfer the cells to a new tube
Prepare and label a 50 mL tube. (If you have a small aliquot, you may choose to use a 15 mL tube.) Pour the PBMCs from the cryo-vial into the new tube.
Step 5. Add warmed cell medium
Measure 8 mL of the warmed cell medium. Slowly add it to the tube, swirling as you go to help the cells mix with the medium.
Step 6. Rinse the original vial
To increase the amount of cells you recover, rinse the cryo-vial with 1 mL warm medium and pour it into the 50 mL tube.
Step 7. Pellet the cells and discard the supernatant
Centrifuge the cells at 400xG for 10 minutes. If there is no pellet, centrifuge the cells again at 400xG for 15 minutes more. After centrifuging, discard the supernatant, as it contains the new cell medium mixed with the cryo-protective medium.
Step 8. Add more cell medium and pellet the cells again
To ensure that all of the cryo-protective medium is removed from the cells, give the cells an extra rinse. To do so, add 10 mL more of the warmed cell medium and centrifuge the vial at 400xG for 10 minutes. Again, discard the supernatant when you are finished centrifuging and re-suspend the cells in whatever volume of the warm medium you need for your cell counting protocol. Be sure to mix (carefully) the cell solution to ensure that there are no clumps.
Step 9. Count the cells
Use your laboratory’s preferred procedure to count the cells.
Step 10. Rest the cells overnight
Before using the cells in cell-based assays, it is recommended that you rest the cells overnight at 37°C ± 3°.
There you have it—a 10-step method for thawing PBMCs. Precision for Medicine wants to help ensure you get the full value from your cryopreserved PBMCs. By following these procedures you’ll find that cells have a greater than 90% average lot viability and purity post-thaw.
Click here to view our full-length video on how to thaw PBMCs and to learn more about our products, visit buypbmcs.com.