VIDEO: Thawing Cryopreserved PBMCs for Cell Recovery and Viability

 

At Precision, we want to help ensure you get the full value from you cryopreserved PBMCs. So, we’ll show you how to thaw for the best cell recovery and viability. By following these procedures you’ll find that our cells have a greater than 90% average lot viability and purity post-thaw.

  1. Transfer frozen vials to your lab on dry ice to keep them cold. We recommend only thawing 4 vials at a time.
  2. Warm the medium for cell dilution to 37°C ± 3°.
  3. Hold vials in a water bath that is also 37°C ± 3°, being careful not to immerse the vial below the level of the cap.
  4. Watch your vials carefully, and just before the last ice crystal has melted, remove the vial from the water and wipe the vial with a sterile alcohol pad, focusing on the cap area.
  5. Gently pour the PBMCs from the cryo-vial into a pre-labeled 50 mL tube (or 15 mL if you have a small cell aliquot).
  6. Drop by drop, add 8 mL of warmed medium to the tube, gently swirling the tube as you go, to mix the cells with the medium. This allows the cells to adjust to the change in environment.
  7. Rinse the original vial with 1 mL of warm culture medium and add the rinse media to the new tube to make sure you recover all of the cells.
  8. Pellet the cells by centrifugation at 400xG for 10 minutes with rapid acceleration and brake on. If no pellet is observed, centrifuge again at 400xG for an additional 15 minutes.
  9. When you are finished, discard the supernatant.
  10. Add sufficient warmed medium to bring the cell volume back to 10 mL, then pellet the cells again by centrifugation at 400xG for 10 minutes, to make sure all the cryo-protective medium is removed
  11. Re-suspend the cells in the desired volume for counting using warm medium, mixing the cell solution carefully to make sure there’s no clumps
  12. Count the cells using your preferred laboratory-specific procedures. We recommend resting your cells overnight at 37°C ± 3°C before using them in cell-based assays, but it is not always necessary to do this and we are happy to advise.

Apply these procedures and you’ll benefit from a greater than 90% average lot viability and purity post-thaw!

About The Author

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Deborah Phippard

Pharma industry veteran and expert at biomarker-driven clinical trial design and execution. Led biomarker and drug development programs for pharmaceutical and diagnostics companies, as well as the National Institutes of Health. Spearheaded the discovery of pharmacodynamic biomarkers and novel targets for inflammatory disease therapy.

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