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Updated: Feb 19, 2026
Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners
Published on: October 25, 2024
Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry
Bo Langhoff Hønge1,2, Mikkel Steen Petersen1, Rikke Olesen3
1Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark.
Introduction:
Live peripheral blood mononuclear cells (PBMCs) can be frozen and thawed for later analyses by adding and removing a cryoprotectant, such as dimethyl sulfoxide (DMSO). Laboratories across the world use various procedures, but published evidence of optimal thawing procedures is scarce.
Materials And Methods:
PBMCs were separated from blood collected from healthy Danish blood donors, and stored at -80°C after adding of DMSO. The essential steps in the thawing procedure were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable PMBCs.
Results:
The best-performing washing medium was Roswell Park Memorial Institute (RPMI) 1640 at 37°C with 20% fetal bovine serum. When using 10 mL washing medium in a 15-mL Falcon tube, samples should be centrifuged for at least 10 minutes at 500 g. We failed to detect any differences between the tested methods of mixing PBMCs with washing medium. Likewise, neither the thawing duration nor centrifugation temperature (20°C and 37°C) had any effect. PBMCs could be incubated (rested) for up to eight hours in a 37°C 5% CO2 incubator without affecting cell counts, but incubating PBMCs for 16 hours significantly decreased viability and recovery. In general, high viability was not necessarily associated with high recovery.
Conclusion:
Changing the thawing procedure significantly impacted PBMC viability and live cell recovery. Evaluating both viability and live PBMC recovery are necessary to evaluate method performance. Investigation of differential loss of PBMC subtypes and phenotypic changes during thawing and incubation requires further evaluation.
Insights
Optimizing the thawing of cryopreserved peripheral blood mononuclear cells (PBMCs) is crucial for accurate downstream analysis. The study identified specific washing media, centrifugation times, and incubation periods that maximize PBMC viability and recovery.
Area of Science:
- Immunology
- Cell Biology
- Cryobiology
Background:
- Live peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved using dimethyl sulfoxide (DMSO) for later analysis.
- Standardized, evidence-based protocols for thawing PBMCs are lacking, leading to variability in laboratory procedures.
Purpose of the Study:
- To evaluate and optimize the thawing procedure for cryopreserved PBMCs.
- To identify critical parameters affecting PBMC viability and recovery after thawing.
Main Methods:
- PBMCs from healthy donors were cryopreserved with DMSO and thawed using modified procedures.
- Cell viability and total yield were assessed using flow cytometry.
- Washing media, centrifugation, mixing techniques, and incubation times were systematically varied.
Main Results:
- RPMI 1640 at 37°C with 20% fetal bovine serum was the optimal washing medium.
- A 10-minute centrifugation at 500 g in a 15-mL tube was recommended.
- Incubation up to 8 hours at 37°C did not affect cell counts, but 16 hours significantly reduced viability and recovery.
Conclusions:
- Thawing procedures significantly influence PBMC viability and live cell recovery.
- Both viability and recovery metrics are essential for assessing thawing method performance.
- Further research is needed to understand subtype-specific changes during thawing and incubation.
