Comparison of Three Isolation Techniques for Human Peripheral Blood Mononuclear Cells: Cell Recovery and Viability, Population Composition, and Cell Functionality

Biopreserv Biobank. 2016 Oct;14(5):410-415. doi: 10.1089/bio.2015.0104. Epub 2016 Apr 22.


Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments, these three PBMC isolation techniques were compared for cell recovery and viability, PBMC population composition, and cell functionality, aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors, applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures, with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability, assessed by trypan blue exclusion, was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures, for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities, these data may support selection of a specific PBMC isolation technique for downstream analysis.

Keywords: cell viability; cytokines; density centrifugation; peripheral blood mononuclear cells (PBMCs).

Publication types

  • Comparative Study

MeSH terms

  • Cell Proliferation
  • Cell Separation / instrumentation*
  • Cell Separation / methods*
  • Cell Survival
  • Cytokines / metabolism
  • Healthy Volunteers
  • Humans
  • L-Lactate Dehydrogenase / metabolism
  • Leukocytes, Mononuclear / cytology*
  • Leukocytes, Mononuclear / metabolism
  • Male


  • Cytokines
  • L-Lactate Dehydrogenase