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

Abstract

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.

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

“…For MB, there are no differences between blood sample lots regarding platelet contamination while for ST, the 1ml lot was significantly less contaminated than its 2ml and 3ml counterparts. Regarding cell recovery, our results are consistent with previous studies reporting an average cell recovery by Ficoll isolation between 0.6 and 3 million cells per ml of blood [19][20][21][22]. However, we consider that when assessing performance, "cells per ml of blood" is an inappropriate manner of reporting cell recovery since it provides no information about initial cell count and cell population composition.…”

Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes

“…Another study found the overall performance of PBMCs isolated with CPTs and SepMate Tubes (comparable to LP tubes) to be similar, but superior than those isolated using Ficoll-Paque alone [15]. For example, in that study, cells isolated with either CPT or SepMate resulted in a greater number of PBMCs and higher IFNγ expression after staphylococcal enterotoxin B (SEB) stimulation than PBMCs isolated using only Ficoll-Paque gradient [15].…”

Streptococcus thermophilus alters the expression of genes associated with innate and adaptive immunity in human peripheral blood mononuclear cells

“…Calcium and magnesium free PBS, pH7.2, (Invitrogen, Pty Ltd. Australia) was used after adding 2 mM EDTA and 2% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Pty Ltd. Australia); PBS buffer. SEPMATE tubes (50 ml) with inner inserts (STEMCELL technology, Canada) were used to isolate PBMC following Ficoll-Paque density gradient protocol [29,30]. PBMCs were washed, counted and the required number of PBMC were co-cultured with S. thermophilus 285 and the remaining PBMC were stored in freeze mix and transferred into liquid nitrogen for future use.…”