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

Abstract

Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body's immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation. Methods: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol. Results: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes. Conclusions: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

“…Using the same isolation and viability staining protocols, we have previously shown that the post-isolation viability of monocytes is low (17%), while lymphocytes have very high viabilities (86-91%) (30). However, in the present study, we report an average post-isolation viability of only 82%.…”

“…Isolation and activation of PBMC Isolation of PBMC was performed using an already tested protocol for which we have previously reported high yields of 90% for 3mL blood samples (30). Nevertheless, our subsequent experience with PBMC density gradient isolation (this study included) showed us that processing blood volumes higher than 3mL will in fact significantly lower the cell yield.…”

“…The main steps of the study are summarized in Figure 1A. PBMC were isolated by density gradient centrifugation using a high-yield protocol that was previously tested and implemented in our laboratory (30). PBMC were suspended in RPMI 1640 culture medium (Sigma Aldrich, cat.…”

“…This delay between isolation and staining may have also negatively impacted overall cell viability. Generally, monocytes account for about only 10% of the isolated PBMCs and we have previously shown that less than 20% of these monocytes are viable (30). Moreover, during the 4h activation period, most of the already few viable monocytes will either die or adhere to culture flasks.…”

Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

“…Blood samples were processed within 2hrs from collection. PBMCs were isolated using a high-yield density gradient centrifugation protocol that was already tested and implemented in our laboratory [17]. In between the centrifugation steps, cells were resuspended in complete RPMI medium (cRPMI) consisting of: RPMI 1640 culture medium Sigma Aldrich (Cat.…”