Density Gradient Centrifugation Compromises Bone Marrow Mononuclear Cell Yield

Abstract

Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.

Expression of multidrug resistance 1 and multidrug resistance-related protein 1 in C57BL/6 mice treated with benzene

“…The 2-week benzene gavage (200 mg/kg, once daily, 5 days per week, for 2 weeks) does not give rise to an obvious change of NF-κB p65 expression with the reduction in blood cell counts and ABCB1B inhibition. One possible explanation is that the BMNC are not actually 100% hematopoietic cells, in which some impurities could disturb the NF-κB p65 expression (Pösel et al, 2012). Another potential explanation is that the phosphorylation of the p65 subunit of NF-κB might genuinely be required for its transcriptional activation (Nazar et al, 2012).…”

Circulating Tumor Cells: A Review of Non–EpCAM-Based Approaches for Cell Enrichment and Isolation

“…Despite its long history of use in lab-oratories, there are some pitfalls associated to this technique, such as the possible loss of tumor cells that migrate either to the plasma fraction or to the bottom of the gradient due to the formation of aggregates (22 ). It has been suggested that this cell loss may be due to the cytotoxicity of the density medium (23 ). An alternative to Ficoll is Percoll density (GE Healthcare Life Sciences) gradient medium made of a colloidal silica particle suspension.…”

“…An alternative to Ficoll is Percoll density (GE Healthcare Life Sciences) gradient medium made of a colloidal silica particle suspension. The main advantages over Ficoll include reduced toxicity and a wider density gradient range (23 ). There are certain discrepancies in the literature regarding the use of Percoll because some studies have demonstrated a high purity rate (24 ) while others have shown low isolation efficiencies compared to Ficoll (25 ).…”

Further Characteristics of Pregnant Вone Marrow Cells Action on Rat Development during Early Pregnancy

“…In accordance with multiple studies such cell mixture promotes distinct angiogenic properties, mediates vascular repair, express several cytoprotective growth factors and cytokines [13]. Practically BMC intravenous transplantation dramatically increases level of blood BMC concentration up to concentration which can't be reached by another experimental procedure.…”