A Safe and Convenient Method to Isolate Bone Marrow Mononuclear Cells in Clinical Practice

Background: Bone marrow mononuclear cells (BMMNCs) are becoming a promising cell therapy in regeneration medicine. BMMNCs are now obtained by density gradient centrifugation (DGC) in clinical practice, which is complicated and greatly influenced by human manipulation.

Objective: Our objective is to develop a simple and safe method to isolate BMMNCs.

Methods: Bone marrow was aspirated from nine minipigs. The optimal hypotonic sodium chloride (NaCl) concentration was first investigated based on the BMMNCs viability and lysis efficiency tests. Afterward, three different methods (ammonium chloride (NH₄Cl) lysis, hypotonic NaCl lysis, and DGC) were used for BMMNCs isolation. Nucleated cell yield, residual red blood cells (RBCs) level, BMMNCs viability, apoptotic cell percentage, and colony-forming ability were measured in three groups. Cell morphology, cell phenotype, proliferative capacity, and osteogenic, adipogenic, and chondrogenic lineage differentiation potential of the bone marrow mesenchymal stem cells (BMSCs) were compared in three groups.

Results: 0.3% NaCl lysis group had optimal cell viability and lysis efficiency and the 0.3% NaCl lysis group had higher cell yield and lower RBCs remaining in BMMNCs compared to the DGC group. The BMSCs harvested from the NH₄Cl lysis group had the worst proliferation ability. The NaCl lysis group was not inferior to the other two groups in terms of other biological characteristics of BMMNCs/BMSCs.

Conclusions: The optimal concentration for hypotonic NaCl lysis to obtain BMMNCs is 0.3%. Compared with NH₄Cl lysis and DGC, the 0.3% NaCl lysis may be a safe, appropriate, and low-cost method for BMMNCs isolation.

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