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.

No Level Assigned

This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.