Optimization of standard protocols for subculturing mesenchymal stem cells from primary mouse bone marrow culture

Abstract

The therapeutic and immunomodulatory potential of bone marrow multipotent mesenchymal stem cells (BMSCs) attracts special interest of the scientific community. In this regard, the search for an effective cell culture technique is an urgent task for experimental medicine.

The aim of the study was to optimize the standard protocols for subculturing murine MMSCs obtained from primary bone marrow culture.

Methods. The cell yield was evaluated by trypsin and accutase passaging using different methods after 14 days of culturing bone marrow suspensions of ICR line mice in seed concentrations of primary culture: 10, 15-20, 30 million cells per 25 cm².

Results. With the help of treatment for 15 min with 2 ml of trypsin and 2 ml of accutase, it was possible to save up to 300 thousand adherent and dividing cells with an initial inoculation of 10 million. Using the same method, 30–35% fewer cells were removed from inoculation of treatment in 15–20 in 15 min and 30 million cells in 30 min.

Conclusion. Seeding density of primary bone marrow culture of 10 million cells per 25 cm² is optimal for obtaining an adherent culture of mesenchymal stem cells of mice of ICR line. Subculturing with 2 ml of trypsin and 2 ml of accutase for 15 min is most effective for obtaining the first passage.