To establish the rat bone marrow mesenchymal stem cells(BMSCs) were isolated and cultured, purified and modified optimization method in vitro, to observe cell morphology, and to assess surface markers and differentiation capacity detection.

The bone marrows of 4-week-old, male Sprague Dawley rats were used to obtain mMSCs. Rats were euthanized via cervical dislocation. After 72 hours, the medium was replaced and fresh medium was provided every 3 days. BMSCs were identified via flow cytometry, multi-directional differentiation capacity, and morphology.

Primary culture: Bone marrow cells were seeded in round culture dishes of different sizes and suspended in culture medium. After 24 hours, the part of the cells adhered to the culture dish were visible as round, fusiform, or polygonal. After removal of the non-adherent cells by media replacement, the adherent cells were found to be short, spindle or star shaped, and scattered on the plastic surface. After 4 or 5 days, radially arranged cell colonies were visible, which differed in length and thickness. Spindle cells comprised the main colonies, possessing abundant cytoplasm and a large nucleus. After a week, the cells showed colony growth, were fused at approximately 80%-90%, and were reminiscent of a whirlpool, all with the same directionality. After 10 days, the cells were arranged in close proximity to one another, gradually integrating into sheet form. Culture passage: After digestion and passage, the cells adhered to the plastic surface within 24 hours. The cells were homogeneous, spindle-shaped, and displayed strong cell growth. Each passaging was performed after 4 or 5 days of growth. Cell morphology and growth rate did not significantly differ after 10 generations of stable and continuous passage. The results of flow cytometry revealed a positivity rate of CD90 expression of 96.9%, and of 96.6% for CD29. Thus, the negativity rate of CD45 was 7.56%, and that of CD34 was 0.395% in fourth generation rat mMSCs. The mMSCs were added into an adipocytes-inducing agent, where they were cultured for 18 days, during which time, lipid and lipid droplet accumulation was induced with a string of beads. The oil red O staining was bright red.

Without centrifugation of whole bone marrow adherent culture method has the advantages of simple operation, is not easy to pollute, highly active cell, bulk separation, purification and amplification of BMSCs, cells with a mesenchymal stem cell biological characteristics. After cultured with multilineage differentiation potential.