Abstract:

BACKGROUND: Bone marrow mesenchymal stem cell content is less under normal conditions, and easily confounded with other cells. Therefore, to establish a simple feasible in vitro cultured amplification method and to obtain a large number of stable bone marrow mesenchymal stem cells are of important theoretical significance and application value.
OBJECTIVE: To establish a simple feasible in vitro culture and amplification method and to obtain numerous stable bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and cultured in vitro using density gradient centrifugation and adherence separation screening. Cell morphological changes were observed under an inverted light microscope. Submicroscopic structure of cells was observed under a transmission electron microscope. Living cell number was counted using Trypan blue exclusion method. Cell growth curve was drawn. Cell cycle was analyzed using flow cytometry. The expression of c-kit and CD45 was measured using immunocytochemistry. CD45 expression was analyzed using flow cytometry.
RESULTS AND CONCLUSION: (1) Cell adhesion and pseudopodia could be seen under an inverted light microscope at 24 hours after inoculation; cell colony formed at 4 days; 90% cell confluence was found at 14 days. After passage, the cells were tended to homogenous and became fibroblast-like cells, which showed whirl-like growing or flamboyancy growing. (2) The size of passage 3 bone marrow mesenchymal stem cells was small and nucleolus was large, with clear nucleoli under a transmission electron microscope, with the presence of sparse chromatin and low electron density. There was microvillus on the surface of cells, abundant ribosomes could be seen in the cytoplasm with few other cellular organs such as endoplasmic reticulum, mitochondria and Golgi complex. These showed that ultrastructural structure had undifferentiated features. (3) The number of passage 3 bone marrow mesenchymal stem cells was reduced at 1 day after inoculation. The cells began to grow at 2 days, and entered the period of exponential growth at 3 days, entered the flat period at 7 days and the number of cells began to decrease at 9 days. Growth curve exhibited “S” shape. (4) The percentage of S phase cells was 21.1% as detected by flow cytometry after passage 3 bone marrow mesenchymal stem cells were stained by DNA. (5) Immunocytochemistry and flow cytometry had proved that the c-kit positive rate of cells was 53.3% and CD45 positive rate was 1.68%. (6) After osteogenic induction of mesenchymal stem cells for 16 days, cells became oval, and short processes connected each other. The cytoplasm was dark, suggesting that it may be rich in rough endoplasmic reticulum and Golgi complex. They can secrete osteoid. Results verified that obtained bone marrow mesenchymal stem cells stably grew, and actively proliferated.

Key words: bone marrow, mesenchymal stem cells, cells, cultured, rats, Sprague-Dawley