Abstract:

BACKGROUND: Isolation and culture of highly purified bone marrow mesenchymal stem cells are the premise to study their function.
OBJECTIVE: To investigate the biological characteristics of rat bone marrow mesenchymal stem cells isolated and cultured by whole bone marrow adherence method.
METHODS: Sprague-Dawley rats weighing 80-100 g were selected, and the bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats by whole bone marrow adherence method. During isolation, the time period from sacrificing to putting the cell suspension into the incubator should be controlled strictly, generally less than 40 minutes. Moderate intensity was preferred to flush the bone marrow cavity with basal culture medium, and rotation for several times could make the bone marrow cells fully dropped off and ensure the quantity of cells.
RESULTS AND CONCLUSION: The primary cultured bone marrow mesenchymal stem cells were the adherent fibroblast-like cells, and after passage, the cells were uniform and showed spindle shaped and typical swirl morphology. The growth curve of passage 3 bone marrow mesenchymal stem cells resembled S shape and had three periods: latent period, logarithmic growth phase and lag phase. Flow cytometry detection showed the content of surface antigen: CD29⁺ (99.45%), CD34⁺ (1.45%), CD44⁺ (99.52%), CD45⁺ (1.41%). After osteogenic and adipogenic differentiation, mineralized nodules were orange under alizarin red staining, and lipid droplets were red under oil red O staining. Bone marrow mesenchymal stem cells have the multiple differentiation potential and can differentiate into osteoblasts and adipocytes. The multiple differentiation potential of bone marrow mesenchymal stem cells corresponds to the minimum standards for identifying the animal bone marrow mesenchymal stem cells raised by International Society of Cell Therapy and Mesenchymal and Tissue Stem Cells Committee.