Study On Isolation, Culture And Identification Of SD Rat Bone Mesenchymal Stem Cells (BMSCs) In Vitro

Objective To investigate the method for isolation and amplification of the bone mesenchymal stem cells (BMSCs) in vitro and identified it.Methods Male SD rats around 100 g, the bone marrow cells were obtained from hind femur and tibia, whole bone marrow primary culture, purified by repeated passage in vitro amplification. cell morphology was observed, and expressions of CD34, CD90, CD105 cell factors were examined by flow cytometry, to identify whether it's the BMSCs.Results sells appear spindle-shaped and show characteristic swirling growth. The surface marker CD34 is negative, CD90, CD105 are positive.Conclusion We successfully isolated the bone marrow mesenchymal stem cells (BMSCs) by the whole bone marrow culture method, and found that cells had high purity and good activity within 10th generation. The whole bone marrow culture is simple and easy. Objective To explore the feasibility of the bone marrow mesenchymal stem cells (BMSCs) as the seed cell for Construction of small diameter blood vessel, and the induced mechanisms.Methods Male SD rats around 100g, the bone marrow cells were obtained from hind femur and tibia, whole bone marrow primary culture, purified by repeated passage in vitro amplification. cell morphology was observed, and expressions of CD34, CD90, CD105 cell factors were examined by flow cytometry, to identify whether it's the BMSCs. Two groups were divided: the experimental group and control group.The experimental group was induced to the vascular smooth muscle-like cell by the DMEM-LG with all-trans retinoic acid and db-cAMP, and the control group was cultured by the normal DMEM-LG. we observed the morphological characteristics of BMSCs and detected the expressions of SM-α-actin, calponin, SMMHC by immunofluorescence and flow cytometry with the fifth generations cells after induction.Results The obtained cells appear spindle-shaped and show characteristic swirling growth. The surface marker CD34 is negative, CD90, CD105 are positive. After induction, the cell in the experimental group grew slowly and were slightly oval shaped. The expressions of SM-α-actin, calponin, SMMHC is significant in the experimental group. In the control group, cell morphology and cell growth is similar with the BMSCs, and the expressions of SM-α-actin, calponin, SMMHC is negative.Conclusion The BMSCs can be induced to vascular smooth muscle cells for phenotypic differentiation by all-trans-retinoic acid,the induced cells can be used as seed cells for tissue engineering construction of small diameter blood vessel. Objective To investigate the possibility of the vascular smooth muscle-like cells static culture in vitro.Methods Adult male dogs were sacrificed, taking thoracic aorta, about 6cm long. Using Triton X-100 and trypsin Removed cells in the blood vessel, in order to obtain acellular matrix scaffold.Detection of acellular effect by quick frozen staining. Acellular matrix scaffold sheet was cut into the size of 2x2cm. The induced vascular smooth muscle-like cells were grown in the sheet, the medium was changed every other day, we detected the sheet by quick frozen staining and electron microscope to observe the growth state.Results Quick frozen staining shows the induced vascular smooth muscle-like cells can grow in the surface of the acellular matrix scaffolds of vascular, scanning electron microscopy showed cells in the scaffold complex.Conclusion Completed induced vascular smooth muscle-like cells can grow in the vascular acellular matrix materials by static culture.