Isolation, Culture, And Chondrogenic Induction Of Rabbit Mesenchymal Stem Cells In Vitro

Objective1 .To establish a effective method for the isolation and culture of rabbit bone marrow mesenchymal stem cells (BMSCs) , and to observe their chondrogenic capabilities in definite culture.2. To explore how to isolate and culture the G-CSF Mobilized rabbit peripheral derived mesenchymal stem cells (PBMSCs).Methods1. The bone marrow was taken from Zelanian rabbits aged 4 months. BMSCs were cultured with the density gradient centrifugation, the whole blood culture and NH₄Cl red blood celllysis methods. Cell survival rate, the cloning formation rate, primary passage time, cell count after 15 days and success rate of each method were observed.2. BMSCs were induced into chondrocytes in defined culture medium containing bone morphogenetic protein (BMP)-2 in monolayer culture in vitro. Toliudine blue staining, Masson staining, immunohistochemical staining, Alcian blue chromatomery of glycosaminoglycan (GAG) were performed to detect differentiation. Statistical analysis of the proliferation and chondrogenic differentiation abilities of induced BMSCs was deployed among 2nd, 4th and 7th passages. S.Granulocyte-colony stimulating factor (30μg/ kg·day) was injected into New Zealand White rabbits subcutaneously for 4 days ,then mesenchymal stem cells(MSCs ) were isolated from peripheral blood using the density gradient centrifugation method.Results1. For the cell survival rate, the density gradient centrifugation and the whole blood culture were more preferable than NH₄Cl red blood celllysis. For other four above mentioned indexs, the density gradient centrifugation method was the best.2. Induced BMSCs have characteristics of the chondrocyte. There was no statistical difference in the proliferation and chondrogenic differentiation abilities between the P₂ and P₄ BMSCs (P >0.05). Compared with the P₂ and P₄ BMSCs, the proliferation and chondrogenic differentiation abilities of the P₇ BMSCs was decreased (P<0.01).3. There were no spindle shaped cells in the unmobilized group. While in the mobilized group, there were spindle shaped cells and proliferated in small colonies, but unable to expand quickly and abundantly.Conclusion1. For the isolation of BMSCs in adult rabbits, the density gradient centrifugation method is more preferable than the whole blood culture and NH₄Cl red blood celllysis methods.2. BMSCs can be induced into chondrocytes in monolayer culture containing BMP-2 in vitro. Cell passage is an important factor in the proliferation and chondrogenesis differentiation of BMSCs.3. It is not sure that the spindle shaped cells in the mobilized group were MSCs .The isolation and culture of PBMSCs need to further be explored.