Isolation and cultivation of bone marrow mesenchymal stem cells of rats and intracephalic tracing of the stem cells after CM-Dil labeling / 解放军医学杂志

ABSTRACT

Objective To establish a method to isolate and cultivate rat bone marrow mesenchymal stem cells (BMSCs),and explore the feasibility of labeling in vitro,and tracing in vivo,BMSCs with chloromethyl-benzamidodialkylcarbocyanine (CM-Dil). Methods BMSCs were obtained and subsequently cultured with whole bone marrow cell culture system,and the third generation of BMSCs was harvested. The positive rates of CD34,CD44 and CD29 were detected by flow cytometry. CM-Dil was used to label BMSCs in vitro and the efficiency of labeling after 24 hours and at day 21 and 30 were examined under fluorescent microscope. Moreover,the growth curves were sketched to determine the negative effects of CM-Dil on the vitality and proliferation of BMSCs cultured in vitro. Rat model of focal cerebral ischemia was reproduced,CM-Dil labeled BMSCs were implanted into corpus striatum of rats' brain with computer-guided stereotaxis thereafter. Brain tissues were obtained to prepare frozen sections at 7th,14th and 21st day post-implantation,and the survival and distribution of labeled cells were observed with fluorescent microscopy. Results The third generation passage of cultured BMSCs grew orderly,with shape of desmocytes,and homogeneous in morphology. The positive rates of CD34,CD44 and CD29 expressions in BMSCs were 1.71%,80.32% and 84.89%,respectively. Red fluorescence was observed in CM-Dil labeled BMSCs in vivo 24 hours after labeling,with a positive rate of 100%. The fluorescence intensity of passage cultured BMSCs observed on day 21 was similar to that observed at 24 hours after labeling,but diminished on day 30. The growth,proliferation and morphology of BMSCs,were not influenced by CM-Dil labeling. On day 7,14 and 21 post-implantation,BMSCs decreased in quantity,appearing in oval or irregular shapes,and most of the cells were found around the needle tract,with a tendency of diffusion to peripheral area. Conclusion High purity of BMSCs may be obtained with whole bone marrow cell culture system from bone marrow of rats. CM-Dil labeling is easy to handle and effective,with no cytotoxicity to cells. The latest labeling period of CM-Dil is 21 days in vitro,therefore it seems to be an effective method for in vivo tracing of BMSCs in brain after implantation.