OBJECTIVENoting the morphological and cytobiology characteristics and phenotypes of MMSCs, to establish an isolation and culture method for fetal MMSCs in order to provide a source of marrow mesenchymal stem cells (MMSCs).

METHODSFetal MMSCs were isolated and cultured with in vitro cell culture technique; the characteristics of the proliferating and growing fetal MMSCs were studied with MTT and image analysis; the phenotypes of MMSCs were identified by flow cytometry and immunohistochemistry.

RESULTSBone marrow of 12 fetuses was isolated within 0.5-2 h, and about 300+/-80 adherent cells were obtained at 24 h. Colonies with more than 5 cells were 15+/-6, growth detention period of culture cell was at 1-3 d after planting, log phase growth period was at day 4, and the amount of disintegration phase cells was reduced significantly. Original culture and serial subcultivations showed that cells divided prosperiously; unequal divisions special for stem cells were observed, and the amount of MMSCs harvested from each fetus was as much as 10(11)-10(12) cells after 10 serial subcultivations. The phenotype of MMSCs was CD166 positive and CD34 negative. Serial subcultivated MMSCs expressed a microamount of AFP and did not express albumine or CK18.

CONCLUSIONFetal MMSCs are easily isolated and proliferate prosperouly. Serial subcultivated MMSCs did not differentiate into hepatocyte-like cells under common culture condition and are feasibile as seed cells for tissue engineering reconstruction.