Kuang Wen-Yong, Zhou Xin-Fu, Zhang Guang-Sen, Liu Li-Hua, Chen Shao-Fang, Li Rui-Juan, Xiao Le
Department of Hematology, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Jun;16(3):633-8.
This study was aimed to investigate the efficiency of 4 different culture media for in vitro culture and expanding adult human bone marrow mesenchymal stem cells (ahBM-MSCs) so as to establish a protocol of culturing and expanding hBM-MSCs and provide exprimental basis for hematopoietic blood stem cell transplantation combined with BM-MSCs. BM-MSCs were obtained from 16 fresh adult human bone marrow aspirate by gradient centrifugation with Ficoll Paque, then cultured in DMEM/F12 with 10% umbilical cord blood serum, 10% fetal calf serum (FCS), human blood serum, and MesenCult culture medium. The surface antigens of BM-MSCs were detected by flow cytometry. BM-MSCs were differentiated into osteoblasts and adipocytes under culture in the conditioned medium special for osteogenesis, and adipogenesis and the differentiated MSCs were identified by morphological observation, immunophenotype and immunohistochemical staining. The results showed that BM-MSCs could be isolated from adult human bone marrow and cultured by all culture media. The effect of umbilical cord blood serum on BM-MSC proliferation and their purity were similar to that of MesenCult culture medium, but better than that of FCS and human blood serum. The positive rate of CD29, CD73, CD105 on BM-MSCs cultured in umbilical cord serum and MesenCult medium was higher than that in FCS and adult human serum (p < 0.05), and the positive rate of CD31 was lower than that in FCS and adult human serum (p < 0.05). The positive rate of BM-MSCs differentiated into osteoblasts and adipocytes under culture in the conditioned medium for osteogenesis and adipogenesis with umbilical cord blood serum and MesenCult culture medium was also higher than that in FCS and adult human serum (p < 0.05). It is concluded that BM-MSCs can be obtained by all the four methods. DMEM/F12 with 10% umbilical cord blood serum and MesenCult culture medium are better than the others for the purification and differentiation potency of BM-MSCs in vitro. The medium with umbilical cord serum is valuable for clinical application in HSCT.
本研究旨在探讨4种不同培养基对成人骨髓间充质干细胞（ahBM-MSCs）进行体外培养和扩增的效率，从而建立hBM-MSCs的培养和扩增方案，并为造血干细胞移植联合BM-MSCs提供实验依据。通过Ficoll Paque梯度离心法从16份新鲜成人骨髓抽吸物中获取BM-MSCs，然后将其培养于含有10%脐带血血清、10%胎牛血清（FCS）、人血清和MesenCult培养基的DMEM/F12中。采用流式细胞术检测BM-MSCs的表面抗原。在成骨和成脂条件培养基中培养时，BM-MSCs分化为成骨细胞和脂肪细胞，并通过形态学观察、免疫表型和免疫组织化学染色对分化后的MSCs进行鉴定。结果显示，BM-MSCs可从成人骨髓中分离出来，并能用所有培养基进行培养。脐带血血清对BM-MSC增殖及其纯度的影响与MesenCult培养基相似，但优于FCS和人血清。在脐带血清和MesenCult培养基中培养的BM-MSCs上，CD29、CD73、CD105的阳性率高于FCS和成人血清中的阳性率（p<0.05），而CD31的阳性率低于FCS和成人血清中的阳性率（p<0.05）。在含脐带血血清和MesenCult培养基的成骨和成脂条件培养基中培养时，BM-MSCs分化为成骨细胞和脂肪细胞的阳性率也高于FCS和成人血清中的阳性率（p<0.05）。结论是，所有这四种方法均可获得BM-MSCs。含有10%脐带血血清的DMEM/F12和MesenCult培养基在体外对BM-MSCs的纯化和分化能力方面优于其他培养基。含脐带血清的培养基在造血干细胞移植的临床应用中具有价值。
