Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, China.
J Neurosci Methods. 2012 Jul 30;209(1):241-9. doi: 10.1016/j.jneumeth.2012.05.032. Epub 2012 Jun 9.
Oligodendrocyte progenitor cell (OPC) culture has provided a powerful approach to mechanistically investigate the proliferation and differentiation of oligodendroglia. However, existing culture methods (including the traditional shake-off method) have limitations, particularly their low productivities. Therefore, we developed a simplified and highly efficient method to produce a large yield of OPCs with low expense by using specialised modified media, in which B104-conditioned medium (B104-CM) instead of growth factors was used as a mitogenic source for OPC propagation, while a modified OPC isolation-medium was applied to improve the isolation of OPCs. First, we withdrew foetal bovine serum when primary mixed glial cultures were 65-75% confluent and substituted with modified oligodendrocyte growth medium to enrich OPCs. Second, we employed a chemical-based method to isolate and purify OPCs from mixed glial cultures using a modified oligodendrocyte isolation medium. As a result, our approach produced a high yield of purified OPCs, approximately 90-fold higher than that produced via the traditional shake-off method. Importantly, the purified OPCs produced via our modified approach maintained typical capacities of proliferation and differentiation observed in oligodendrocyte lineage cells. Together, our modified method provides a highly efficient approach to OPC culture for oligodendrocyte research.
少突胶质前体细胞(OPC)培养为研究少突胶质细胞的增殖和分化提供了一种强有力的方法。然而,现有的培养方法(包括传统的摇落法)存在局限性,特别是其产量低。因此,我们开发了一种简化且高效的方法,使用专门的改良培养基,以低费用产生大量 OPC,其中 B104 条件培养基(B104-CM)代替生长因子作为 OPC 增殖的有丝分裂源,同时应用改良的 OPC 分离培养基来提高 OPC 的分离效率。首先,当原代混合神经胶质培养物达到 65-75%汇合时,我们撤去胎牛血清并用改良的少突胶质细胞生长培养基代替,以富集 OPC。其次,我们采用基于化学的方法,使用改良的少突胶质细胞分离培养基从混合神经胶质培养物中分离和纯化 OPC。结果,我们的方法产生了高产量的纯化 OPC,比传统的摇落法高约 90 倍。重要的是,通过我们的改良方法产生的纯化 OPC 保持了在少突胶质细胞谱系细胞中观察到的典型增殖和分化能力。总之,我们的改良方法为少突胶质细胞研究提供了一种高效的 OPC 培养方法。