【摘要】 为了构建一种新型的造血干细胞和基质细胞动态共培养体系,脐带血单个核细胞和包埋有兔骨髓间充质干细胞的海藻酸钠-壳聚糖-海藻酸钠(alginate-chitosan-alginate,ACA)微胶囊在旋转壁式生物反应器(rotating wall vessel,RWV)中进行了7d动态悬浮共培养。培养液不含血清,补充多种生长因子(SCF50ng·mL-1,FL50ng·mL-1,TPO50ng·mL-1及IL-325ng·mL-1)。每24h进行总有核细胞计数,并测量培养液的pH和渗透压,在0h、72h和168h进行流式CD34+细胞分析以及集落形成能力检验。结果表明在RWV动态共培养过程中,培养液的pH保持在7.2~7.4,渗透压保持在280~310mmOsmol·kg-1,均适合造血干/祖细胞的体外扩增。经过7d的无血清动态共培养,总有核细胞、CD34+细胞以及混合集落(colony-formingunitsinculture,CFU-Cs)分别扩增了107.05±6.08,26.52±1.5和19.2±3.18倍。这种新型的动态微囊化共培养体系支持了造血干/祖细胞的大规模体外扩增,基质细胞抑制了造血干/祖细胞的分化,微胶囊对造血细胞和基质细胞进行了免疫隔离,RWV则为细胞和微胶囊提供了优良的低剪切力的悬浮培养环境。更多还原

【Abstract】 Coculture of cord blood mononuclear cells (CB MNCs) and alginate-chitosan-alginate (ACA) microcapsules containing rabbit bone marrow (BM) mesenchymal stem cells (MSCs) was carried out in a rotating wall vessel (RWV) bioreactor for 7 days to construct a novel dynamic coculture system. The medium was supplemented with conventional doses of purified recombinant human cytokines (SCF 50 ng;/ mL-1, FL 50 ng·mL-1, TPO 50 ng·mL-1 and IL-3 25 ng·mL-1) without any serum. The total nucleated cell density, pH and osmolality of the culture medium in the bioreactor were measured every 24 hours. Flow cytometric assay for CD34+ cells and methylcellulose colony assays (colony-forming units in culture, CFU-Cs) were carried out at 0h, 72 h and 168 h, respectively. Results show that the pH of the medium in the bioreactor maintains between 7.2~7.4 and the osmolality is between 280~310 mmOsmol·kg-1, both of which are in the proper ranges for ex vivo expansion of hematopoietic stem cells (HSCs) and progenitors during the dynamic coculture. And at the end of 168 h culture, the total nucleated cell number is multipled by 107.05±6.08 fold, CD34+ cells 26.52±1.5fold, and CFU-Cs 19.2±3.18 fold. This novel dynamic microencapsulated coculture system supports the large-scale ex vivo expansion of HSCs and progenitors. In this system, the stromal cells inhibit the HSCs differentiation, the microcapsules protect cells from immunreaction and the RWV provides a better suspension culture environment with low shear force for cells and microcapsules.更多还原