中文摘要：

目的为利用生物反应器,实现脐带血（Umbilical cord blood,UCB）来源的造血干细胞（Hematopoietic stem cells,HSCs）与间充质干细胞（Mesenchymal stem cells,MSCs）的同时扩增与收获。方法为在不添加血清、只添加细胞因子组合（SCF15ng·mL-1,FL5ng·mL-1,TPO6ng·mL-1,IL-315ng·mL-1,G-CSF1ng·mL-1,GM-CSF5ng·mL-1）及海藻酸钙壳聚糖胶珠包被基质细胞支持的条件下,采用玻璃包被的聚苯乙烯（Glass coated styrene copolymer,GCSC）微载体与生物反应器相结合的策略,考察了UCB-HSCs与UCB-MSCs在转瓶及旋转壁式生物反应器（Rotating wall vessel bioreactor,RWVB）内的共培养。结果RWVB中的扩增效果最佳,12天内有核细胞（Nuclear cells,NCs）扩增了3.7±0.3倍;集落形成细胞（Colony-forming units in culture,CFU-Cs）扩增了5.1±1.2倍;CD34＋CD45＋CD105-（HSCs）细胞扩增了5.2±0.4倍;CD34-CD45-CD105＋（MSCs）细胞扩增了13.9±1.2倍。培养结束后,通过自由沉降的方法分离UCB-HSCs和粘附在GCSC微载体表面的UCB-MSCs。同时,细胞多向诱导分化及免疫表型分析结果显示,粘附在GCSC微载体表面上的细胞能够向骨、软骨及脂肪细胞分化;并能够表达间质细胞相关表面标志CD13,CD44,CD73和CD105,而不表达造血细胞的相关表面标志CD34,CD45及HLA-DR,与骨髓MSCs相一致。结论为添加细胞因子、基质细胞及微载体支持的条件下,在生物反应器内能够实现UCB-HSCs和UCB-MSCs的同时扩增与收获。

英文摘要：

Simultaneous expansion and harvest of hematopoietic stem cells（HSCs） and mesenchymal stem cells（MSCs） derived from umbilical cord blood（UCB） were carried out in a bioreactor.The coculture of UCB-HSCs and UCB-MSCs were investigated respectively in spinner flasks and rotating wall vessel bioreactor（RWVB） together with glass coated styrene copolymer（GCSC） microcarriers.The medium is IMDM without serum but supplemented with the combination of cytokines including SCF 15 ng·mL-1,FL 5 ng·mL-1,TPO 6 ng·mL-1,IL-3 15 ng·mL-1,G-CSF 1 ng·mL-1 and GM-CSF 5 ng·mL-1.The stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate chitosan（AC） beads and used as feeding cells.Immunophenotype analysis,methylcellulose colony assay and multi-lineage differentiation of UCB-MSCs were applied to evaluate the quality of the harvested UCB-HSCs and-MSCs.After 12 days culture,the expansions of total cell number,CFU-Cs,CD34＋CD45＋CD105-（HSCs） cells and CD34-CD45-CD105＋（MSCs） cells in RWVB are（3.7±0.3） fold,（5.1±1.2） fold,（5.2±0.4） fold and（13.9±1.2） fold,respectively,which is better than those cocultured in spinner flasks.Since UCB-MSCs are only adhered on the microcarriers,the UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after coculture.At the same time,the fibroblast-like cells appeared on the surface of GCSC microcarriers can be induced and differentiated into osteoblasts,chondrocytes and adipocytes,and can express MSCs-related surface markers of CD13,CD44,CD73 and CD105 and not hematpopietic cells-related surface markers of CD34,CD45 and HLA-DR,which is similar to MSCs derived from bone marrow.The results show that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs can be realized in a feasible bioreactor culture system such as RWVB with GCSC microcarriers.