免疫隔离动态共培养环境中多参数支持和调控CD34+细胞有效扩增的协同作用机制

A handful of CD34+ cells play an increasingly important role in the fields of fundamental research and cell therapy. Up to now, the key problem is how to effectively achieve the effective expansion of CD34+ cells while maintaining their stemness. This project aims to design and construct an immunoisolated co-culture bioreactor system (ICBS) with different shear stress environment for the co-culture of human-derived osteoblasts (OB) and umbilical cord blood CD34+ cells. To design, optimize and prepare a polycarbonate intersaeptum for the immunoisolation and mass transfer enhancement, the effect of the intersaeptum on the flow field distribution, shear stress variance and transfer efficiency of cell factors in both sides of chamber will be integratively investigated from both simulative calculation and experimental testing. The relationship between the releasing efficiency of BMP factors and the mixture rate of bone powder/PLGA microsphere, the surface and internal structure as well as the culture environment will be further considered. Based on this, the intrinsic correlation between the variation of above-mentioned factors and the status of hemopoietic enhancing factor secreted by OB will be further explored. Moreover, we also need to confirm the protection of lower shear stress and the immunoisolation of special double-wall design and calcium alginate/gelatin microbeads in ICBS towards CD 34+ cells, and to confirm the synchronization effect of the presence of bio-derived bone powder for accelerating the secretion of hemopoietic enhancing factor secreted by OB and maintaining the stemness of CD 34+ cells effectively. The synergistic effect of the coupled multiparameter on the effective expansion of CD34+ cells will be explored in the way of design of experiment.

数量稀少的CD34+细胞，在基础研究和细胞治疗中起着愈加重要的作用。如何在维持其干性的基础上实现有效扩增，是亟待解决的关键问题。本项目拟设计和构建一种在不同剪切应力环境中成骨细胞(OB)支持和调控CD34+细胞免疫隔离动态共培养的生物反应器系统(ICBS)。设计、优化和制备起免疫隔离和强化传质作用的聚碳酸酯隔膜，从模拟计算和试验两方面综合探究隔膜对两侧培养室的流场分布、剪切应力和动压变化及细胞因子传递效率的影响。考虑衍生骨粉/PLGA复合微球载体的复合比例、表面和内部结构的变化等与BMP因子释放效率的关系；在此基础上，探究上述因素变化与OB分泌促造血因子状态的内在关联，验证ICBS的双筒设计和海藻酸钙/明胶微珠对CD34+细胞的低剪切力保护和免疫隔离效果，证实衍生骨粉对OB分泌促造血因子和CD34+细胞干性维持的促进作用；通过试验设计探究多参数整合对CD34+细胞有效扩增的协同作用机制。

数量稀少的CD34+细胞，在基础研究和细胞治疗中起着愈加重要的作用。如何在维持其干性的基础上实现有效扩增，是亟待解决的关键问题。本研究从成骨细胞和CD34+细胞的免疫隔离、对载体要求和剪切力耐受程度不同等因素综合考虑，制备了一种用于两种细胞在不同切应力及动压环境中免疫隔离共培养的生物反应器系统(ICBS)。分别完成了骨粉/PLA复合微球粘附成骨细胞和海藻酸钙/明胶微珠包埋CD34+细胞在ICBS低氧环境，对照组中ICBS常氧、RWVB低氧(无基质细胞)和静态共培养等体系中的试验和数值模拟。结果发现：成骨细胞因ICBS产生的周期性应力刺激加速分泌促造血因子，利于CD34+细胞的干性维持并显著降低其对细胞因子的依赖性。一周共培养后，ICBS低氧系统中CD34+细胞大型集落致密、分布集中且均匀。CD34+细胞仍分别具有88.5±4.6(**P<0.01)、82.1±4.1、77.2±6.6和62.6±3.9的活性；成骨细胞具有94.2±1.5、89.4±1.6和92.2±1.8的活性。CD34+细胞的扩增倍数分别为：76.3±7.4(ICBS低氧，**P<0.01)、23.7±6.8(3D常氧)、26.9±4.4(RWVB低氧无基质细胞)和7.8±1.5(2D静态)。CD34+细胞的阳性细胞含量分别为：14.3±2.6(3D低氧ICBS，**P<0.01)、26.9±4.3(RWVB低氧，无基质细胞)和40.1±5.7(2D静态)。而CFU-Cs扩增倍数分别为：32.86±4.24(3D低氧ICBS，**P<0.01)、14.53±3.20(RWVB低氧无基质细胞)和9.86±1.83(2D静态)。本研究进一步证实整合了免疫隔离动态共培养、衍生骨粉/PLA微球控释细胞因子、基质细胞支持与调控和低氧环境等多种参数协同作用的动态培养系统可以实现CD34+细胞的有效扩增。