Development of three-dimensional cell culture bioprocesses

固定化细胞培养的应用范围从益生菌的稳定到组织工程和细胞治疗。材料内细胞的3D组织影响营养和废物浓度梯度，从而影响产品性能。此外，制造复杂的3D结构，同时优化细胞功能和材料特性目前是具有挑战性的。拟议研究计划的长期目标是开发生物工艺，以在复杂的3D环境中扩大细胞的生产。该计划的5年目标是开发将胰腺细胞簇包封在水凝胶珠（如藻酸盐珠）中的方法。藻酸盐是一种带负电荷的线性多糖，用于治疗糖尿病的囊化胰岛移植的临床试验，并广泛用于其他几种细胞治疗应用。藻酸盐乳化和内部凝胶化是一种简单而强大的藻酸盐珠生产方法，我以前已经适应哺乳动物细胞固定化。该工艺具有高度可扩展性，但生产的珠粒具有宽的珠粒尺寸分布，这导致珠粒之间在机械性质和性能上的差异。到目前为止，作为工艺参数的函数的细胞存活和珠特性的优化主要依赖于经验观察。为了获得具有良好控制的机械性能和对包封细胞的影响的单分散珠，所提出的方法是开发一种新的基于微通道包封的细胞包封过程。短期目标是（1）模拟水凝胶珠粒形成动力学，（2）开发和（3）优化用于细胞包封的基于微通道微囊化的工艺，然后（4）测试所得珠粒性质对胰腺细胞功能的影响。这些新的细胞固定化策略代表了在具有良好控制的物理性质的水凝胶中进行可缩放的三维细胞培养的显著进步。这些新的生物过程可以应用于其他需要三维支架的细胞类型，以最大限度地发挥所需的生物功能。这项研究计划解决了细胞固定化生物过程的关键限制。

Applications of immobilized cell culture range from the stabilization of probiotic bacteria to tissue engineering and cellular therapy. The 3D organisation of the cells within the materials affects nutrient and waste product concentration gradients, and hence product performance. In addition, manufacturing complex 3D constructs while optimizing both cellular function and material properties is currently challenging. The long-term goal of the proposed research program is to develop bioprocesses to scale up the production of cells in complex 3D environments. The 5-year goal of this program is to develop methods to encapsulate pancreatic cell clusters in hydrogel beads such as alginate beads. Alginate is a negatively charged linear polysaccharide used in clinical trials of encapsulated pancreatic islet transplantation to treat diabetes, and is broadly used in several other cellular therapy applications. Alginate emulsification and internal gelation is a simple and robust alginate bead production method that I have previously adapted to mammalian cell immobilisation. The process is highly scaleable, but produces beads with a broad bead size distribution, which leads to between-bead differences in mechanical properties and performance. So far, the optimisation of cell survival and bead properties as a function of process parameters has relied mainly on empirical observations. To obtain monodisperse beads with well-controlled mechanical properties and effects on the encapsulated cells, the proposed approach is to develop a novel microchannel emulsification-based cell encapsulation process. The short-term objectives are to (1) model hydrogel bead formation kinetics, (2) develop and (3) optimise a microchannel emulsification-based process for cell encapsulation, and then (4) test the effect of the resulting bead properties on pancreatic cell function. These novel cell immobilisation strategies represent a significant advance towards scaleable three-dimensional cell culture in hydrogels with well-controlled physical properties. These novel bioprocesses could be applied to other cell types that require three-dimensional scaffolds to maximize desired biological functions. This research program addresses key limitations of cell immobilisation bioprocesses.