Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
Department of Chemical Systems Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
Commun Biol. 2021 Nov 19;4(1):1316. doi: 10.1038/s42003-021-02848-x.
Three-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.2 to 4×10 cells/mL. The high-density expansion was supported by reduction of excessive shear stress and agglomeration mediated by the addition of the functional polymer FP003. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous Nodal, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, the cost reduced in inverse proportional manner with the cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility of cost-effective mass production of hiPSCs in high-density culture.
三维聚集体悬浮培养是一种潜在的生物制造方法，可以大量生产人诱导多能干细胞（hiPSC）；然而，昂贵的生长因子的使用和方法诱导的机械应力分别可能导致低效的生产成本和难以保持多能性。在这里，我们开发了一种简单、微型化的双室透析培养装置，该装置基于带有深孔板的常规膜培养插入物。该装置将细胞扩增提高到约~3.2 至 4×10 个细胞/ml。通过添加功能聚合物 FP003 减少了过高的剪切应力和团聚，从而支持高密度扩增。结果表明，几种生长因子（包括成纤维细胞生长因子 2 和胰岛素）的积累，以及作为耗尽的转化生长因子-β1 的替代物的内源性 Nodal，作用是维持多能性。由于我们在上层培养室中每体积使用相同的生长因子配方，因此成本随细胞密度成反比降低。我们表明，在低剪切应力环境中生长因子积累的动力学成功地提高了 hiPSC 的增殖、多能性和分化潜能。这种微型透析培养系统证明了在高密度培养中以经济高效的方式大规模生产 hiPSC 的可行性。
