Department of Physiology and Biomedical Engineering, Mayo Clinic.
Department of Physiology and Biomedical Engineering, Mayo Clinic;
J Vis Exp. 2021 Oct 13(176). doi: 10.3791/62944.
Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 µm diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with >95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had <50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.
三维（3D）或球体培养人多能干细胞（hPSCs）提供了改善分化结果和可扩展性的优势。在本文中，我们描述了一种策略，用于稳健且可重复地形成 hPSC 球体，其中同轴流聚焦装置用于将 hPSCs 困在核壳微胶囊内。核心溶液包含单细胞悬浮 hPSCs，并通过掺入高分子量聚乙二醇（PEG）和密度梯度介质使其粘稠。壳流由 PEG-4 臂马来酰亚胺或 PEG-4-Mal 组成，并与核心流一起流向两个连续的油接头。在第一个油接头处形成液滴，壳溶液包裹在核心周围。在第二个油接头处通过向这些液滴中引入二硫醇交联剂（1,4-二硫苏糖醇或 DTT）发生壳交联。交联剂通过点击化学与马来酰亚胺官能团反应，导致在微胶囊周围形成水凝胶壳。我们的封装技术以每秒 10 个胶囊的速度产生 400 µm 直径的胶囊。所得的胶囊具有水凝胶壳和水核，允许单细胞迅速组装成聚集体并形成球体。封装过程不会对 hPSCs 的活力产生不利影响，封装后 3 天观察到活力>95%。相比之下，封装在固体凝胶微球中的 hPSCs（没有水核）没有形成球体，封装后 3 天活力<50%。hPSCs 在核壳微胶囊内的球体形成在封装后 48 小时内发生，球体直径是细胞接种密度的函数。总体而言，本文所述的微流控封装技术非常适合 hPSC 封装和球体形成。
