Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors

Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome the scale-up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need for large-scale culture in regenerative medicine. Despite constant improvements, current protocols that use microcarriers or generate cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies significantly improve viability and expansion rates while maintaining pluripotency compared to standard hPSC culture platforms such as 2D cultures, microcarriers, and aggregates. By further tuning capsule size and culture conditions, we scale up this method to industrial-scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 277-fold in 6.5 days. In brief, our findings indicate that our 3D culture system offers a suitable strategy both for basic stem cell biology experiments and for clinical applications.