Scaling iPSC production in different 3D platforms in suspension culture for their use in allogeneic regenerative therapies

Human induced pluripotent stem cells (iPSCs) exhibit significant potential for regenerative medical treatments due to their pluripotency and proliferation capacity. However, the large-scale production required for allogeneic therapies necessitates efficient and scalable culture systems. Conventional 2D culture techniques are inadequate for this, driving interest for innovative bioreactor-based expansion techniques such as microcarrier-based or aggregate cultures. In this study, we compare iPSC expansion and quality in microcarrier and aggregate cultures in spinner flasks, aiming to ascertain which 3D growth configuration yields superior cell mass while maintaining cellular quality. We demonstrated comparable expansion of iPSCs on Cytodex 1 and Cultisphere G microcarriers, as well as in aggregates by day 6 in culture, achieving a final cell density of $2.6*{10}^6$cells/ml and $5.67*{10}^6$ cells/ml on microcarriers respectively, and $9.76*{10}^6$ cells/ml in aggregates, yielding 4- and 9-fold expansion on microcarriers and 15-fold in aggregates. The efficiency in the recovery of iPSCs as single cells was over 91.5 % for all cultures. iPSCs cultured on microcarriers and as aggregates exhibit OCT3/4, SSEA4, TRA-1–60, and SOX2 pluripotency marker expression, as well as retention of differentiation potential into three germinal layers. This work supports the utilization of microcarriers and aggregates as efficient 3D expansion platforms for suspension cultures of iPSCs.