Billion‐Scale Expansion of Functional hiPSC‐Derived Cardiomyocytes in Bioreactors Through Oxygen Control and Continuous Wnt Activation

Abstract Generation of upscaled quantities of human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM), for therapeutic or testing applications, is both expensive and time‐consuming. Herein, a scalable bioprocess for hiPSC‐CM expansion in stirred‐tank bioreactors (STB) is developed. By combining the continuous activation of the Wnt pathway, through perfusion of CHIR99021, within a mild hypoxia environment, the expansion of hiPSC‐CM as aggregates is maximized, reaching 4 billion of pure hiPSC‐CM in 2L STB. In particular, the importance of i) controlling the dissolved oxygen at 10% O 2 to reduce reactive oxygen species production and upregulate genes involved in cell proliferation, resulting in higher expansion rates (tenfold) compared to normoxic conditions, and ii) maintaining constant power input per volume as a scale‐up criteria is demonstrated. After expansion, hiPSC‐CM further mature in culture, revealing more mature transcriptional signatures, higher sarcomere alignment and improved calcium handling. This new bioprocess opens the door to time‐ and cost‐effective generation of hiPSC‐CM.