Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes

Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 10⁶ hiPSCs per layer yielded 7.2 × 10⁸ hiPSCs in 4-layer CPs and 1.7 × 10⁹ hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 10⁸ cells (4-layer) and 1.5–2.8 × 10⁹ cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future. In this article, Fujita and colleagues show that human induced pluripotent stem cells (hiPSCs) and hiPSC-derived cardiomyocytes (hiPSC-CMs) are stably and efficiently produced using multilayer culture plates with an active gas ventilation system because of stable CO₂ and O₂ concentration in culture media. Large numbers of obtained hiPSC-CMs are metabolically purified under glucose- and glutamine-depleted and lactate-supplemented conditions.