A prototype closed-system platform for iPSC culture process design

Abstract To facilitate translation of induced pluripotent stem cell (iPSC) processes from laboratory development to commercial manufacturing, we developed a prototype closed-system platform that enables two-dimensional culture inside a manufacturing-compatible vessel. The system incorporates multilayer plates within a closed G-Rex vessel, allowing cell seeding, iPSC establishment, differentiation induction, and culture-supernatant analysis under closed conditions. Seeding 1 × 10 6 cells yielded 1.64 × 10 5 adherent cells across 19 plates, corresponding to 16.4% adhesion, with 18.0% variation among plates. TRA-1-60-positive iPSC colonies were detected on day 19, with 0.79 ± 1.17 colonies per plate, indicating successful reprogramming in the closed vessel. Next, the functional requirements for the culture plates used in the commercial production of therapeutic cells were designed. For passaging, thermo-responsive Smart Surface Culture Ware (SSCW) supported strong adhesion at 37 °C and efficient detachment at 25 °C; for example, at 0.69 µL/mL iMatrix-511, adhesion scores changed from 5.00 ± 0.00 to 1.50 ± 0.52. iPSCs cultured on SSCW maintained quality through five temperature-responsive passages, showed OCT3/4 positivity above 93%, and exhibited no detectable karyotypic abnormalities. These results demonstrate that this platform supports iPSC establishment, expansion, and differentiation in a closed culture format while preserving key pluripotency and quality characteristics.