Morais, Fernando Jorge Teodoro Duarte GarciaEl-Guendouz, SoukainaNeves, RafaelaDuarte, AndreiaRodrigues, Miguel A.Pinho Melo, Eduardo2025-03-212025-03-212025-03-128756-7938http://hdl.handle.net/10400.1/26940Induced pluripotent stem cells (iPSCs) hold large potential in regenerative medicine due to their pluripotency and unlimited self-renewal capacity without the ethical issues of embryonic stem cells. To provide quality-controlled iPSCs for clinical therapies, it is essential to develop safe cryopreservation protocols for long-term storage, preferably amenable to scale-up and automation. We have compared the impact of two different freezing geometries (bottom-up and conventional radial freezing) on the viability and differentiation potential of human iPSCs. Our results demonstrate that bottom-up freezing under optimized conditions significantly increases iPSC viability, up to 9% for cell membrane integrity and up to 21% for cell metabolic state, compared to conventional freezing. The improvement achieved for bottom-up versus conventional freezing was maintained after scale-up from cryogenic vials to 30 mL bags, highlighting its potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their application in regenerative medicine.engBottom-up freezingCryopreservationInduced pluripotent stem cellsScale-upjournal article10.1002/btpr.700191520-6033