Teodoro Duarte Garcia Morais, Fernando JorgeEl-Guendouz, SoukainaNeves, RafaelaDuarte, AndreiaRodrigues, Miguel A.Pinho Melo, Eduardo2025-02-132025-02-132024-11-038756-7938http://hdl.handle.net/10400.1/26786Induced pluripotent stem cells (iPSCs) hold large potential on 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, preferable amenable for 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 capability of human iPSCs. Our results demonstrate that the bottom-up freezing under optimized conditions significantly increases iPSCs viability, up to 9% for the cell membrane integrity and up to 21% for the 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 the method’s potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their future use in regenerative medicine.engCryopreservationBottom-up freezingInduced pluripotent stem cellsScale- upjournal article10.1101/2024.10.30.621091