Browsing by Author "Rodrigues, Miguel A."
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- Enhancing cryopreservation of human induced pluripotent stem cells: bottom‐up versus conventional freezing geometryPublication . Teodoro Duarte Garcia Morais, Fernando Jorge; El-Guendouz, Soukaina; Neves, Rafaela; Duarte, Andreia; Rodrigues, Miguel A.; Pinho Melo, EduardoInduced 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.
- Enhancing cryopreservation of human iPSCs: Bottom-up vs Conventional freezing geometryPublication . Teodoro Duarte Garcia Morais, Fernando Jorge; El-Guendouz, Soukaina; Neves, Rafaela; Duarte, Andreia; Rodrigues, Miguel A.; Pinho Melo, EduardoInduced 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.
- Stability of protein formulations at subzero temperatures by Isochoric CoolingPublication . Tavares, Evandro; Lopes, Carlos; Silva, Joana G.; Duarte, Andreia; Geraldes, Vitor; Rodrigues, Miguel A.; Melo, Eduardo; Correia, CátiaOptimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP β-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.