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Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

This study establishes effective clinically compatible strategies for cold (4°C) storage of human pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) cultured as two‐dimensional (2D) monolayers and three‐dimensional (3D) aggregates. hPSC‐CMs are more resistant to prolonged hypothermic storage‐induced cell injury in 3D aggregates than in 2D monolayers, showing high cell recoveries (>70%) and typical ultrastructure, phenotype, and function, after 7 days of storage. To fully explore the potential of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical‐compatible preservation formulation and investigated the time frame that hPSC‐CMs could be subjected to hypothermic storage. We showed that two‐dimensional (2D) monolayers of hPSC‐CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC‐CMs are more resistant to prolonged hypothermic storage‐induced cell injury in three‐dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC‐CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. Significance The applicability of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC‐CMs cultured as two‐dimensional (2D) monolayers and three‐dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC‐CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC‐CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC‐CMs that could be valuable in improving global commercial distribution of hPSC‐CMs.

The production of cardiomyocytes (CMs) from induced pluripotent stem cells (iPSCs) presents great potential for patient-specific regenerative therapies and cardiotoxicity drug evaluation. The successful translation of iPSCs to these fields requires the development of robust bioprocesses capable of producing CMs in high quality, quantity and purity. Traditional protocols for CM differentiation of iPSCs lack control and robustness and are thus inefficient. Furthermore, efficient cryopreservation and hypothermic storage strategies are a demand, as cell banking and transport is a prerequisite for clinical and industrial applications. The main aim of this thesis was the evaluation of different bioreactor systems for the production and purification of miPSC-derived CMs. Novel strategies for CM cryopreservation were tested. Also, CMs were used to study the cardioprotective effect of antioxidant compounds.The wave bioreactor was the most suitable system for CM differentiation, allowing high differentiation yields (60 CMs/input of miPSC) and the production of clinically relevant numbers of CMs (2.3x109 CMs), simultaneously reducing bioprocess duration when compared to stirred tank bioreactors. Produced CMs presented typical structural and functional features. Moreover, CryoStorTMCS10 and FBS+10%DMSO (with ROCKi pretreatment) revealed to be suitable solutions for cryopreservation of miPSC-derived CMs, achieving high cell recoveries after thawing. In addition, HypoThermosol®-FRS enabled hypothermic storage of CMs for up to 7 days. Finally, it was shown that CMs derived from miPSCs present potential to be used in the development of cardioprotective assays.This work demonstrates the establishment of a fully integrated bioprocess, capable of producing high quality miPSC-derived CMs in environmentally controlled bioreactors and ensuring efficient cryopreservation and storage of the produced cells. Hopefully, the knowledge acquired with this work can be translated to human iPSCs, presenting a relevant step forward towards the application of human CMs to clinical and industrial applications, such as cardiac regeneration, disease modeling and cardiotoxicity and cardioprotective cellbased assays.

Portuguese historiography has mostly adopted a pessimistic view regarding the contribution of the railways to the development of country. Yet, railway access helped to increase population concentration and economic development, favoring migration into towns, the growth of pre-existing urban centers, and the emergence of new centers. But railways tended to be more beneficial to regions that were already prosperous and to aggravate the conditions unfavorable to development in areas with greater structural weaknesses.