Bewaring van een geavanceerd menselijk hartmodel bij kamertemperatuur voor biomedisch en ruimteonderzoek: verder dan cryogene bewaarstrategieën

Current preservation strategies of biological cells and advanced three-dimensional (3D) in vitro models rely predominantly on cryopreservation, a method that, despite its widespread use, has significant limitations. These include dependence on specialized infrastructure, strict temperature control, and the use of cryoprotective agents (CPAs) that can induce cytotoxicity, osmotic stress, and structural damage. Such challenges are particularly pronounced in complex multicellular systems, such as cardiac organoids, where structural heterogeneity and diffusion constraints lead to inconsistent preservation outcomes and high post-thaw variability. These limitations highlight the need for alternative, scalable, and low-resource preservation strategies, especially for applications requiring robust transport and storage solutions, including space-related biological research. This thesis explores ambient temperature-based preservation as a promising alternative to conventional cryopreservation. The study addressed two primary objectives: (1) to evaluate whether human 3D cardiac organoids can be preserved at ambient temperature while maintaining viability and functional performance upon recovery, and (2) to assess the impact of simulated space stressors, specifically microgravity and ionizing radiation, on preserved versus non-preserved organoids. Following model validation, cardiac organoids were identified as a robust platform due to their stable phenotype, sustained proliferative capacity, and intrinsic contractile activity, enabling real-time functional assessment. Comparative analyses revealed that conventional cryopreservation using CryoStor®CS10 resulted in poor recovery, characterized by reduced organoid size, decreased adenosine triphosphate (ATP) levels, and increased apoptosis, with no functional improvement observed even after extended recovery time. In contrast, ambient preservation for up to seven days using CellShip® transport medium maintained organoid structural integrity, viability, and functionality, with rapid recovery of beating activity upon re-incubation. Extended preservation for fourteen days was feasible but required longer recovery times, suggesting the induction of a reversible low-metabolic state during preservation. Under simulated space conditions, microgravity alone had minimal impact on organoid viability and function, whereas ionizing radiation emerged as the primary driver of cellular damage. Notably, ambient-preserved organoids showed delayed and reduced functional impairment upon exposure to space stressors compared to non-preserved controls and retained contractile activity following irradiation. Combined exposure to ionizing radiation and simulated microgravity did not produce synergistic effects beyond those observed for individual stressors. These findings suggest that ambient preservation does not compromise, and may even transiently buffer, organoid responses to simulated space-related environmental stress. In conclusion, this work demonstrates that ambient temperature preservation is a viable and effective alternative to cryopreservation for complex 3D cardiac models. By maintaining viability and functional properties upon recovery while reducing logistical constraints, this strategy offers significant advantages for experimental reproducibility, scalability, and transportability. Importantly, it enables the development of ready-to-use biological systems for space biology applications, supporting future research on human physiology in extraterrestrial environments. This thesis contributes to advancing preservation technologies towards more practical and versatile solutions for both terrestrial and space-related biomedical research.