Evaluating the Response to Cryopreservation of Ovine Fibroblast Spheroids

Cell spheroids are widely studied for their potential applications in tissue engineering and regenerative medicine. The present work investigated the effects of cryopreservation on spheroids derived from ovine fibroblasts, depending on spheroid size (140 or 220 µm). Specifically, it explored how cryopreservation impacted several biological and physical parameters including cell damage, viability, metabolism, adhesion, proliferation, and spheroid mass density, weight, and diameter at three time points after thawing. A Live/Dead assay provided a visual assessment of cell damage, cell viability and metabolic activity were assessed by an Alamar Blue assay, and a replating assay evaluated cell adhesion and proliferation capabilities. Spheroid mass density, weight, and diameter were quantified by the W8 Biophysical Analyzer, creating accurate biophysical profiles. Real-time PCR (RT-PCR) analysis was employed to uncover gene expression changes following cryopreservation. Our findings indicate that spheroids measuring 140 µm in diameter largely maintained their biophysical features and cell viability post-cryopreservation, whereas those at 220 µm exhibited a decline in both vitality and mass density. The reduced vitality of 220 µm spheroids likely reflects size-related limitations in cryoprotectant diffusion and stress within the core. Overall, this study provides a comprehensive understanding of how cryopreservation affects ovine fibroblast spheroid biophysics and cellular integrity, laying the groundwork for improved preservation techniques for cell spheroids.