# Evaluating the Response to Cryopreservation of Ovine Fibroblast Spheroids

## TL;DR

This study shows that small sheep skin cell spheroids survive freezing better than larger ones, which suffer damage and cell death in their centers.

## Contribution

The study identifies a size threshold for cryopreservation success in ovine fibroblast spheroids.

## Key findings

- Small spheroids (140 μm) retained viability and function after freezing, while large ones (220 μm) showed core cell death.
- Cryopreservation altered gene expression, upregulating stress genes and downregulating anti-apoptotic genes.
- Biophysical properties like mass density and weight were preserved in small spheroids but declined in large ones.

## Abstract

Long-term storage of three-dimensional cell cultures is essential for tissue engineering and regenerative medicine, yet freezing can damage these complex structures. We produced two sets of spherical aggregates of sheep skin cells, measuring about 140 and 220 μm in diameter, and preserved them by slow freezing in a solution containing dimethyl sulfoxide to limit ice formation. After thawing we quantified survival, energy metabolism, capacity to attach and spread on a surface, weight, mass density and diameter, and we examined genes that signal stress or cell death. Small aggregates rapidly regained normal metabolism, formed continuous cell layers within twenty-four hours and maintained both physical integrity and balanced gene activity. Large aggregates lost compactness and weight, showed extensive cell death in their centers and produced stable layers in only seventy-five per cent of samples. At the molecular level, cryopreservation upregulated stress-related genes such as HSPA1A (HSP70) and HSP90AB1, while downregulating the anti-apoptotic gene BCL2, These findings reveal a clear size threshold beyond which cryopreservation compromises viability, offering a practical guideline for building reliable cell banks that can advance wound-repair research, implant design and veterinary therapies.

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.