Cryopreservation includes a set of techniques aimed at storing biological samples and preserving their biochemical and functional features without any significant alterations. This study set out to investigate the effects induced by cryopreservation on cultured sheepskin fibroblasts (CSSF) through cryomicroscopy and gene expression analysis after subsequent in vitro culture. CSSF cells were cryopreserved in a cryomicroscope (CM) or in a straw programmable freezer (SPF) using a similar thermal profile (cooling rate -5°C/min to -120°C, then -150°C/min to -196°C). CSSF volume and intracellular ice formation (IIF) were monitored by a CM, while gene expression levels were investigated by real-time polymerase chain reaction in SPF-cryopreserved cells immediately after thawing (T0) and after 24 or 48 hours (T24, T48) of post-thaw in vitro culture. No significant difference in cell viability was observed at T0 between CM and SPF samples, while both CM and SPF groups showed lower viability (p < 0.05) compared to the untreated control group. Gene expression analysis of cryopreserved CSSF 24 and 48 hours post-thawing showed a significant upregulation of the genes involved in protein folding and antioxidant mechanisms (HPS90b and SOD1), while a transient increase (p < 0.05) in the expression levels of OCT4, BCL2, and GAPDH was detected 24 hours post-thawing. Overall, our data suggest that cryostored CSSF need at least 24 hours to activate specific networks to promote cell readaptation.

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