Studio sulla crioconservazione: dalle cellule somatiche alle cellule staminali

The challenging theme of cryopreservation was the focus of this PhD project. Different experimental approaches explored this topic in three ovine cell types of somatic, stem and germinal origin.Skin fibroblasts were cryopreserved at 5°C/min. Cryomicroscopy analysis allowed to observe dynamics of ice formation and to calculate the probability of intracellular ice formation. Gene expression levels of some markers involved in metabolism, apoptosis and pathways of thermal stress were investigated at 0, 24 and 48 hours after thawing, showing that post-thaw in vitro culture could improve cryopreservation outcome.The second experiment evaluated stem properties and differentiation capacity of primary isolated Wharton’s jelly cells and their ability to keep the original characteristics after cryopreservation. Three freezing methods were compared. According to gene expression and immunohistochemistry analysis, the slow and controlled freezing better retained the original cell characteristics than the other two studied methods.Final experiment was a pilot study aimed to compare standard sperm freezing with a partial freeze-drying method (PFD). Cell motility and physical events as ice formation and the amount of the unfrozen fraction were evaluated via cryomicroscope and environmental scanning electron microscocope. Data showed that PFD was better in preserving motility and ensuring a higher unfrozen fraction, limiting severe damages related to ice growth and mechanical pressure.