A Simple Cryopreservation Method for Efficient Isolation of Live Cells from Dead Animals

Abstract. Cryopreservation of somatic tissues and cells can be applied to biodiversity conservation. Although vitrification is widely used for tissue cryopreservation, it is challenging to obtain viable cells in facilities that lack adequate experimental tools, such as zoos. In this study, we established a simple tissue cryopreservation method for obtaining viable cells. Using mouse tissues of the ears and skin, we explored the conditions suitable for cryopreservation. After freezing, the tissues were thawed, and the cells were isolated. The tissues were then cut into small pieces to obtain viable cells. The use of a cryopreservative solution and freezing at –80°C increased the probability of obtaining viable cells. Viable cells were obtained and cultured even after the ear tissues were stored at room temperature for 24 h. Our method allowed primary cells to be isolated and cultured from ear tissues of dead animal. Further, we examined whether cells isolated from cryopreserved tissues could be studied in vitro. We found that treatment with lipopolysaccharides and Poly I:C increased the mRNA expression of pro-inflammatory cytokines in wild boar cells. These data suggest that the simple cryopreservation method developed here can be applied to biodiversity conservation and basic science studies of wild animal cells.