Vitrification of Dog Skin Tissue as a Source of Mesenchymal Stem Cells

Abstract

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.

Testing of various protocols for human skin cryopreservation by vitrification method

“…The results obtained are generally consistent with the literature data, since a similar combination of extracellular and intracellular CPA had previously been successfully used in several studies of skin vitrification (Silvestre et al, 2002; Son et al, 2021). However, in this work, PEG polymer was used as an extracellular CPA for skin cryopreservation for the first time.…”

“…For vitrification of the epidermis, the combination of DMSO, PEG and sucrose was less effective, Δ(MTT value) = -40%, but the viability of thawed samples remained at an acceptable level. The results obtained are generally consistent with the literature data, since a similar combination of extracellular and intracellular CPA had previously been successfully used in several studies of skin vitrification (Silvestre et al, 2002;Son et al, 2021). However, in this work, PEG polymer was used as an extracellular CPA for skin cryopreservation for the first time.…”

“…In several other studies, PEG in combination with other substances had also been successfully used for vitrification (O’Neil et al, 1997; Derakhshan et al, 2017; Jung et al, 2021). Sucrose also belongs to non-penetrating CPA and is often used in combination with other substances for skin vitrification (Fujita et al, 2000; Silvestre et al, 2002; Son et al, 2021).…”

“…Due to their relatively thinness and relatively low density, human skin and its components, such as epithelium, are a common biological object, for which various cryopreservation techniques can be effectively applied, and many cryopreservation protocols have been developed. In this work, several types of cryopreservants were tested, which included combinations of CPA often used for skin vitrification (Tian et al, 2019; Zhu, 1991; Zieger et al, 1997; Son et al, 2021).…”

“…various cryopreservation techniques can be effectively applied, and many cryopreservation protocols have been developed. In this work, several types of cryopreservants were tested, which included combinations of CPA often used for skin vitrification (Tian et al, 2019;Zhu, 1991;Zieger et al, 1997;Son et al, 2021).…”