Cryopreservation of heat-shocked canine adipose-derived mesenchymal stromal cells with 10% dimethyl sulfoxide and 40% serum results in better viability, proliferation, anti-oxidation, and in-vitro differentiation

Abstract

Cryopreserved canine adipose-derived mesenchymal stromal cells (Ad-MSCs) can be used instantly in dogs for clinical uses. However, cryopreservation results in a reduction of the cellular viability, proliferation, and anti-oxidation of post-thawed Ad-MSCs. Therefore, there is a need for in-vitro procedure to improve post-thawed Ad-MSCs’ viability, proliferation, anti-oxidation, and differentiation capacity. In this study, fresh-Ad-MSCs were activated with heat shock, hypoxia (5% O2), or hypoxia (5% O2) + heat shock treatments. The results showed that compared to the other treatments, heat shock significantly improved the proliferation rate, anti-oxidation, heat shock proteins and growth factors expressions of canine-fresh-Ad-MSCs. Consequently, fresh-Ad-MSCs were heat-shocked and then cryopreserved with different combinations of dimethyl sulfoxide (Me2SO) and fetal bovine serum (FBS) to determine the combination that could effectively preserve the cellular viability, proliferation, anti-oxidation and differentiation capacity of Ad-MSCs after cryopreservation. We found that C-HST-Ad-MSCs cryopreserved with 10% Me2SO + 40% FBS presented significantly (p < 0.05) improved cellular viability, proliferation rate, anti-oxidant capacity, and differentiation potential as compared to C-HST-Ad-MSCs cryopreserved with 1% Me2SO + 10% FBS or 1% Me2SO alone or control. We concluded, heat shock treatment is much better to enhance the characteristics of fresh-Ad-MSCs than other treatments, moreover, C-HST-Ad-MSCs in 10% Me2SO + 40% FBS showed better results compared to other cryopreserved groups. However, future work is required to optimize the expression of heat shock proteins, which would further improve the characteristics of fresh- and cryopreserved-HST-Ad-MSCs and reduce the dependency on Me2SO and FBS.