Cryopreservation Strategy for Adipose Tissue and Adipose Tissue Derived Stem Cells by Developing Non-Toxic Freezing Solutions

Abstract

The present research focuses on the development of a Me2SO and serum-free non-toxic freezing solution from natural cryoprotective agents (CPAs) in a suitable carrier media for preserving adipose tissue and adipose tissue-derived stem cells (hADSCs) with long shelve-life. The efficiency of the various hydrocolloids and organic osmolytes as CPAs and individual PBS components such as NaCl, Na2HPO4, KCl and KH2PO4 were evaluated to select the potential CPAs and carrier media. Among these, trehalose and NaCl were found to be the most efficient extracellular CPAs and carrier media. The freezing solution comprising of 160mM NaCl/90mM trehalose achieved adipose tissue viability of 84%. The efficiency of the freezing solution (89% viability) was increased by the addition of curcumin as antioxidant. The cryopreservation efficiency was further improved by optimizing the control freezing parameters, which resulted in 93% cell viability. Thus, the formulated freezing solution comprising of 160mM NaCl/90mM trehalose/1mg/ml curcumin has been proven to have the ability in maintaining the viability for a long time and provides the isolated hADSCs from cryopreserved adipose tissue with desired proliferation and differentiation potential.
An effort has also been given to isolate hADSCs from adipose tissue and develop a cryopreservation strategy for their preservation by formulating an effective freezing solution similar to adipose tissue. A cell viability of 81% was achieved with 10%PVP/0.9%NaCl/60mM ectoin. The cryopreservation efficacy of the formulated freezing solution was optimized by following Taguchi orthogonal design method thereby optimal composition of the freezing solution was obtained as 160 mM NaCl/10% PVP/90mM ectoin/100μg/ml catalase, providing the post-thaw viability of 85%. The viability was further enhanced to 89% in control rate freezing with the optimized condition at -1°C/min. The freezing solution has the ability to maintain cell viability, proliferation and differentiation capability of hADSCs for long storage time.
Thus, it has been demonstrated that the formulated serum-free and non-toxic freezing solutions comprising of 160mM NaCl/90mM trehalose/1mg/ml curcumin and 160 mM NaCl/10% PVP/90mM ectoin/100μg/ml catalase are effective for long-term preservation of adipose tissue and hADSCs respectively. These solutions may provide effective cryopreservation strategy for the supply of these products for clinical application in future.