The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis

Abstract

The use of dimethyl sulfoxide (Me 2 SO) as a cryoprotectant agent (CPA) is controversial. Indeed, this cryoprotectant agent (CPA) is cytotoxic and potentially mutagenic. Therefore, other cryoprotectants must be used to reduce the proportion of Me 2 SO in slow-freezing solutions. In this chapter, we propose to present the first evaluation of new non-penetrating cryoprotectants: the chitooligosaccharides (COS). These molecules are chitosan oligomers, which are biocompatible, antioxidant, and bacteriostatic. We first review the use of saccharides through cryopreservation processes. We question the possibility to reduce penetrating CPA during slow-freezing procedures. We propose to use COS as extracellular CPA to reduce the use of Me 2 SO. We question the biocompatibility of COS on mouse embryos through the analysis of the cells' development. Next, we evaluate these molecules in slow-freezing solutions with a reduced quantity of Me 2 SO. Our experimental approach is a physical method often used to characterize slow-freezing solutions. Differential scanning calorimetry (DSC) allows to evaluate the crystallization and melting processes, the amount of crystallized water, and the equilibrium temperature and consequently to evaluate the impact of different cryoprotectants. This study gives a better understanding on how slow-freezing protocols could be improved with extracellular CPA.