DMSO-Free Cryopreservation of Therapeutic Cells by Agarose Hydrogel Encapsulation

Abstract

In recent decades, the rapid advancement of cell therapy and regenerative medicine has generated an urgent need for efficient and safe cell/tissue cryopreservation techniques on both laboratory and industrial scales. The current use of dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA) presents toxicity concerns, prompting the development of alternative methods. The primary objective of this research is to develop a novel cryopreservation method for therapeutic cells using hydrogel encapsulation, which aims to minimize cryoinjuries while eliminating the need for toxic penetrating CPAs such as DMSO. The dissertation consists of three main parts. In the first part, molecular changes associated with warming injury in cryopreserved human white blood cells (WBCs) were analyzed. It was observed that slow warming led to irreversible dehydration of cell membrane lipids and denaturation of cellular proteins. Also, WBCs were found to be very susceptible to kinetic processes during warming, including eutectic crystallization/melting, devitrification, and ice recrystallization. The second part focuses on the development of an encapsulation cryopreservation method using the combination of agarose hydrogel and trehalose as an alternative to membrane-permeable CPAs. A comprehensive analysis of the kinetic and thermodynamic changes within the agarose-trehalose hydrogel during freeze/thaw was conducted. The combination of agarose and hydrogel was found to reduce ice phase volume with a less ordered structure, eliminate eutectic crystallization and melting, and inhibit ice recrystallization during warming. The third part of the dissertation involves the validation of the DMSO-free hydrogel encapsulation method for the cryopreservation of natural killer (NK) cells. High post-thaw cell viability was achieved, while decreased viability with compromised cytotoxicity was observed after cells were extracted from the hydrogel. Ongoing efforts are focused on optimizing the cell extraction process from the hydrogel.