Ice Inhibition for Cryopreservation: Materials, Strategies, and Challenges

Abstract

Cryopreservation technology has developed into a fundamental and important supporting method for biomedical applications such as cell‐based therapeutics, tissue engineering, assisted reproduction, and vaccine storage. The formation, growth, and recrystallization of ice crystals are the major limitations in cell/tissue/organ cryopreservation, and cause fatal cryoinjury to cryopreserved biological samples. Flourishing anti‐icing materials and strategies can effectively regulate and suppress ice crystals, thus reducing ice damage and promoting cryopreservation efficiency. This review first describes the basic ice cryodamage mechanisms in the cryopreservation process. The recent development of chemical ice‐inhibition molecules, including cryoprotectant, antifreeze protein, synthetic polymer, nanomaterial, and hydrogel, and their applications in cryopreservation are summarized. The advanced engineering strategies, including trehalose delivery, cell encapsulation, and bioinspired structure design for ice inhibition, are further discussed. Furthermore, external physical field technologies used for inhibiting ice crystals in both the cooling and thawing processes are systematically reviewed. Finally, the current challenges and future perspectives in the field of ice inhibition for high‐efficiency cryopreservation are proposed.

Engineering mammalian living materials towards clinically relevant therapeutics

“…Recently, researchers have demonstrated that alginate-based hydrogels are able to confine ice crystal nucleation/ growth and decrease osmotic stress during the cryopreservation of cell-biomaterial constructs as well as individual pancreatic islets [78]. Despite promising, this strategy is still insufficient for the cryopreservation of large-volume constructs, particularly those containing complex bioarchitectures interwoven in vascular systems [79]. A major roadblock is attaining uniform and rapid volumetric warming to inhibit ice recrystallization and devitrification during thawing, which causes deleterious injuries and undermines their viability.…”

“…Concerning this, recent innovations in magnetic field thawing have been demonstrated to be attractive for overcoming intrinsic heterogeneities in the characteristics of cells and their microenvironment, thus reducing the amount of cryoprotectants and improving cryopreservation outcomes in dental pulp microtissues [78]. In the upcoming future, it will be paramount to improve and scale-up precision preservation to large-volume constructs (i.e., by combining multiple technologies to attain synergistic thawing), as well as to develop biosensing tools to monitor the success rates and the biological outcomes (i.e., changes in cellular functions, chromosomal instabilities/ aberrations, and epigenetic alterations) of thawed living materials to ensure that their cytogenetic status remains unaffected [78,79]. The path that living materials must travel to benefit from rapid and widespread clinical applications, will require a multidisciplinary effort from the scientific community to ensure safety, feasibility and reliability of the final products, while considering affordability and overcoming the necessary regulatory and ethical challenges.…”

Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep

“…Recently, the development of chemical ice-inhibition molecules, including cryoprotectant, antifreeze protein, synthetic polymer, nanomaterial, and hydrogel, and their applications in regenerative devices and cryopreservation, has progressed. Additionally, advanced engineering strategies, including trehalose delivery, cell encapsulation, and bioinspired structure design for ice inhibition, are also amazingly developed [ [41] , [42] , [43] ]. Through the combination of SCK and these novel products or advanced engineering techniques, we expect to improve cryopreservation methods.…”

Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

“…Defining the suitable exposure duration of explants to the vitrification solutions is essential for cell dehydration, necessary to avoid the formation of intracellular ice crystals during freezing and thawing, and to prevent injury by chemical toxicity of cryoprotectants [56,57] or excessive osmotic stress [30]. In the current study, cryopreservation by DV was applied to axillary buds of C. avellana, the Italian cultivated variety Tonda Gentile Romana, collected from in vitro grown shoots.…”

“…The PVS2 treatment applied for 90 min induced a greater reduction in regrowth (60.0%) in the control (−LN) explants than the same duration of PVS3 application (70.0%), further suggesting that for the hazelnut axillary buds PVS3 is less toxic than PVS2. This response could be related to the presence of DMSO in the composition of PVS2, which can cause damage at cellular levels [56,59,60].…”