Natural zwitterionic betaine enables cells to survive ultrarapid cryopreservation

Abstract

Cryoprotectants (CPAs) play a critical role in cryopreservation because they can resist the cell damage caused by the freezing process. Current state-of-the-art CPAs are mainly based on an organic solvent dimethyl sulfoxide (DMSO), and several DMSO-cryopreserved cell products have been brought to market. However, the intrinsic toxicity and complex freezing protocol of DMSO still remain as the bottleneck of the wide use for clinical applications. Herein, we reported that betaine, a natural zwitterionic molecule, could serve as a nontoxic and high efficient CPA. At optimum concentration of betaine, different cell types exhibited exceptional post-thaw survival efficiency with ultrarapid freezing protocol, which was straightforward, cost efficient but difficult to succeed using DMSO. Moreover, betaine showed negligible cytotoxicity even after long-term exposure of cells. Mechanistically, we hypothesized that betaine could be ultra-rapidly taken up by cells for intracellular protection during the freezing process. This technology unlocks the possibility of alternating the traditional toxic CPAs and is applicable to a variety of clinical applications.

Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants

“…Similarly, Sharp et al observed lower cell densities after 24 h compared to immediately post-thaw. 40 Yang and co-workers measured cell survival over time (after cryopreservation) and found it peaked at 1–2 h post-thaw but decreased after 24 h incubation, 41 highlighting that immediate post-thaw measurements lead to significant overestimation of cryoprotectant activity. Mercado et al showed that adding an amphiphilic polymer to SAOS-2 cells along with 200 mM trehalose gave a cryoprotective benefit but found significant differences between the two assessment methods (trypan blue and MTS assay) when the cells were analyzed immediately post-thaw.…”

Supplement of Betaine into Embryo Culture Medium Can Rescue Injury Effect of Ethanol on Mouse Embryo Development

“…Moreover, Wang et al .’s work shows embryos would be arrested in the late of 2-cell stage when they are stressed by physiological osmolarity in the absence of organic osmolytes 43 . Besides above, being a natural zwitterionic molecule, betaine had been found to serve as a nontoxic and high efficient cryoprotectant for it presented negligible cytotoxicity even after long-term exposure of cells 44 . But, our study seems not to support this hypothesis of betaine protecting embryos against ethanol by regulating osmolarity in that glycine did not function as expected in this study, although it had been proven that glycine could participate in the process of osmolarity regulation in vitrification of mouse GV oocytes 30 .…”

A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation

“…CB has been used in a range of biotechnological applications, above all as cryoprotectant [8,9]. It has been suggested that this modified amino acid has the capacity to form hydrogen bonds with a range of organic and inorganic acids that are likely to be responsible for the ascertained CB properties in supporting the adhesion and proliferation of several types of cells [10][11][12].…”