Synthetic zwitterions as efficient non-permeable cryoprotectants

Cryopreservation is crucial for long-term cell storage, but some cell lines are difficult to cryopreserve even with optimized commercial cryoprotectants. Previous research by the authors showed that a low-toxic synthetic zwitterion solution improved cryopreservation, but not for all cell lines. This study aimed to understand the cryoprotective mechanisms of zwitterions and optimize their use. The research question focuses on identifying the optimal zwitterion structure and concentration, and whether combining zwitterions with other cryoprotectants can improve the cryopreservation of even the most sensitive cell types. The study is important because improving cryopreservation techniques will greatly benefit various fields including medicine, biotechnology, and agriculture, expanding the possibilities for cell-based therapies and research. The efficient cryopreservation of vulnerable cells, which have previously proved difficult to preserve, represents a significant advancement in the field. This paper focuses on understanding the detailed mechanisms of action and determining the optimal conditions for using zwitterions as non-permeable cryoprotectants.

The existing literature highlights the challenges in cryopreservation, particularly for cells that are highly sensitive to freezing. Conventional cryoprotectants, such as DMSO and glycerol, have limitations like toxicity and permeability issues. The paper references previous studies on ice inhibition strategies and the use of trehalose and other novel cryoprotectants. The authors build upon their earlier work on synthetic zwitterions, presenting a more comprehensive analysis of their cryoprotective properties and mechanisms. The literature review also covers the osmotic pressure effects in cryopreservation and how they impact cell volume and viability. The review lays the groundwork for the current study by establishing the need for new cryoprotectants with improved efficiency and reduced toxicity.

The researchers synthesized 18 zwitterion species with varying structures and properties. They assessed the cryoprotective effects of these zwitterions on several cell lines including hNF, mNF, BOSC, WM, MDA, PC9, B16F10, 4T1, HL-60, K562, Vn1919, and OVMANA. Cryopreservation was performed by mixing cells with different concentrations of zwitterions, with and without the addition of DMSO, and using a commercial cryoprotectant as a control. Cell viability was assessed after freezing and thawing using hemocytometry and trypan blue staining. The optimal zwitterion concentration was determined by assessing cell viability after cryopreservation. The researchers also investigated the effects of other factors, such as the osmotic pressure of the zwitterion solutions, cell dehydration, and cell volume changes. To further understand the mechanisms involved, they performed molecular dynamics simulations to study the interaction of zwitterions with cell membranes in the presence and absence of DMSO. The simulations focused on membrane stability and DMSO penetration behavior. Osmotic pressure measurements were also conducted. The paper details the synthesis of zwitterions, and mentions the use of various methods including DSC (Differential Scanning Calorimetry) to analyze the physical states of the cryoprotectant solutions at cryogenic temperatures.

The study found that cell dehydration was a key factor in successful cryopreservation. While non-permeable zwitterions effectively inhibited extracellular ice formation, insufficient cell dehydration limited their efficacy, especially for the BOSC cell line. The optimal concentration of OE2imC3C was determined to be in the range of 90/10 and 85/15 (v/w). The combination of zwitterions (OE2imC3C and C1imC3S) and DMSO (90/10/15, v/w/w) significantly enhanced cryopreservation efficiency across different cell lines, including highly freezing-sensitive K562 and OVMANA cells. The relative number of living cells after cryopreservation using the zwitterion/DMSO mixture was 1.8-fold higher than with the commercial cryoprotectant for K562 cells. The molecular dynamics simulation showed that OE2imC3C protected the cell membrane from DMSO-induced collapse, and the interaction between OE2imC3C and DMSO shifted the DMSO penetration behaviour. The presence of OE2imC3C seemed to reduce the toxicity of DMSO. The study demonstrated that the zwitterion/DMSO mixtures were effective cryoprotectants for a broad range of cells.

The findings address the research question by demonstrating the effectiveness of the zwitterion/DMSO mixture as a novel cryoprotectant. The successful cryopreservation of freezing-vulnerable cells highlights the significant improvement over commercial alternatives. The superior performance is likely due to the synergistic effects of the non-permeable zwitterion (inhibiting extracellular ice formation and promoting dehydration) and the cell-permeable DMSO (inhibiting intracellular ice formation). The molecular dynamics simulations provided mechanistic insights into the protective effect of zwitterions on cell membranes in the presence of DMSO. This study opens up possibilities for optimizing cryopreservation protocols for various cells and tissues, including those previously challenging to preserve. This has considerable implications for cell-based therapies and storage of biological materials.

This study demonstrates that a mixture of synthetic zwitterions and DMSO is a highly effective cryoprotectant, surpassing commercial alternatives, especially for freezing-sensitive cells. The non-permeable zwitterion inhibits extracellular ice formation and promotes dehydration, while DMSO inhibits intracellular ice formation. The synergistic effect of both significantly improves cryopreservation efficiency. Future research could focus on further optimizing the zwitterion structure and exploring other cell-permeable cryoprotectant combinations to further enhance cryopreservation. Further investigation on the interaction between zwitterions, DMSO, and cell membranes will also enhance the understanding of the mechanism involved.

The study focused on a limited number of cell lines, although it included freezing-vulnerable ones. While the results suggest broad applicability, further testing on a wider variety of cells and tissues is needed to validate the generalizability of the findings. The molecular dynamics simulations, while providing valuable insights, are simplifications of complex biological systems. Further experimental validation of the simulated interactions will be necessary. The study does not detail the cost-effectiveness of synthesizing the zwitterions compared to using existing commercial cryoprotectants.