Takeru ISHIZAKI1, Daisuke TANAKA2, Kojiro ISHIBASHI3, Kenji TAKAHASHI1, Eishu HIRATA3,4, Kosuke KURODA1,5
1Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan
2Research Center of Genetic Resources, National Agriculture and Food Research Organization, Tsukuba, Japan
3Cancer Research Institute, Kanazawa University, Kanazawa, Japan
4WPI-Nano Life Science Institute, Kanazawa University, Kanazawa, Japan
5NanoMaterials Research Institute, Kanazawa University, Kanazawa, Japan
Cryopreservation is an essential technique in a wide range of fields. For example, cell lines, sperm and oocytes have been cryopreserved. Cryopreservation usually involves the use of cryoprotectants (CPAs) to prevent damage to cells induced by ice crystals. Dimethyl sulfoxide (DMSO) is a typical CPA but is cytotoxic and causes DNA methylation and protein denaturation.
We have proposed a zwitterion (OE2imC3C) (Fig. a) as an alternative CPA to DMSO [1]. Since zwitterions have both positive and negative charges in the same molecule, they interact strongly with water molecules and inhibit ice crystal formation. They are also less toxic than DMSO because they do not penetrate the membrane and thus do not affect the intracellular domain. Therefore, zwitterions have the potential to be better CPAs than DMSO. However, we have found some cell lines whose cryopreservation has been difficult with only a zwitterion [2].
In this study, the main reason for damage during cryopreservation was studied. It is known that the high osmotic pressure caused by the solutes concentrated during freezing is lethal to cells. After freezing an aqueous solution containing 10 wt% OE2imC3C, the solution is concentrated to approximately 40 wt%. When several cell lines were immersed in a 40 wt% OE2imC3C solution for 1 hour at 0 °C, the cells which are difficult to cryopreserve with OE2imC3C showed a lower number of living cells. These results supposed that cryopreservation of cells with OE2imC3C is difficult because the cells cannot withstand the osmotic pressure after freezing OE2imC3C.
A more suitable zwitterionic CPA should have low osmotic pressure. We synthesized a zwitterion with a high molecular weight named N(OE2)3C3S (Fig. b) to achieve a lower molar concentration, which is expected to have a lower osmotic pressure, at the same mass concentration. Since an increase in the hydrophobic part of the zwitterion increases cytotoxicity, the molecular weight was increased by introducing a polar oligoether. The molecular weight of N(OE2)3C3S synthesized in this study is 445.6, which is 1.7 times larger than that of OE2imC3C.
N(OE2)3C3S solution was used to cryopreserve human kidney cells (BOSC), which are challenging to cryopreserve with the OE2imC3C solutions. The relative number of living cells (the number of live cells cryopreserved with commercial CPA is set at 1.00) with OE2imC3C was 0.16. N(OE2)3C3S showed a higher relative number of living cells in 10, 15 and 25 wt% aqueous solution (0.56, 0.88 and 0.81) and the 15wt% N(OE2)3C3S solution was optimal (Fig. c). Cryoprotecting effect of the N(OE2)3C3S was lower than the commercial CPA but the commercial CPA includes DMSO, leading to cytotoxicity as mentioned above. Therefore, N(OE2)3C3S has the potential to be a low toxic CPA superior to commercial CPAs after optimization.
References
[1] K. Kuroda, E. Hirata et al., Commun. Chem., 2020, 3, 163.
[2] Y. Kato, K. Kuroda, E. Hirata et al., Commun. Chem., 2021, 4, 151.