Yuya MATSUDA1, Ishizaki TAKERU1, Eishu HIRATA1, Kenji TAKAHASHI1, Kuroda KOSUKE1
1Kanazawa University, kanazawa, Japan
Cryopreservation is a process in which cells and tissues are preserved at low temperatures for long periods of time. However, cell damage can occur during this process. To prevent cryopreservation-related damage, dimethyl sulfoxide (DMSO) is one of the most commonly used cryoprotectants (CPAs). Nevertheless, DMSO is known to be cytotoxic and can induce apoptosis in the worst case. Recently, we proposed zwitterionic compounds (ZIs) [1] as cryoprotectants, which have a high affinity to water due to their charge and effectively prevent ice crystal formation. Importantly, ZIs are membrane-impermeable and less toxic than DMSO, while their anionic portion strongly interacts with cell membranes [2]. Therefore, ZIs are considered to be promising, next generation cryoprotectants, however, some cell types can not be effectively cryopreserved with the compounds.[3] On the other hand, polyampholytes proposed as CPAs by Matsumura et al. [4,5] prevent the ice crystals from entering the cells by forming a polymer matrix around the cell. To take advantage of the characteristics of ZIs and polyampholytes, we have developed a new zwitterionic polymer (poly(ZI), Fig. 1a) to enhance the polymer matrix formation rather than the polyampholyte because of the strong interaction of zwitterions to the cell membrane. As a result, the poly(ZI) has exhibited high cryopreservation capacity, even for which are freeze-sensitive cells.
In this work, we developed a poly(ZI) modified with anchors that insert into the cell membrane to further strengthen the matrix formation. To this end, we synthesized poly(ZI) containing C16 — alkyl chain imidazolium (poly(ZI- C16), Fig. 1a). C16 — alkyl chain imidazolium cations with a long alkyl chain are able to form hydrophobic interactions with cell membranes, which allows the incorporation of the polymer into the membrane surface [6]. After, poly(ZI-C16) was tested as CPA for cryopreservation of leukocytes (K562) and ovarian (OVMANA) cell lines, as these cells are freeze-sensitive. The culture sure freezing medium, a commercial CPA, was also tested for comparison. The results showed that poly(ZI-C16) exhibited a stronger cryoprotective effect than poly(ZI) in both cell lines K562 (Fig. 1b) and OVMANA. The cryoprotecting effect of poly(ZI-C16) was similar to a DMSO – based commercial CPA. In addition, we examined the cellular localization of poly(ZI-C16) before and after cell freezing by confocal microscopes, and the results indicate that poly(ZI-C16) was concentrated on the cell membrane surface. In summary, the results suggest that the modification of the alkyl chains of the zwitterionic polymer contributes to successful cryopreservation through membrane protection.
References
1) K. Kuroda et al., J. Am. Chem. Soc., 139, 45 (2017), 16052-16055
2) K. Kuroda et al., Commun. Chem., 3, 163 (2020), 1-7.
3) Y. Kato et al., Commun. Chem., 4, 151 (2021),1-10.
4) K. Matsumura et al., Commun Mater., 2, 15, (2021), 1-12.
5) K. Matsumura et al., Biomaterials, 30, 27 (2009), 4842-4849.
6) Y. Teramura et al., Biomaterials, 29, 10 (2008), 1345-1355.