Cryopreservation of tissues by slow-freezing using an emerging zwitterionic cryoprotectant

Abstract

Cryopreservation of tissues is a tough challenge. Cryopreservation is categorized into slow-freezing and vitrification, and vitrification has recently been recognized as a suitable method for tissue cryopreservation. On the contrary, some researchers have reported that slow-freezing also has potential for tissue cryopreservation. Although conventional cryoprotectants have been studied well, some novel ones may efficiently cryopreserve tissues via slow-freezing. In this study, we used aqueous solutions of an emerging cryoprotectant, an artificial zwitterion supplemented with a conventional cryoprotectant, dimethyl sulfoxide (DMSO), for cell spheroids. The zwitterion/DMSO aqueous solutions produced a better cryoprotective effect on cell spheroids, which are the smallest units of tissues, compared to that of a commercial cryoprotectant. Cryopreservation with the zwitterion/DMSO solutions not only exhibited better cell recovery but also maintained the functions of the spheroids effectively. The optimized composition of the solution was 10 wt% zwitterion, 15 wt% DMSO, and 75 wt% water. The zwitterion/DMSO solution gave a higher number of living cells for the cryopreservation of mouse tumor tissues than a commercial cryoprotectant. The zwitterion/DMSO solution was also able to cryopreserve human tumor tissue, a patient-derived xenograft.

Figure 1

Chemical structure of the imidazolium/carboxylate zwitterion used in this study.

Figure 2

Relative cell recoveries of cryopreserved mouse melanoma cell spheroids post-thaw using different mixtures of zwitterion and DMSO. (a) Initial tests on non-optimized mixtures with cell recoveries measured immediately post-thaw (n = 1, experimental triplicates). (b) Optimization of mixture concentrations with cell recoveries measured immediately post-thaw (n = 3, biological triplicates). (c) Optimization of mixture concentrations with cell recoveries measured after 24 h of incubation post-thaw (n = 3, biological triplicates). The bars show standard errors. The commercial CPA employed is CultureSure® freezing medium (Fujifilm Wako Pure Chemical Corporation).

Figure 3

Relative cell recoveries and invasion ability of cryopreserved 5555/MAF1 co-cultured spheroids post-thaw using optimized mixtures of zwitterion and DMSO. (a) Relative cell recoveries of cryopreserved 5555/MAF1 co-cultured spheroids measured immediately post-thaw (n = 3, biological triplicates). (b) Typical images for invasion of spheroids after 24 h of incubation post-thaw. All images are in Fig. S11. Scale bar represents 500 µm. (c) Invasion radius of spheroids after 24 h of incubation post-thaw (n = 1, experimental quadruplicates). The bar shows standard errors. The commercial CPA employed was CultureSure® freezing medium (Fujifilm Wako Pure Chemical Corporation).

Figure 4

The relative number of living cells in cryopreserved mouse tumor tissues post-thaw using commercial CPA and ZD-10/15. Each tumor explant was quantified with IVIS and plotted. The commercial CPA employed was Cell Reservoir One (Nacalai Tesque).

Figure 5

Tumor growth and its speeds of cryopreserved patient-derived xenografts using commercial CPA and ZD-10/15. (a) Tumor formation and growth of PDXs after cryopreservation with the commercial CPA or ZD-10/15. PDXs were resected at the final marked days. (b) The number of elapsed days from transplantation to resection (resection was carried out after growing by the long diameter of approximately 10 mm), from transplantation to tumor formation, and from tumor formation to resection. The bars show standard errors. The commercial CPA employed was Cell Reservoir One (Nacalai Tesque).