Cryopreservation

The zwitterion and zwitterion/DMSO solutions were prepared via mixing with ultrapure water. Thereafter, cells (1 × 10⁶ cells) were collected and centrifuged (100 × g, 5 min at room temperature). After removing the supernatant, 100 μL of cryoprotectants were added and pipetted slowly. The samples were stored in a box (Mr. Frosty, Thermo Fisher Scientific Inc.) in a –85 °C freezer for 3–5 days. For sample thawing, a culture medium incubated at 37 °C was added to the frozen samples. The relative number of living cells was counted using a hemocytometer (Fukaekasei Corporation and Watson Corporation) with trypan blue (Fujifilm Wako Pure Chemical Corporation).

For the proliferation studies, K562 and OVMANA cells after cryopreservation were seeded in a six-well plate. K562 cells were counted sequentially. OVMANA cells were counted after trypsin treatment when the most-grown cells were at 80% confluent.

The commercial cryoprotectant employed was Culture Sure freezing medium (DMSO-containing, Fujifilm Wako Pure Chemical Corporation). This is one of the typical commercial cryoprotectants and is suitable for comparison in cryopreservation efficiency. In the present study, the relative number of living cells was employed because the absolute number of living cells was variable, based on biological variation, even when using the commercial cryoprotectant. The absolute numbers of living cells given by the commercial cryoprotectant were roughly associated with those given by the sample solutions. In addition, most experiments were experimentally triplicated in this study. The results therefore still contain a certain amount of error based on the biological variation but are sufficient to suggest rough trends and relations.

When preparing the freezing medium, the amount of culture medium, FBS, and water were measured by volume using pipettes. The number of zwitterions, DMSO, an ionic liquid, glycerol, and sucrose were measured by weight using an electronic balance unless we note.