In this study, we developed a novel approach to preserving and reviving neuronal cells at low temperatures, which offers the potential for on-demand access to transplantable biologics. Our innovative experimental protocol involves isochoric cooling, which enables us to preserve the cells at sub-zero centigrade temperatures without altering their volume. The process begins by preparing a UW organ preservation solution and chilling an isochoric chamber with a 12.5% NaCl solution. We then carefully detach the N2A cells from the tissue culture vessel, count them using flow cytometry, centrifuge them, and resuspend them in the chilled UW solution before placing them in the isochoric chamber. The chamber is gradually cooled to -4°C and held there for a period of 24 to 72 hours. To revive the cells, we warm the chamber to 2°C, retrieve the cells, and immediately centrifuge and resuspend them in a 37°C DMEM solution to minimize exposure to temperatures above +8°C. After imaging and incubating the cells for 24 hours, we assess the cell count using flow cytometry. Our experimental protocol has yielded promising results and could have significant applications in the neuroscience field.
