Cell cryopreservation procedure

Conditions for freezing the hNPC-derived, MACS-purified immature neurons involved two comparative cryoprotective groups; the conventional 10% DMSO in regular cell culture media (described above) and the animal component-free Cryo-Store 10 from BioLife Solutions, Inc. Cell solutions of both groups were placed in cryovials (8 cryovials per group), which were placed into a Thermo Scientific Mr. Frosty freezing container with 100% isopropyl alcohol. The Mr. Frosty was then placed in −80 °C for various durations, ranging from 1–60 days. Viability was measured upon thaw using trypan blue (TB) uptake using a hemocytometer, as well as by propidium iodide (PI) uptake using flow cytometry. Survival frequency was calculated by dividing total TB-negative or PI-negative cells by the total number of cells.