I am interested in freezing primary nasal epithelial cells for subsequent functional analyses. Do you have any suggestions on at which step to freeze cells (passage#), what the best thawing procedure is, whether complete ALI differentiation is still possible after freeze-thawing and what number of cells you need to freeze to be able to regrow and differentiate successfully?

Answer

We recommend freezing the cells at the end of P0 (The cells will be at P1 when they are cultured after thaw). The cells should be frozen at approximately 1 X 10^6 cells/mL in PneumaCult™-Ex with 10% DMSO. The best thawing procedure is to quickly thaw the cells in a 37℃ water bath and seed 1 mL directly into a T75 flask containing 20 mL warm PneumaCult™-Ex. After an overnight incubation (~16 hours) at 37℃, exchange the medium with fresh PneumaCult™-Ex to remove the DMSO. The freeze-thaw procedure will affect downstream ALI differentiation quality; however, if you follow the proper protocol, good ALI differentiation should be achievable for P2 and P3 cells.