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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.