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?

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Accepted 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.

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Can this ALI system also be used for murine airway epithelial cells? And if yes do you have to change the protocol (as compared to human cells)? How many cells do you need per transwell?

For your 3D models of the airway epithelium air-liquid interface (ALI) cultures, is it possible to grow epithelial cells on fibroblasts? Or do you have other methods to generate stromal cells?

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