Enhanced cell culture medium improves airway epithelial cell transplantation and allows the generation of clonally derived disease models.

Primary human bronchial epithelial cells can be expanded in co-culture with mitotically-inactivated 3T3 fibroblasts in FAD medium (Rheinwald, J.G. & Green H. Cell 1975; 6(3):331-343). However these cell cultures have limited proliferation capacity, their differentiation potential declines over time and the derivation of successful clonal cell cultures remains challenging. We have previously shown that the addition of Y-27632 to FAD medium improved the survival and proliferation of airway basal cells (Butler, C. et al. AJRCCM 2016; 194(2):156-168). Here, we discover that the further addition of commercially available small-molecule ‘Compound E’, to be disclosed at the meeting, enhanced the clonogenicity, proliferation rate and longevity of cell cultures. We investigate the mechanism of action and effect on genome integrity over prolonged culture periods. In these cell culture conditions, we were able to reliably expand clonal basal cells. This allowed the isolation and expansion of isogenic clones of CRISPR knockout primary cell cultures to develop disease models of primary ciliary dyskinesia and epidermolysis bullosa. Finally, in vivo cell transplantation in immunocompromised mice demonstrated that addition of Compound E to medium improves human airway basal cell transplantation. Overall, this new epithelial cell culture medium formulation- applicable to airway and also other stratified epithelia - allows primary clonal cell cultures and opens up avenues for fundamental respiratory research, disease modelling and regenerative medicine.