Hyperosmotic and hypoosmotic conditions induce opposing forces on ASL hydration. Left, differentiated ALI cultures were exposed to hyperosmotic (+77 mOsm) and hypoosmotic (−77 mOsm) basolateral media for 24 h and ASL height was measured at 0 and 24 h following small volume addition to the apical surface. Data are mean ± s.e.m. Statistical significance by paired two-tailed Student’s t -test ( n = 4 donors, each using 2 cultures). Right, quantification of ionocyte numbers in 21 days ALI cultures maintained under hyperosmotic and hypoosmotic conditions throughout basal cell differentiation. Data are mean ± s.e.m. Statistical significance by paired two-tailed Student’s t -test (hyperosmotic: n = 5 donors, 1 culture per donor, quantified as FOXI1 -Cre ERT2 EGFP + cells; hypoosmotic: n = 4 donors, each using 2–3 cultures, quantified as ATP6V1G3 + cells).
