MDCK cells are cultured using wide-ranging conditions and can produce variable results. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII-PGP) and MDCKII wild-type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 x 10(5) or 5 x 10(4) cells/cm2), substratum (polycarbonate +/- collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport. The saquinavir efflux ratio (ratio of BL --> AP/AP --> BL permeability) for MDCKII-PGP cells (6.9 x 10(5) cells/cm2) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 x 10(4) cells/cm2 on polycarbonate (78) or collagen-coated polycarbonate (126). The MDCKII/wt saquinavir efflux ratio was 9. Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen-coated membranes. Collagen coating caused increased Pgp expression and saquinavir efflux ratios correlated (r2 = 0.96) with Pgp expression levels [MDCKII-PGP (on collagen-coated polycarbonate) > MDCKII-PGP (on polycarbonate) > MDCKII/wt (on collagen-coated polycarbonate)]. These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models.
Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association