The Effect of Cell Culture Conditions on Saquinavir Transport Through, and Interactions with, MDCKII Cells Overexpressing hMDR1

Abstract

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 × 105 or 5 × 104 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 × 105 cells/cm2) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 × 104 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. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1957–1967, 2003