Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system

• Published in: International journal of pharmaceutics Vol. 200; no. 1; pp. 41 - 51
• Main Authors: Lentz, Kimberley A, Hayashi, Jun, Lucisano, Leo J, Polli, James E
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.04.2000; Elsevier
• Subjects: Adrenergic beta-Antagonists - pharmacokinetics; Alkaline Phosphatase - metabolism; Animal cells; Animals; Biological and medical sciences; Biomarkers; Biopharmaceutics - classification; Biopharmaceutics Classification System; Biotechnology; Caco-2 Cells; Cattle; Cell Division - physiology; Cell Membrane Permeability - physiology; Cholagogues and Choleretics - pharmacokinetics; Culture Media; Cytological Techniques; Diuretics, Osmotic - pharmacokinetics; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Mannitol - pharmacokinetics; Methods. Procedures. Technologies; Metoprolol - pharmacokinetics; Microscopy, Electron; Oral absorption; Permeability; Reduced-serum cell culture; Taurocholic Acid - pharmacokinetics; Oral absorption; Caco-2 cells; Permeability; Biopharmaceutics Classification System; Reduced-serum cell culture; Hormone; Cell culture; Biological transport; Calf; Oral administration; Ox; In vitro; Vertebrata; Mammalia; Absorption; Enzymatic activity; Animal; Established cell line; Active ingredient; Serum; Models; Artiodactyla; Ungulata; Growth factor
• ISSN: 0378-5173; 1873-3476
• DOI: 10.1016/S0378-5173(00)00334-3

The objectives were: (1) to develop a more rapid, reduced serum culture system for Caco-2 monolayers, relative to the traditional 21-day, 10% fetal bovine serum (FBS) system; and (2) to determine the biopharmaceutical drug classification of an oral therapeutic agent using this new system. Caco-2 cells were grown in the six well format on polycarbonate filters, in medium containing 2% iron supplemented calf serum (sCS) and a combination of growth factors and hormones. After 4 days in culture, permeabilities of three marker compounds (metoprolol, mannitol, and taurocholate) across monolayers were determined, and compared to permeabilities from the traditional 21-day, 10% FBS system, using cells at similar passage number. Cell morphology, degree of cell differentiation, and the presence of two efflux pumps were assessed. The 2% sCS model was also used to classify the permeability of an oral therapeutic agent as high or low. No difference in permeability was observed for metoprolol transport ( P=0.38) between the two culture methods, and the values obtained were independent of passage number of the cells. Mannitol permeability was about 2-fold higher from the 2% sCS system, as compared to the 10% FBS system. Taurocholate permeability was low indicating the 2% sCS culture at 4 days lacked this particular active transporter capability. Electron micrographs of cells grown in the 2% sCS system at 4 days revealed the presence of microvilli and tight junctions. P-glycoprotein and an efflux pump for furosemide were functionally present. The 2% sCS system indicated the oral therapeutic agent as highly permeable, which agreed with the 10% FBS system. This new system provides a rapid, accurate, and economical option for passive permeability determination, and appears to be applicable to the proposed Biopharmaceutics Classification System (BCS).