Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers?
Despite the fact that different laboratories have reported large differences in permeability for actively transported substrates, Caco-2 cell monolayers are widely used as in vitro model to study small intestinal drug transport. Therefore, we investigated the effect of cell culture conditions, such as time in culture, membrane support, seeding density and supplements to the medium, on the morphology, the formation of tight junctions, as well as the expression of two peptide transporters (PepT1, HPT1) and the efflux pump, P-glycoprotein (Pgp), in Caco-2 cell monolayers. Tight junction formation was assessed by transepithelial electrical resistance measurements; multi-cell layer formation by confocal laser scanning microscopy, the expression of transporters by RT-PCR and the permeability of the PepT1 substrate, cephradine. Both morphology and the expression of carrier-mediated transporters, varied strongly as a function of culture conditions. An increase of differentiation, as documented by tight, homogeneous cell monolayer formation displaying a strong expression of all carrier-mediated transporters, was found up to 3 weeks post seeding. One week later, multi-layer structures were observed and the expression of Pgp decreased. Polyester and polyethylene terephthalate membrane supports decreased the paracellular transport rates substantially, while collagen-coating of PC inserts showed no influence on the morphology and even increased carrier-mediated transporter expression. An average seeding density of 6x10(4) cells/cm(2) seemed to be most favorable, since lower seeding densities led to thin monolayers with altered tight junctions and higher seeding densities to the formation of multilayers. In summary, the expression of carrier-mediated transporters was strongly affected by the culture conditions. The full differentiation was reached after 21 days on collagen-coated polycarbonate inserts at an initial seeding density of 6x10(4) cells/cm(2).
Behrens
Isabel
I
Department of Pharmaceutics and Biopharmacy, University of Marburg, Philipps-University, Ketzerbach 63, D-35032, Marburg/Lahn, Germany.
Kissel
Thomas
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000199:Actins / Q000378:metabolism; D000465:Algorithms; D001693:Biological Transport, Active; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002515:Cephradine / Q000378:metabolism; D003470:Culture Media; D017097:Electric Impedance; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal; D000074011:Peptide Transporter 1; D010455:Peptides / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters; D019108:Tight Junctions / Q000187:drug effects
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D002352:Carrier Proteins; D003470:Culture Media; D005456:Fluorescent Dyes; D000074011:Peptide Transporter 1; D010455:Peptides; D012333:RNA, Messenger; C082186:SLC15A1 protein, human; D027981:Symporters; D002515:Cephradine; D016650:Fluorescein-5-isothiocyanate