title: In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model.
abstract: The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [14C]-glycylsarcosine ([14C]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [3H]-mannitol permeabilities. Tissue morphology and hPEPT1 expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until approximately day 21. Apical hPEPT1 uptake activity appeared to be maximal in cells cultured for >21 days, whereas basolateral uptake reached a maximum already after 12 days in culture. In some of the passages studied, a secondary increase in hPEPT1 transport activity was observed in cells grown for >25 days. A large carrier-mediated transepithelial peptide flux component was evident from day 14.
authors.affiliation: Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Silvina A
authors.initials: SA
authors.lastname: Bravo
authors.forename: Carsten Uhd
authors.initials: CU
authors.lastname: Nielsen
authors.forename: Jan
authors.initials: J
authors.lastname: Amstrup
authors.forename: Sven
authors.initials: S
authors.lastname: Frokjaer
authors.forename: Birger
authors.initials: B
authors.lastname: Brodin
mesh_terms: D001692:Biological Transport / Q000502:physiology; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D006801:Humans; D000074011:Peptide Transporter 1; D027981:Symporters; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine