In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model
- 作者：Bravo ; Silvina A. ; Nielsen ; Carsten Uhd ; Amstrup ; Jan ; Frokjaer ; Sven ; Brodin ; Birger
- 关键词：Caco-2 cell model ; Immunohistochemistry ; Gly-Sar uptake ; hPEPT1 ; Characterization
- 刊名：European Journal of Pharmaceutical Sciences
- 出版年：2004
- 期刊代码：15_09280987
- 类别：pha
- 出版时间：January, 2004
- 卷：21
- 期：1
- 页码：77-86
- 文件大小：476 K
摘要
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 
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.
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.