In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model
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In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model. / Bravo, Silvina A.; Nielsen, Carsten Uhd; Amstrup, Jan; Frokjaer, Sven; Brodin, Birger.
In: European Journal of Pharmaceutical Sciences, Vol. 21, No. 1, 01.01.2004, p. 77-86.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model
AU - Bravo, Silvina A.
AU - Nielsen, Carsten Uhd
AU - Amstrup, Jan
AU - Frokjaer, Sven
AU - Brodin, Birger
PY - 2004/1/1
Y1 - 2004/1/1
N2 - 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.
AB - 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.
KW - Caco-2 cell model
KW - Characterization
KW - Gly-Sar uptake
KW - hPEPT1
KW - Immunohistochemistry
U2 - 10.1016/S0928-0987(03)00205-7
DO - 10.1016/S0928-0987(03)00205-7
M3 - Journal article
C2 - 14706814
AN - SCOPUS:0347989382
VL - 21
SP - 77
EP - 86
JO - Norvegica Pharmaceutica Acta
JF - Norvegica Pharmaceutica Acta
SN - 0928-0987
IS - 1
ER -
ID: 131735180