An improved cell culture model based on 2/4/A1 cell monolayers for studies of intestinal drug transport: characterization of transport routes.
OBJECTIVE
To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line.


METHODS
Two approaches were taken to decrease apoptosis. First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2. Second. normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis. The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy. The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers.


RESULTS
Normal 2/4/A1 cells died by apoptosis at 39 degrees C. Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells. Optimization of the culture conditions for 2/4/A1 cells inhibited cell death. The integrity was comparable to that of the human jejunum (50 omega x cm2), making this approach preferable to Bcl-2 overexpression. Transcriptional analysis showed that some (e.g., MDRI). but not all (e.g., PepT1), transporters were found in 2/4/A1 cells. Studies using substrates for PepT1, P-gp. MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1.


CONCLUSIONS
The improved culture procedure will facilitate the use of 2/4/A1 cells. 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport.
Tavelin
Staffan
S
Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden.
Taipalensuu
Jan
J
Hallböök
Finn
F
Vellonen
Kati-Sisko
KS
Moore
Vanessa
V
Artursson
Per
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001692:Biological Transport; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured / Q000378:metabolism; D017097:Electric Impedance; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D007583:Jejunum / Q000378:metabolism; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D000074011:Peptide Transporter 1; D010539:Permeability; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074011:Peptide Transporter 1; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D027981:Symporters; C000715087:ABCC2 protein, human; C082186:SLC15A1 protein, human; C489940:Slc15a1 protein, rat