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Intestinal Transwell Monolayer

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info@definigen.com
UK +44 (0)1223 497106
USA 617-674-3260

DefiniGEN’s intestinal monolayer product INTReady constitutes a ground-breaking new system to model the permeability of the human intestine and investigate drug efflux. The cells are grown as a monolayer in a similar manner to standard Caco-2 immortalized cell lines with the key benefit of greatly increased biological relevance, as the cells have primary human intestinal cell function, metabolism and genetics.

 

Monolayer formation

Confluent monolayer formation has been demonstrated via TEER, (transepithelial electrical resistance), ZO1 (Zonula Occludens/Tight Junction Protein-1) immunostaining, and Lucifer Yellow analysis. TEER measurement of the monolayer demonstrates tight junction formation with typical values of >150ohm x cm2 being observed after 7 days of monolayer culture. These levels are comparable to those observed with a reference Caco-2 line after 20+ days. ZO1 protein localization immunostaining at the cell boundaries of all cells analyzed indicates a confluent monolayer has been formed. Correspondingly, Lucifer Yellow assays to assess the permeability characteristics of the monolayer has established monolayer integrity with observed values of Papp = 0.87 ± 0.32 × 10-6 cm/sec (A to B, 100 μM, 60 min).

(A)                                                                                                       (B)

  TEER measurement of the INTReady monolayer compared to Caco-2 cells   Immunostaining of Zonula Occludens/Tight Junction Protein-1, ZO1, in the INTReady monolayer

                                                                           

 

Figure 1. Verification of intestinal cell monolayer integrity. (A) TEER measurement of the INTReady monolayer demonstrates tight junction formation with typical values of >150ohm x cm2 being observed after 7 days of monolayer culture. (B) ZO1 localization at the cell boundaries signifies the presence of tight junctions in the monolayer.

Transporter activities

(A)                                                                                                       (B)

INTReady monolayer displays functional trancellular transport of Digoxin and Hoechst 33324 as ABCB1 and BCRP substrates respectively

 

Figure 2. Functional ABCB1 (MDR1) and BCRP transporter activity. INTReady monolayer cells exhibit functional efflux transporter activities when Digoxin and Hoechst 33324 were used as ABCB1 (A) and BCRP substrates respectively (B). These results indicate the presence of a polarized monolayer with functional apical transporter activities being observed.

 

CYP3A4 activity

 

CYP3A4 activity was confirmed in the INTReady monolayer cells via mass spectrometry analysis

 

Figure 3. CYP3A4 activity assay. CYP3A4 activity was confirmed in the INTReady monolayer cells via mass spectrometry analysis. The CYP3A4 activity catalyzed the conversion of the substrate testosterone into the hydroxylated derivative in the presence and absence of a recognized inhibitor Ketoconazole.

Gene expression analysis

 

Key intestinal marker gene expression analysis

Figure 4. Key intestinal marker gene expression analysis. Gene expression analysis of multiple intestinal markers demonstrates that the INTReady intestinal cells generally express similar gene expression profiles to primary human intestinal cells (hint).

Lucifer yellow permeability

INTReady intestinal cells maintain a monolayer with tight junctions

Figure 5. INTReady intestinal cells maintain a monolayer with tight junctions for several days over extended timeframes as assessed by Lucifer yellow analysis.

 

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