Objective

Although the Caco-2 Transwell assay is the accepted industry standard for drug transport in vitro, such 2D monocultures lack in vivo characteristics such as multiple cell types and epithelial-stromal interactions. We used Transwell and Alvetex Scaffold for the co-culture of Caco-2 cells with CCD-18co fibroblasts and goblet-like HT29-MTX cells, to create several models closer in morphology and function to in vivo intestinal mucosa. Caco-2 were differentiated with or without HT29-MTX, either on top of Transwell inserts with or without CCD-18co cultured at the bottom of the well to provide paracrine signalling, or on top of Alvetex Scaffold inserts pre-seeded with CCD-18co to allow for ECM secretion and formation of a sub-mucosa in direct contact with the epithelial cells. Paracrine Transwell bi-culture (CaCo-2 /CCD-18co) decreased TEER values and increased drug transporter protein expression (pgp, MRP2), while Transwell tri-culture (CaCo-2/HT29-MTX/CCD-18co) abrogated the goblet-like properties of HT29-MTX. Alvetex Scaffold bi-culture (Caco-2/CCD-18co) promoted columnar epithelial morphology (H&E) and relevant junctional protein and ECM expression (E-cadherin, collagens, IF). Future characterisation will focus on mRNA and protein expression of drug transporters in Transwell and Alvetex bi-culture models, as well as Ussing chamber drug and ion transport studies.