Objective

Introduction Cell-based culture models of the intestinal epithelium have been developed to better understand intestinal epithelial function in humans. These models use cell lines grown as single layer monocultures on a two-dimensional (2D) substrate. An example of this is the Caco-2 Transwell assay, used for the quantification of drug transport characteristics in vitro. These models include many deficits: 1) lack of multiple cell types; 2) no three dimensional (3D) structures; 3) absence of interactions between the epithelial and stromal compartments. Such limitations result in differences in epithelial permeability, receptor complex and transporter expression involved in absorption and secretion. In this study we are using Alvetex® scaffold for the co-culture of CCD-18co and Caco-2 cells in a 3D environment. We hypothesise that this construct will possess more realistic tissue-like structure and function compared to existing 2D epithelial models.

Methods: CCD-18co cells were seeded onto Alvetex scaffold for 4 weeks. This permits the secretion of ECM proteins into the cellular scaffold and allows for the formation of a fibroblast layer able to support the long term culture of epithelial cells. Caco-2 intestinal epithelial cells were added to the developed sub-epithelial layer and allowed to mature and differentiate for 3 weeks prior to analysis. Constructed tissues were analysed through; H+E staining, SEM and TEM to assess tissue structures. 3D protein localisation was assessed by immunohistochemistry. 2D 21 day mature caco-2 cells on Transwell inserts were used as a control in addition to being a comparison to the industry standard. Results: Caco-2 and CCD-18co intestinal cell lines were successfully cultured in 3D within Alvetex Scaffold^®. SEM and Toluidine blue images show extensive brush border formation indicative of  cellular differentiation. Utilisation of Alvetex^® Scaffold has allowed for the culture of CCD-18co and Caco-2 cell types in combination with one another in a 3D environment, creating a complex structure of both epithelial and stromal compartments able to mimic some of the histological properties of tissue. Immunofluorescence staining resultsshow the expression of key junctional proteins and the formation of a tissue relevant ECM. Transwell co-culture of cells shows significant paracrine effects of CCD-18co cells on Caco-2 phenotype. Discussion: In this study we were able to successfully culture multiple cell type within the 3D environment of Alvetex scaffold allowing for the formation of a more physiological culture environment. It is anticipated that the behaviour of existing intestinal cell lineages supplied by ECACC for this project will be enhanced when part of the 3D intestinal model. Future characterisation will focus on elucidating the effects of a physiologically relevant 3D environment on proteome expression and analysing resultant