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Background

Caco-2 is considered as the gold standard of in-vitro cell-based model for permeability of compounds passing through the intestinal epithelium. However, this cell line exhibits a number of drawbacks, including long culture time, different expression of efflux transporters and especially the absence of mucus. Although Madin-Darby Canine Kidney (MDCK) cell line has shown the potential to overcome part of the disadvantages of Caco-2 cells, the mono-culture of this cell line still suffers from the obstacle of mucus absence. Therefore, in this project, a co-culture between MDCK and HT29 cells, a mucus-secreting cell line, was investigated as an effort to solve the ongoing problems of each cell line in the mono-culture and better simulate the properties of the intestine.

Aim

The objective is to establish an in vitro co-culture model using MDCK and HT29-MTX cell lines that better mimic both the absorptive and mucus-secreting functions of the intestinal epithelium

Method

Culture condition of each cell line was first optimized by using different culture media. Subsequently, different techniques (e.g. treatment with sodium butyrate or forskolin, as well as the culturing at 130 rpm) to enhance mucus secretion were investigated. Afterwards, two designs of experiments (DOE) were established to provide a systematic approach to the investigation of the co-culture model. Total seeding density of the co-culture, along with seeding ratio and seeding timepoint of HT29-MTX-E12 were optimized through the DOE. In the end, the integrity of the co-culture, permeability over the co-culture model and influence of the mucus layer on the permeability of dextran were explored.

Results

No significant difference in the growth rate of both MDCKcMDR1-KO and the HT29-MTX-E12 cell lines between different culture media was observed. In addition, neither sodium butyrate or forskolin treatment, nor culturing during shaking, induced mucus production. From the analysis of DOE, seeding timepoint was the key factor influencing the co-culture model. Functional studies showed a higher integrity for the co-culture in comparison with the MDCKcMDR1-KO monolayer. However, no significant difference in the permeability of fluorescent dextran in the co-culture with and without mucus was reported.