Abstract
An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.
Highlights
In order to mimic the human intestinal environment, all assays so far developed employ in vitro cell lines, since many experimental difficulties hamper in establishing a long-term primary culture of normal small intestinal and colon cells
In the first part of the present study, the right proportion of the two cell lines was identified in order to obtain an in vitro model of the intestinal epithelium as more similar to the physiological one as possible
The ratios 30/70, 50/50, 70/30 of Caco2/HT-29 cells were chosen based on the different percentage between absorptive enterocytes and goblet cells found along the human intestinal epithelium [25,26] and were analyzed for their main features
Summary
In order to mimic the human intestinal environment, all assays so far developed employ in vitro cell lines, since many experimental difficulties hamper in establishing a long-term primary culture of normal small intestinal and colon cells. Both HT-29 and Caco cell lines share their origin from colon adenocarcinoma but, when differentiated, they exhibit similar structural and functional features of enterocytes [3,4,5], and some relevant differences Based on these premises, it is undoubted that one single cell line is not fully representative of the human intestine, neither from a morphological, nor from a permeability point of view. The co-cultures so far proposed in literature were obtained performing two types of methodologies: (i) the use of mucus secreting HT-29 subclones, generally HT29-MTX [6,7,8,9,10]; (ii) the adaptation of these two cell lines to modified growth conditions [11,12] These types of co-culturing present some negative aspects: first, they require time-consuming and long-term growth c 2018 The Author(s).
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