Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium

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.

A Guide to Human Zinc Absorption: General Overview and Recent Advances of In Vitro Intestinal Models

“…Moreover, this co-culture does not only improve the in vitro intestinal Caco-2 model regarding the presence of a mucus layer, but was also reported to optimize the cellular permeability of conventional Caco-2 monocultures [299,300] and is considered a more physiological in vitro model [256,301]. The Caco-2/HT-29-MTX model is well characterized [299,[302][303][304] and was already used to investigate the absorption of different metal species [254,[305][306][307], the effect of nanoparticles on nutrient absorption [308], and bacterial adhesion [309]. Recently, a Caco-2/HT-29-MTX model, optimized with respect to its buffer composition and basolateral serum concentration, was applied to study the absorption of zinc via the human intestinal epithelium in the presence of a mucus layer [102,103].…”

Bovine whey peptides transit the intestinal barrier to reduce oxidative stress in muscle cells

“…After 6 days post confluence, transport studies were performed. After 6 days post confluence, co-culture cells showed the presence of (1) microvilli with active enzymes, (2) mucus and (3) barrier properties characterized by a permeability value similar to the human small intestine in vivo (Ferraretto et al, 2018).…”

“…x 10 4 cells/cm, as previously described by Ferraretto et al (2018). Cell confluence was achieved 4 days after seeding.…”

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

“…Several groups have suggested to coculture Caco-2 cells with mucus-producing HT29 cells to improve this cell model. Publications analyzing the advantages and disadvantages of this co-culture have already been published [76][77][78][79][80]. Whenever interpreting results obtained with the Caco-2 cell model, these limitations should be kept in mind.…”