3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells

Abstract

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications.

Bioengineering Novel in vitro Co-culture Models That Represent the Human Intestinal Mucosa With Improved Caco-2 Structure and Barrier Function

“…Some of these improved models include primary enterocytes or intestinal fibroblasts, both of which present issues of variability and lack of reproducibility (Takenaka et al, 2014;Schweinlin et al, 2016). Many coculture systems also utilize fibroblast layers as a subepithelial compartment, which fail to account for the spatial organization within the mucosal tissue (Li et al, 2013;Matsusaki et al, 2015).…”

“…Both fibroblast lineages have previously used in co-culture studies with Caco-2 cells and have induced changes in the epithelium to better resemble the in vivo intestine. For example, co-culture of dermal fibroblast and Caco-2 cells induced a switch in the predominantly expressed carboxylesterase (CE) expression in Caco-2 cells from CE1 to higher CE2 expression more accurately reflecting the human small intestine (Matsusaki et al, 2015). Co-cultures developed using CCD-18co cells embedded in Matrigel have been shown to reduce TEER and P-gp expression and more accurately predicted the permeability of insulin than Transwell R monolayers (Li et al, 2013;Pereira et al, 2015).…”

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro

“…A number of intestinal in vitro models have now been developed using relevant tissue-specific cell types such as fibroblasts 19,20 or immune cells such as macrophages and dendritic cells 21,22 added to the barrier forming enterocytes such as Caco-2. As previously reviewed, the microvascular endothelium of the intestinal villi also plays a crucial role in the innate and adaptive immune defense and accordingly also in the pathogenesis of IBDs.…”

In Vitro Entero-Capillary Barrier Exhibits Altered Inflammatory and Exosomal Communication Pattern after Exposure to Silica Nanoparticles

“…The present model could be modified to more closely mimic the in vivo conditions of the iMV in vitro through the addition of other tissue-relevant cell-types such as monocytes/macrophages or dendritic cells [1,2,3,4], intraepithelial lymphocytes [5], fibroblasts [6], enteric glial cells [7,8] or intestinal pericytes [49]. These cell types have a critical influence on the iMV.…”

“…Multicellular in vitro barrier models are increasingly being used in studies to evaluate, e.g., immune responses of the intestinal barrier after exposure to externally added stimuli. In vitro models with enterocytes combined with several other tissue-relevant cell-types such as monocytes/macrophages or dendritic cells [1,2,3,4], intraepithelial lymphocytes [5], fibroblasts [6] or with enteric glial cells [7,8] have been described. However, until recently, the intestinal microvasculature (iMV) has been largely ignored.…”