Dissecting stromal-epithelial interactions in a 3D in vitro cellularized intestinal model for permeability studies
Introduction
The growing interest in predicting the properties of incoming drugs in the market has increasingly valuing in vitro tools as they may encompass many characteristics of the tissue in question, and also benefit from standardized conditions and lack of ethical considerations [1]. Caco-2 cell model is the gold standard of intestinal in vitro permeability models [2], since after 21 days in culture, these cells acquire a differentiated and polarized phenotype that, morphologically and functionally, resembles the absorptive enterocytes. Besides many of features found in enterocytes are also characteristic of Caco-2 cells presence of microvilli, tight junctions (TJ”), the expression of enzymes (alkaline phosphatase, sucrase) and transporters (P-glycoprotein (P-gp)) [3], [4], the value of Caco-2 model has been mitigated. In particular, the tighter TJ tremendously underestimate the paracellular transport [5] and the efflux transport is overestimated due to P-gp up-regulation [6]. Added to that, in order to overcome the lack of mucus layer that extensively influences absorption, the new cellular intestinal models include HT29-MTX cells that are responsible for mucus secretion [7], [8], [9], [10].
However, the existing in vitro intestinal models are restricted to ‘petri-dish’-based cell cultures that do not replicate the entire architecture and mechanisms occurring in a living tissue [11]. In turn, the striking similarity between the morphology and behavior of cells in vivo and in 3D culture conditions empower the importance of matrix dimensionality [12]. Moreover, intestinal absorption is not only regulated by the intestinal epithelium and epithelial–stromal interactions have been recognized as important players in the maintenance of intestinal mucosal architecture [13]. In particular, intestinal subepithelial myofibroblasts (ISEMFs) constitute a cellular network that sheaths the lamina propria of the intestinal villi. ISEMFs, defined by phenotypic characteristics of both fibroblasts and smooth muscle cells [14], were originally believed to be restricted to a two dimensional network along the villi. In fact, they are connected to α-smooth muscle actin negative fibroblasts-like cells and pericytes (mural cells that surround the capillaries) located in the lamina propria forming a 3D network [15].
The present study extends a traditional standard intestinal model to a deeper layer of the small intestine - the intestinal mucosa. To faithfully capture the in vivo scenario, the herein model focus on the first intestinal barrier to absorption - the epithelial layer – but simultaneously centered in the crosstalk with the 3D network of the myofibroblats in the lamina propria. To this end, CCD18-Co intestinal fibroblasts were embedded in Matrigel onto which lay Caco-2 and HT29-MTX cells. The aim of this work was to characterize the model architecture and functioning in view to establish a tool feasible to study the permeability of novel drugs. For this, an extensive characterization of the fibroblasts behavior within the Matrigel matrix was performed, following the characterization of the epithelial layer. Permeability studies were further carried out to confirm the efficiency of the model in predict the permeability of a model drug.
Section snippets
Materials
Dulbecco's Modified Eagle's Medium (DMEM), DMEM without phenol red, non-essential aminoacids (NEAA), 0.05% tripsin-EDTA, 0.4% trypan blue and HBSS (Hank's Balanced Salt Solution) were purchased from Gibco. Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from BioWest. Resazurin, human insulin, DAPI (4′,6-diamidino-2-phenylindole), fluoroshield™ mounting medium, hexamethyldisilazane (HMDS), triton X-100 and 4 kDa fluorescein isothiocyanate-dextran molecule were purchased from
Behavior of CCD18-Co myofibroblasts in 2D and 3D
To set-up the triple-culture 3D model (CCD18-Co + Matrigel/Caco-2/HT29-MTX), CCD18-Co cell density was optimized, ranging from 1 × 104 cells/cm2 (D1) to 20 × 104 cells/cm2 (D4).
The metabolic activity of CCD18-Co cells cultured under 2D and 3D conditions was evaluated along 21 days. In 2D conditions (Fig. 1A) at day 7, the metabolic activity presented a stairway profile between D1 and D3, with exception of D4. From day 7–14, a significant increase in metabolic activity was observed for D1 and
Discussion
In the attempt of establish a triple-culture 3D model, myofibroblasts behavior was evaluated bearing in mind that it can substantially differ depending on whether the cells are in 3D or 2D culture conditions [35]. Therefore, metabolic activity and proliferation assays were performed over a range of densities (between 1 × 104 cells/cm2 (D1) and 20 × 104 cells/cm2 (D4)) in both 2D and 3D conditions, to assess the most suitable density to be used over 21 days, which corresponds to the period
Conclusion
In this study, a triple-culture 3D model was successfully established reproducing somewhat the native intestinal mucosa in which myofibroblasts had an active role in the maintenance of the overlying epithelial cells through the production of their own ECM matrix.
This model shown to efficiently predict the insulin permeability, which is related with the less tight character between Caco-2 and HT29-MTX cells, the 3D assembly of myofibroblasts and the presence of mucus layer.
Even though in vitro
Acknowledgments
Raji B cell line was kindly provided by Dr. Alexandre Carmo (IBMC, Porto, Portugal) and CCD18-Co cells were kindly provided by Dr. Sérgia Velho (IPATIMUP, Porto, Portugal).
This work was also financed by ERDF through the Programa Operacional Factores de Competitividade – COMPETE, by Portuguese funds through FCT in the framework of the project PEst-C/SAU/LA0002/2013, and co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte) in the framework of project
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