Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

Summary

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

Abstract

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, “healthy tissue” three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Introduction

The intestinal epithelial barrier, a one-cell-thick internal lining containing different types of epithelial cells, constitutes the first physical defensive barrier or interface between the outside and the internal milieu of the body^1,2. Columnar-type enterocytes constitute the most abundant type of epithelial cells. These are responsible for maintaining epithelial barrier integrity through interactions between several barrier components, including tight junctions (TJs), playing a significant role in barrier tightening^1,3. The TJ structure consist....

Protocol

1. Preparation of the basic 3D reconstructed intestinal mucosa model

NOTE: The entire procedure must be carried out in a sterile laminar flow hood. All steps in the procedure involving the use of the cell incubator signify that cultures are incubated at 37 °C in a humidified atmosphere containing 5% CO₂ (unless stated otherwise in the protocol)_.

1. Prior preparation of the cell lines used in the intestinal model system
   1. Seed L929 mouse fibroblast .......

Discussion

The basic reconstructed intestinal mucosa model system presented here (Figure 6) combines physiological complexity (more physiologically relevant 3D cell cultures containing a Caco-2 monolayer with an ECM-rich lamina propria support containing fibroblasts and monocytes) with experimental simplicity (using commercial human cell lines to produce standardized and easily repeatable system)¹³. As such, this model system is deemed a suitable alternative to murine models aim.......