# Comparison of 2D, 3D In Vitro, and Ex Vivo Platforms for Modeling the Rat Small Intestine

## TL;DR

This study compares 2D, 3D in vitro, and ex vivo models of the rat small intestine, showing that the 3D model better mimics real tissue behavior.

## Contribution

The study validates a 3D bioelectronic platform for modeling the rat small intestine, demonstrating its superior functionality compared to 2D models.

## Key findings

- The 3D platform showed time-dependent increases in barrier resistance over 21 days.
- The 3D model and ex vivo tissue exhibited reversible barrier disruption and recovery in response to EGTA.
- 2D cultures showed limited recovery after EGTA treatment, unlike the 3D and ex vivo models.

## Abstract

Physiologically relevant in vitro intestinal models are essential for studying key physiological processes, including barrier function, drug screening and gut-microbiota interactions. However, conventional 2D culture systems often fail to recapitulate structural and functional complexity. Here, we aimed to validate a 3D bioelectronic transmembrane platform, previously used for monitoring human intestinal epithelium and vascular endothelium, for modeling the rat small intestinal barrier in vitro. The device integrates a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) scaffold supporting co-cultures of rat intestinal epithelial cells (IEC-6) and rat fibroblasts (208F), enabling real-time monitoring of barrier formation through electrical measurements using electrochemical impedance spectroscopy (EIS). Barrier formation was monitored over 21 days and exhibited a time-dependent increase in barrier resistance. The 3D platform was compared with traditional 2D insert-based cultures and ex vivo rat tissue using an Ethylene Glycol Tetraacetic Acid (EGTA)-induced calcium switch assay to evaluate barrier disruption and recovery. EGTA treatment and removal induced reversible barrier disruption in the 3D in vitro and ex vivo models, whereas 2D in vitro cultures showed limited recovery. These findings demonstrate that the 3D platform more faithfully recapitulates native tissue architecture and function, closely paralleling ex vivo responses. Our study highlights the importance of validating advanced 3D in vitro models and establishes this bioelectronic platform as a robust tool for drug screening, barrier studies, and preclinical gastrointestinal research.