Review of available cell-based in vitro models to evaluate intestinal permeability

Gastrointestinal System 3D
David Temporal | BD Department
25 November 2021

In vitro models are commonly used and wildly accepted in research stages that investigate several biological processes, reducing animal experimentation, simplifying discovery projects, and minimizing operational costs.

The gastrointestinal (GI) system is considered a relevant tissue due to the absorption of xenobiotics during digestion. Consequently, in vitro models that reproduce intestinal homeostasis were developed and standardized to correlate to in vivo gastrointestinal kinetics.

These systems are mainly based on immortalized cellular lines forming an adherent monolayer reflecting certain characteristics of the GI epithelium. The cell monolayer grows to confluence on a semi-permeable membrane separating two chambers used to assess the permeability of a compound from the donor to the receiver chamber. This system simulates bioavailability from the gut lumen to the circulating basolateral lymph.

This post reviews the most used in vitro models indicated for drug discovery and scientific research following scientific recommendations, starting with:

  • In vitro models based on intestinal Caco-2 cells are considered the reference model following the main regulatory agencies’ recommendations. These cells create a monolayer that models intestinal enterocytes, the most abundant epithelial cell type in the intestine. Caco-2 cells systems are well known and obtained results are extrapolated to physiological conditions.

Additionally, numerous in vitro cell systems that integrate various intestinal cell types are being developed resembling the complex anatomy of the GI tract epithelium. These systems are indicated to model complex intestinal pathways and chemical mechanisms, reproducing closely physiological conditions, such as:

  • Combination of Caco-2 and HT-29 cells. HT29 cells represent 10-25% of the intestinal cells and are responsible for the mucus barrier of the GI tract. The coculture of Caco-2 and HT-29 cells create an in vitro system closer to physiological conditions that evaluate alternative cellular transport mechanisms observed in the intestine.


  • Caco-2 and Raji B cells. Raji B cells are human cells with immunological activity when the cell line is cocultured with Caco-2 cells. It is considered a reference model to predict intestinal absorption, considering the possible immunological response to the tested compound during intestinal homeostasis. The Raji B cells are added in the apical and basolateral compartment of the transwell plate once the Caco-2 cells are completely differentiated.New biotechnological developments are also allowing alternative in vitro models that reproduce intestinal conditions. Once these systems’ extrapolation to physiological conditions is demonstrated, they will most likely be considered reference systems as well.


  • In vitro3D cell-culture models are emerging that accurately mimic the structure of the GI mucosa, including structural fibroblasts and the reference intestinal cells for scientific research, the Caco-2 cells. The cells are cultured in a 3D scaffold mainly composed of collagen, which replicates the intestinal anatomy.


  • Intestinal organoids, scientific research of Adult Stem Cells is capable to obtain differentiated intestinal tissue, including various cellular types, which mimics in vivo cellular metabolism. Some preeliminar models are being developed in 3D models cocultured with fibroblasts.


  • The gut-on-a-chip in vitro model is designed with fluidic channels plates seeded with a Caco-2 monolayer and engineered with microcircuitry to assess xenobiotics’ kinetics. Once this technology is developed will reduce assay timings and will facilitate test procedures.


Since most drugs are developed as oral dosage products, evaluating the mechanism of action of intestinal absorption is a crucial pharmacokinetic parameter. In vitro kits offer screening optimization of novel compounds, correlating the in vitro permeability with in vivo absorption.

ReadyCell, as a specialized manufacturer of in vitro kits indicated for intestinal permeability assays, is available to share our knowledge in preclinical development. Reach out to us at


Lefebvre, David E., et al. “Utility of models of the gastrointestinal tract for assessment of the digestion and absorption of engineered nanomaterials released from food matrices.” Nanotoxicology 9.4 (2015): 523-542.

Related posts

Select the right well system for your research: A comprehensive guide.

Select the right well system for your research: A comprehensive guide.

In preclinical research, the choice of the appropriate well system format – whether it’s a transwell, non-transwell, or individual transwell – can significantly impact the outcome of experiments. Selecting the correct plate is crucial to working with permeability, toxicity, drug screening, or other assays.