Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - COMIET (Engineering Complex Intestinal Epithelial Tissue Models)

Teaser

What is the problem/issue being addressed?Epithelial tissues form barriers protecting the body against physical, chemical, and microbial offences. Epithelial tissues cover all the surfaces and cavities of our bodies and line most of our organs. Functional in vitro models of...

Summary

What is the problem/issue being addressed?

Epithelial tissues form barriers protecting the body against physical, chemical, and microbial offences. Epithelial tissues cover all the surfaces and cavities of our bodies and line most of our organs. Functional in vitro models of epithelial tissues are key elements in basic biological research, disease modelling, drug discovery, and regenerative medicine. Clinical applications of tissue engineering are hampered by the risk of bacterial infection due to the lack of functional epithelial engineered tissues. In the case of the small intestinal epithelium, functional in vitro models are needed to accurately predict the absorbance of drugs delivered orally.

Why is it important for society?

Almost all our fundamental knowledge about cell biology comes from in vitro assays performed on cell monolayers growing in plastic wells. This simplistic approach leads to in vitro results that are not always useful within the in vivo scenario. For instance, anticancer drugs that were effective in vitro do not work when working with the tissue in vivo. Therefore, there is a trend to shift from the simplistic approaches to organotypic, tissue-like systems for in vitro applications. COMIET aims to accelerate this trend by providing a new cell culture platform for epithelial tissues which can advance the in vitro modelling of diseases, the preclinical screening for drug efficacy and toxicity and the understanding of organ development. Today, advancing a drug candidate to regulatory approval takes roughly 12 to 15 years and costs an estimated $800 to $900 million on average. As over 50% of drug candidates fail due to ADME (absorption, distribution, metabolism, and excretion) or toxicity deficiencies, early discarding of compounds in the in vitro preclinical tests saves costs, time and reduces the ethical concerns of indiscriminate usage of animal testing. On the other hand, the impact of digestive diseases and disorders is huge and have enormous societal and economic costs. The quality of life of people affected with inflammatory bowed disease, Crohn’s syndrome, ulcerative colitis or irritable bowel syndrome is significantly lower than in healthy persons, comparable to those with clinical depression, and lower than in those with type 2 diabetes or who have experienced a heart attack. More than 40% of these patients report avoidance of some activities, including work, travelling, and socializing. Because the aetiology of these disorders is not well established, therapeutic approaches are only symptomatic. In this context, the development of in vitro human intestinal models that faithfully replicate in vivo behaviour might contribute to shed light on the basic biology of these diseases, assessing drug efficacy and toxicity, and proposing new therapeutic strategies. In addition, new intestinal functional models developed from adult stem cells also open new possibilities in regenerating intestinal tissues or personalizing cancer healthcare.

What are the overall objectives?

COMIET proposes the use of microengineering technologies to develop artificial intestinal epithelial tissues that mimic the physiological characteristics found in vivo to open new areas of research on human intestinal diseases. The novel in vitro models of intestinal epithelial tissues will address in vivo intestinal epithelial cell renewal and migration, multicell-type differentiation, epithelial cell interactions with the underlying basement membrane and the interactions with the luminal contents to go beyond the state-of-the-art 3D organoid-based in vitro models. The cell culture platforms proposed in here will provide physiologically relevant and highly reproducible data, and they will be compatible with conventional cell culture assays and high-throughput testing. To achieve this, we will develop an experimental setup that combines microfabrication technologies, tissue engineering and advances in intestinal stem cell research, e

Work performed

Since the beginning of the project, work performed in COMIET has dealt with the technological set up for the microfabrication of scaffolds that faithfully reproduce the anatomical architecture of the human small intestinal tissue. In addition, we had established the culture of intestinal epithelial cells derived from intestinal organoids. The main results achieved so far can be summarized as:

1. We have successfully implemented a relatively simple and cost-effective method to fabricate soft 3D villi-like microstructures with physiological-resembling anatomic architecture and dimensions.

2. Artificial villi-like microstructured hydrogels have proven suitable for epithelial cell growth. Caco-2 cell monolayers formed on the villi-like microstructures showed values closer to in vivo situation than those found in the current standard models. We have also established the co-culture of Caco-2 and goblet cells on these villi-like microstructures, thus recreating the mucus layer. These promising results suggest the suitability of our in vitro model in drug absorption and toxicity tests, as well as biological studies and disease modelling.

3. We have successfully generated intestinal epithelial monolayers that contain major intestinal cell types organized in proliferative and differentiated cell domains as in vivo. We have established a protocol that renders monolayers with tissue barrier function suitable for drug absorption studies.

4. We have established a protocol to trigger ephrin signalling, which is related to cell compartmentalization, from surface-bound ligands and nanopatterns.

Final results

Because most of the drug uptake is delivered orally, drug absorption at the intestine is routinely measured in in vitro preclinical tests. Because the vast majority of these tests are performed with cell lines on flat surfaces, drug absorption values do not correlate with in vivo measurements for drugs with low permeability. In such cases, it has been demonstrated that the presence of the three-dimensional architecture of the small intestinal epithelium is key in the absorption process. In COMIET, we provide an alternative cell culture system that shows as advantages to the standard state of the art: (i) reproduces the three-dimensional architecture of the tissue structure, (ii) it is as soft as the intestinal tissue, (iii) the fabrication process is relatively easy and cost effective and can be adapted to standard cell culture platforms used for drug permeability assays, (iv) provides TEER values closer to those reported in vivo. As over 50% of drug candidates fail due to ADME (absorption, distribution, metabolism, and excretion) or toxicity deficiencies, early discarding of compounds in the in vitro preclinical tests saves costs, time and reduces the ethical concerns of indiscriminate usage of animal testing.

On the other hand, the impact of digestive diseases and disorders is huge and have enormous societal and economic costs. Because the aetiology of these disorders is not well established, therapeutic approaches are only symptomatic. In this context, the development of in vitro human intestinal models that faithfully replicate in vivo behaviour might contribute to shed light on the basic biology of these diseases and contribute to propose new therapeutic strategies. In this sense, the identification of an adult stem cell source in the small intestine able to proliferate and differentiate in vitro, forming self-assembling 3D structures, called organoids, with the multicellularity, functionality and renewal properties of the in vivo tissue, opened a new avenue in the field. COMIET is contributing to this field by providing a new protocol to produce intestinal epithelial monolayers from this adult stem cells. These new systems might be used in basic biology applications, drug absorption tests and disease modelling. It will also open new possibilities in regenerating intestinal tissues or personalizing cancer healthcare.