Faculteit Dierengeneeskunde
November 2015

2REAL-GUTS: innovative non-animal intestinal models

A picture of the intestinal epithelium tissue (by Rob Bleumink, IRAS)

Natural compounds, like food additives and ingredients, could contribute in maintaining or improving human health. An important aspect of the mode-of-action of these compounds concerns their interaction in the gastrointestinal tract. These interactions are especially important in the gut, in which the first-line-of-defense of immune cells reside. This is the research area of the 2REAL-GUTS project1 of Dr. Raymond Pieters, professor at the institute of Life Sciences and Chemistry of HU University of Applied Sciences (UAS) and associate professor in immunotoxicology at the Institute for Risk Assessment Studies (IRAS) of Utrecht University. His research group at UAS started 2REAL-GUTS with funding from the SIA-RAAK program1. This project aims to address the needs of the food industry for relevant intestinal models without the use of animals, to test their products in the company’s Research & Development programs. For example, food company Danone Nutricia Research and animal nutrition company Nutreco are involved in this project.

Better translation: in vitro to in vivo

Currently, the Caco-2 cell line (derived from a colon carcinoma) is widely used across the food industry as an in vitro model to investigate effects on human small intestine. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. The project strives to a better translation of research results from in-vitro intestinal models to in vivo models. Therefore, there is a need to develop new methods to assess biological processes for testing drugs with respect to intestinal health. Two innovative intestinal models are under development, being stem cell-derived organoids (1) and pig explants (2).

1. Organoids

Hans Clevers and his research group showed earlier the capacity of single crypt cells to initiate intestinal organoids in long-term culture conditions2. The advantage of this culture is that stem cells differentiate into different cell types of the intestine, e.g. Paneth cells, goblet cells, endocrine cells. Raymond's research group used this culture method to study effects of natural compounds on organoids of different intestinal origin, from duodenum to colon. However, it would be better to use organoids of human origin to test potential orally administered drugs. Eventually, the 2REAL-GUTS project aims to include human intestinal organoids from the Living Biobank of the Hubrecht Organoid Technology Foundation3 for this research.

2. Pig Explants

To study intestinal tissue functions, including cellular uptake of compounds and cellular interactions, intestinal pig explants are obtained via pig offals. The advantage of explants would be that they have the original set-up of the in vivo intestinal tissue. A variety of culture conditions is required to compare the different outcomes of biological processes. According to dr. Pieters, it should be possible to get functional explants in culture for up to 2-3 hours. The aim is to optimize culture conditions to improve the function of the intestinal explants in vitro.
No animal testing?

The development of these innovative models could reduce and replace animal experiments. However, dr. Pieters admits that laboratory animals are still used: “the validation process, to determine the safety and efficacy of products, still requires animals at some point, although maybe not necessarily mice or rats. If we use more sophisticated in vitro pre-testing, fewer animals are needed. Animal testing in toxicology is often conducted because researchers just follow standard test procedures to comply with governing regulations, even without knowing whether they will provide the right answers with regard to safety".

This reliance on animal models in toxicology is a key issue and a matter of discussion between researchers, policymakers, regulatory agencies, and research-funding organizations around the world. For toxicity testing, the policymakers seem to be the ones that can change regulations. But it should also be kept in mind that some animal models are really useful, of course as long as results can be translated to the clinic or real-life situations. At the same time, those who have already begun to think along animal-free research lines should be encouraged, in particular when research questions approach a mechanistic point of view.

1: Read more about the 2REAL-GUTS project and the SIA-RAAK program here 
2: Sato T. et al., Nature 459 (2009): Single Lgr5 stem cells build crypt-villus structures in-vitro without a mesenchymal niche
3: Living biobanks are initiated by the Hubrecht Organoid Technology (HUB) Foundation, read more here