Organoids as a research tool in animal breeding and nutrition
Organoids are an important innovative key technology that can have a large impact in animal breeding, nutrition and health. In this project we will develop high throughput tools to generate and use organoids from pig intestines. Multiple studies will be performed focusing on intestinal organoids to test intestinal functionality. This project will result in the development of organoids as an in vitro key technology to contribute to different Topsector missions with less animal testing.
This project contributes to mission ST2 “Biotechnology and Breeding”, priority 44, especially SUBDOMAIN “Phenotyping”. In this PPP project we will use the innovative in vitro organoid key technology to generate organoids on a large scale in a high-throughput manner so that we can confidently and conveniently measure phenotypes as reflected in the in vivo conditions. A high throughput in vitro intestinal organoid model will be developed for pigs. The intestinal organoids will be tested as a screening tool for functional dietary ingredients that support intestinal health, nutrient absorption and epithelial (barrier and immune) response in order to improve health and efficiency of pigs. The in vitro phenotypes will be linked to the in vivo phenotype (health, efficiency) of the animal where the organoid is derived from. The organoid key technology will enable breeding and nutrition companies to contribute to improve the missions and priorities related to animal health and welfare (mission D, priority 28) and circular agriculture (mission A) of the pig husbandry. Furthermore, using organoids as an in vitro model system contributes towards the 3R’s, in particular to the refinement of animal experiments with the potential to reduce animal testing.
Currently, the in vitro organoid system is only used for small scale research involving a small number of animals. To apply the in vitro organoid system in practice, there is a need to develop a robust high throughput system. This will enable breeding companies to produce large units of “mother/feeder” organoids from a large number of slaughter animals to use as a phenotyping tool. Furthermore, it will enable nutrition companies to develop a screening tool of functional ingredients on a large number of “daughter” organoids (many organoids from a single pig), which will reduce the number of animal experiments.
Methods developed in the project will result in the high throughput application of the in vitro organoid system in animal breeding, nutrition, and health. Topigs Norsvin Research Center B.V. (TNRC) will use this key technology to phenotype selection candidates on a large scale, resulting in effective breeding goals and breeding strategies that contribute to the mission(s), i.e. sustainable livestock farming. Furthermore, methods developed in this project will enable Nutrition Sciences N.V. (NuS) to increase the speed of screening functional ingredients in order to optimize the feed of the future and therefore contributing to sustainable livestock farming and a further decrease in the use of antibiotics. The unique key to the success of this project is the combination of the development of key technologies for high throughput and the in-depth studies, which will support the application of the in vitro tool in practice. This project will strengthen the top position of TNRC, NuS and Wageningen Livestock Research (WLR) in in vitro research and the development of new phenotyping and screening tools. Furthermore, the project is crucial to have impact in the Topsector missions and to reduce the number of animal experiments.
This project will deliver technological tools to phenotype animals (mission ST2). In the long term, this project will contribute to the Topsector missions A and D. More specifically, this project will contribute to the improvement of animal health and welfare, and circular agriculture, with less animal testing.
The strength of this consortium is that implementation of the key technology and the generated knowledge is ensured by the direct involvement of a breeding and nutrition company. Tools developed in this project will help the animal breeding and nutrition industry to improve the health, welfare and efficiency of their animals. TNRS will use organoids to improve the efficiency and health of their pigs, contributing sustainable pig husbandry in The Netherlands and worldwide. NuS will use organoids as a screening tool for health supporting functional ingredients to include in the feed of the future, with less animal testing. WLR will use organoids to strengthen their research position and work on a better quality of life.
Aim of the project
The main aim of this PPP is to pave the road for high throughput in vitro organoid systems of the pig intestinal tract to use as a key technology in animal breeding and nutrition. The objectives are:
- Develop a robust key technology to generate pig intestinal organoids in a high throughput manner
- Use organoids to measure nutrient uptake and epithelial (barrier and immune) response thereby connecting to an animal (in vivo) phenotype
- Employ organoids to screen different functional ingredients, including glycolipids (e.g. glucose caproate) and ligand receptors (e.g. galactooligosaccharides)
The corresponding work packages and activities
WP1. Detailing Standard Operating Procedures to generate pig intestinal organoids in a high throughput manner
1.1 Collect tissue samples from a large number of pig intestines
1.2 Produce “mother/feeder” organoids on a large scale
1.3 Store tissue and organoids
1.4 Optimize the high throughput 2D Transwell organoid system using “daughter” organoids
In WP1 we foresee a phasing of collecting the relevant tissues samples and producing organoids in activity 1.1 and 1.2. Both activities will consist of three phases. (1) In phase 1 we will use 10 (slaughter) pigs, of which we will collect tissue samples from both the small and large intestine. Phase 1 will be used to optimize the current standard operating procedure (SOP) to collect tissue and produce organoids on a large scale, using state-of-the-art laboratory technologies. (2) In phase 2 we will collect tissue from 40 pigs of which (performance) phenotypes are available. Phase 2 will be the testing phase of the high throughput key technology. (3) In Phase 3 another 120 pigs will be sampled. During the collection of the relevant tissue samples, we will also examine different freezing methods (activity 1.3), i.e. collecting the tissue and generate organoids and subsequently freeze them or freeze the tissue directly and generate organoids at a later stage. In activity 1.4 “daughter” organoids (many organoids from a single pig) will be used to optimize the 2D Transwell organoid system.
D1. Standard operating procedure (SOP) collection of tissue samples
D2. SOP production of organoids
D3. SOP storing of tissue and organoids
D4. SOP 2D monolayer transwell system
Go / no-go: After completing the activities in WP1, there will be a go/no-go moment, to evaluate whether to initiate or to re-design the activities of WP2.
WP2. Studies to measure nutrient uptake, response to microbiota challenge, and screening of functional ingredients in organoids and relate them to the phenotype
2.1 Measuring nutrient uptake in organoids
2.2 Measuring epithelial response to bacterial challenge in organoids
2.3 Screening functional ingredients using organoids
2.4 From organoid to phenotype
In WP2 the activities are cascading, in the sense that activity 2.1 will be initiated first, because the technique/outcome can also be applied in the activities 2.2 and 2.3. Similarly, for activity 2.2 the technique/outcome can also be applied in activity 2.3. Activity 2.4 will investigate to what extend the in vitro organoid model can be linked to the in vivo phenotype of interest. Activities 2.1 to 2.3 will be carried out on “daughter” organoids. Furthermore, activities 2.1, 2.2., 2.3, and 2.4 will deliver a report/scientific paper.
D5. Report/scientific paper measuring nutrient uptake
D6. Report/scientific paper measuring epithelial response to bacterial challenge in organoids
D7. Report/scientific paper screening functional ingredients using organoids
D8. Report/scientific paper from organoid to phenotype
- Monthly: project team – research from WLR, TN, NS
- Quarter: Steering committee meeting