Gene editing in breeding

Organoid immune-cell co-culture platform to advance animal breeding research

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Abstract

Organoids are 3-dimensional (3D) structures that represent the organ from which they originate, containing most of the cells that are present in the organ (Sato and Clevers, 2013). An in vitro intestinal porcine organoid model can be a powerful tool to 1) study complex phenotypes, like (gut) health, 2) screen functional ingredients or pharmaceutical components, and 3) reduce animal testing. However, this in vitro model typically lacks immune competence. A major intestinal immune cell population are macrophages. These cells are both responsible for first line of protection and crucial for maintenance and stability of the organ. Moreover, macrophages adjust their local phenotype based on nutrients and microbial metabolic products, making them a valuable immune cell type for animal research. The aim of this study was to develop a 2D co-culture of pig intestinal organoid-derived cells and blood-monocyte-derived macrophages to investigate the biological mechanism of gut health and immune response in porcine. Intestinal tissue and blood samples were collected from three pigs (3 technical replicates/pig). From the blood samples, monocytes were isolated and differentiated into macrophages, using established, hM-CSF-based protocols. In this study, a large variation between animals was found in immune cell viability prior to the introduction of immune cells to the co-culture. Immune cells from only one animal were viable and could be used in the co-culture. Simultaneously, ileal organoids were cultured on the apical side of Transwells. After 7 days of culture, macrophages were added to the basolateral side of the organoid culture. To test immune cell response of the co-culture, on day 7, Lipopolysaccharide (LPS) was incubated overnight to the apical side of the organoid immune-cell co-culture. On day 8, medium and cells were collected for characterization. Immune cells were characterized by flow cytometry. Co-culture functionality was assessed through barrier permeability (measuring the trans-epithelial electrical resistance (TEER)) and immune response to LPS was measured with cytokine levels. In general, a confluent monolayer was formed after one day of organoid culture (Figure 1A). Adding the immune cells and LPS to the organoids did not affect the TEER values (Figure 1B) of the co-culture. Preliminary flow cytometry results show significant higher MFI levels of immune cell marker CD172a when macrophages were cultured in the co-culture system relative to macrophage monocultures. Additionally, macrophages cultured in a co-culture system showed a tendency towards an anti-inflammatory polarisation based on changes in CD1, CD80 and CD163 MFI levels in comparison to the macrophage monoculture. Additional analysis and repeat of the experiment are needed to confirm these results. However, first results are promising, indicate the potential for a stable co-culture model, and suggest that this model may provide deeper insights into the immune system, which could help to improve porcine gut health.

Keywords: 2026

How to Cite:

van der Valk, E., Kruijt, L., Van Laar, N., Govers, C., de Wit, A., Söllner, J., Aydin, E., Verstringe, S., Zaccaria, E., Kar, S. & Ellen, E., (2026) “Organoid immune-cell co-culture platform to advance animal breeding research”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2286562. doi: https://doi.org/10.31274/wcgalp.24050

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