Abstract Gut integrity is a major concern as it plays a crucial role in nutrient absorption, immune defense and the overall health status in young piglets. Among weaning itself, other common stressors occurring in swine intensive conditions have also an impact on the intestine, exacerbating the problems. In this sense, physiological and nutritional studies have become very widespread in the search for innovative solutions to support piglet’s gut health. However, ethical concerns about the practice of samplings from live animals are becoming more critical nowadays. In this sense, alternative techniques avoiding the in vivo methodologies are gaining interest. Three-dimensional organoids are among these alternatives, constituting a more complex and physiologically relevant system that mimics the gut structure better than the traditional in vitro models using classical monolayer cultures. Olive Bioactives (OBE) exhibit great antioxidant and anti-inflammatory activities, potentially improving gut health in weaned piglets. The present study aimed to evaluate the potential OBE to prevent the negative effects on gut integrity of an immune challenge using jejunum organoids as a model, trying to mimic the real complexity of the gut, and minimizing the use of live animals. Porcine intestinal organoids were generated from epithelial crypts isolated from the jejunum, of 30-day-old healthy pigs. Pooled Jejunum organoids from four independent derivations were used to generate two-dimensional organoid-derived monolayers onto Matrigel-coated Transwell inserts. Organoids and monolayers were characterized by assessing the expression of cell-type–specific markers using qPCR and immunofluorescence. Finally, an intestinal barrier challenge model was develop using lipopolysaccharide (LPS) stimulation to evaluate the potential protective effect of OBE, with transepithelial electrical resistance (TEER) and DEAE-dextran permeability as readouts of barrier integrity. The successful establishment of jejunal organoids was demonstrated by the robust expression of markers for stem cells (Lgr5, Olfm4, Sox9, EphB2), proliferative cells (Atoh1, Hes1, Ki67), apoptosis (Casp3), goblet cells (Muc2), enteroendocrine cells (CgA), enterocytes (SGLT1), and epithelial integrity (Villin, E-cadherin). LPS stimulation of two-dimensional organoid-derived monolayers significantly disrupted epithelial barrier function, reducing TEER by 40% compared with controls (2080.8±173.9 vs. 1241.0±102.5Ω/cm²; p 0.0001). OBE pre-treatment partially prevented this decline, maintaining TEER at 82.2% of control values, corresponding to a 22.6% protection against LPS-induced dysfunction (1710.4±130.2Ω/cm²; p 0.0001 vs. LPS). Consistently, LPS challenge markedly increased DEAE-dextran permeability across the monolayers, whereas OBE pre-treatment significantly attenuated this response (Control=5833.6±1523; LPS=18435±1440.2; OBE+LPS=12856.8±1723.3; p 0.005). These results demonstrate the potential of OBE bioactives to mitigate the detrimental effects of immune challenge on intestinal barrier integrity. Moreover, intestinal organoids may represent a reliable and ethical in vitro model to reduce the use of live animals in experimental research. Further studies are required to validate these techniques under various stress conditions and the correlation between these in vitro findings and in vivo data.
Müller-Sánchez et al. (Wed,) studied this question.