Initial pig developmental stage influences intestinal organoid growth but not phenotype

Intestinal organoids are promising tools in the context of animal experiment reduction. Yet, a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig, a valuable model of Humans. Organoids were derived from jejunal and colonic ISC obtained from 1, 7, 28, 36 and 180-day old pigs and passaged three times. We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (P<0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, P<0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (P<0.05), spheroids were larger during the primary culture but smaller after two passages (P<0.05) and organoids smaller after one passage (P>0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) were observed between organoids originating from 7 or 180-day old pigs, while difference between intestinal site origin were noticed. In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence their phenotype.