Establishment of a broiler jejunal organoid model based on optimized activators or optimized two-step media and its assessment

Intestinal organoids (IOs), as a novel three-dimensional (3D) in vitro model, have been widely applied for studying intestinal physiology, metabolism and health in human, rodents and farm animals. However, the culture methods for poultry, especially for broiler IOs, need to be improved due to their low differentiation to form complex, self-organized 3D structures and intact barrier functions. This study aimed to establish the broiler jejunal organoid (JO) model by optimizing the culture conditions of JOs and evaluating the cellular components and barrier integrity of JOs via a two-step media [TSM, including both JO growth medium and JO differentiation medium (JODM)]. The results showed that JODM containing 50 or 100 ng/mL of R-spondin 1 and 2.5 μM of Chiron 99021 increased (P < 0.001) the budding percentage of the JOs up to about 80% and maintained their passaging and cryopreservation capabilities. Furthermore, a variety of intestinal functional cells differentiated into JOs as evidenced by the expressions of different marker proteins, and JOs displayed evidence of barrier integrity. Compared with conditioned medium-dependent medium (CMDM) and IntestiCult™ Organoid Growth Medium (IOGM), TSM increased (P < 0.001) the budding percentage of JOs up to about 80%. Moreover, IOGM and TSM up-regulated (P < 0.001) marker genes expression of different intestinal functional cells compared with CMDM, and jejunal crypts sourced from broilers of different ages had no effect (P = 0.792) on the differentiation and budding percentage of JOs. In conclusion, the broiler JOs model established using the TSM-culture protocol developed in this study has the advantages of high differentiation, enriched budding structures and stable passaging and cryopreservation, providing a superior experimental model for studying the physiological responses of the broiler intestine to nutrients, feed additives, pathogens, drugs and their mechanisms.