Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM used at low concentration represent a suitable model to study differentiated rabbit epithelium.

肠类器官是由单层极化上皮细胞形成的自组织3维（3D）结构。这种创新的体外模型与研究肠道上皮的生理及其在营养和屏障功能中的作用高度相关。但是，这种模型从未在兔子中得到开发，尽管它在生物医学和兽医研究中具有潜在的应用。在这里，我们用药理抑制剂（2Ki培养基）或L-WRN细胞条件培养基（L-WRN CM）培养了兔盲肠类器官，以在体外重建肠道干细胞的生态位。。用2Ki培养基获得了较大的球形类器官，这种形态与高水平的增殖和干细胞标记基因表达有关。相反，用L-WRN CM培养的类器官直径较小；较高的像元高度，其中一部分不是球形。当以低浓度（5％）使用L-WRN CM两天时，干细胞和增殖标志物的基因表达非常低，而吸收和分泌细胞标志物和抗菌肽则升高。用2Ki培养基或L-WRN CM（顶端朝向内腔）在3D培养物中极化类器官内的上皮细胞。我们在2D单层中培养了分离的类器官细胞，从而可以进入根尖区。在这些条件下，使用2Ki培养基观察到肌动蛋白应力纤维，而在L-WRN CM中观察到肌动蛋白的周向定位，这表明在2D培养物中，在L-WRN CM存在下，分化水平也更高。总之，与用L-WRN CM培养的类器官相比，用2Ki培养基培养的兔盲肠类器官具有更高的增殖性和更低的分化能力。我们建议用2Ki培养基培养的类器官可以用来快速产生在体外，大量的兔肠上皮干细胞，而用低浓度的L-WRN CM培养的类器官代表了研究分化的兔上皮的合适模型。