One organoid model system was developed by Ootani et al. [20], whereby pieces of whole intestine or colon (including mesenchymal cells and enteric nerves) are grown in a liquid–air interface, which allows for long-term growth of organoid structures. These organoids contain the major differentiated intestinal cell types found in vivo. Furthermore, the presence of a mesenchyme within these organoids allows growth of the organoids without supplementation with niche factors such as Noggin and R-spondin. Thus, this system would be advantageous for examining the effects of epithelial–mesenchymal interactions in the context of inflammation or regeneration; however, to date it has not been used to study IBD. However, the system has been used to effectively investigate the effects of oncogene activation and inactivation of tumor suppressor genes in mouse models and could be used to study these changes in the context of IBD. For example, loss of the APC tumor suppressor gene allows for faster recovery in response to DSS-mediated epithelial injury [21].

The most commonly used intestinal organoid model was developed in the lab of Hans Clevers [9], and these will be referred to as enteroids because they are purely epithelial. Sato and colleagues demonstrated that single Lgr5+ stem cells could be grown into 3D enteroids in Matrigel culture in medium containing EGF, Noggin (BMP antagonist), and R-spondin (ENR). Interestingly enteroids were able to proliferate and differentiate spontaneously despite the lack of any mesenchymal cells , suggesting that ENR media was sufficient to replace the signals derived from supporting mesenchymal cell types. In addition, this system was used to demonstrate that the Paneth cells act as an ISC niche cell by supplying Wnt3a [5]. Although there are numerous studies that have used the mouse enteroid system to address questions about epithelial biology in an IBD context, we focus on several key examples.

One study by Günther and coworkers examined the role of caspase 8 in epithelial cell death and inflammation of the ileum [22]. This work was based on the observation that mice deficient in caspase 8 within the intestinal epithelium (Casp8^ΔIEC) have depleted Paneth and goblets cells and develop spontaneous ileitis. Examination of enteroids derived from the intestines of Wild Type (WT) and Casp8^ΔIEC mice revealed no differences in the number of Paneth cells, suggesting that a factor present in vivo affected the differentiation or survival of this cell type. To further explore this possibility, the authors examined the effect of TNF-α stimulation on organoids from WT and Casp8^ΔIEC mice. Interestingly, 24 h after stimulation, WT enteroids appeared normal while Casp8^ΔIEC enteroids underwent nec-1-dependent necrosis. In addition, the authors presented evidence that RIP-mediated necroptosis of Paneth cells was also present in samples from patients with Crohn’s disease and suggested that this may be the cause of the defective antimicrobial defense that is observed in these patients.

In another study, Farin and Karthaus et al. used enteroids to examine the effect of bacterial antigens on Paneth and goblet cells [23]. Interestingly these antigens had little effect on these cell types regardless of whether they were administered apically or basolaterally. However, when inflammatory cytokines were applied to these organoids, only IFN-γ caused degranulation of Paneth cells. In addition, IFN-γ stimulation led to loss of mucus containing goblet cells and mature enterocytes. As a consequence of the loss of Paneth cells, organoid growth was severely compromised likely due to the role of Paneth cells as a niche cell for intestinal stem cells. Taken together, these studies demonstrate how this organoid system can be used to interrogate the effect of inflammatory cytokines on various cell types.

Human enteroids (as designated by the NIH Intestinal Stem Cell Consortium [19]) can be grown from isolated intestinal crypts or CD44⁺CD166⁺CD24^lo single cells in Matrigel based 3D culture [9, 24]. In the case of colon, single cells can be grown into colonoids by FACS sorting based on the expression of ephrin type-B receptor 2 (EPHB2) [7]. Enteroids have been generated from small and large intestine and require a unique set of growth factors. Cultured human intestinal enteroids (and colonoids) require Wnt3a, gastrin, nicotinamide, A-83-01 (Alk4/5/6 inhibitor) and SB202190 (p38 inhibitor) in addition to basic growth media containing EGF, Noggin, and R-spondin-1 [8]. These enteroids display remarkable similarity to intestinal tissue in vivo. Enteroids self-organize into structures containing a central lumen surrounded by a single cell layer of polarized epithelial cells. In addition, this single cell layer is organized into crypt-villus like structures which can differentiate into the main differentiated cell types following withdrawal of Wnt3a, nicotinamide, and SB202190. Interestingly, like their mouse counterparts, these organoids lack mesenchymal cells, suggesting that growth factors produced by mesenchymal cells (rather than physical interaction with these cells) are necessary for maintenance of the intestinal stem cell niche.