Gut organoid culture was first described using mouse small intestinal segments (Sato et al., 2009). This approach was further expanded to other portions of the intestine and was referred to as “enteroid” culture when the small intestinal origin was used or “colonoids” when the colon was used as a source (Stelzner et al., 2012). One crucial cellular component driving the formation of enteroids is the Lgr5+ stem cell lineage, which leads to the production of polarized and differentiated enterocytes, goblet, enteroendocrine, and Paneth cells (Sato et al., 2009). Lgr5+ cells form a layer that initially resembles a spheroid with subsequent formation of invaginations, simulating the fission of the crypts and intestinal architecture observed in vivo (Sato and Clevers, 2013). A study reported that Paneth cells are crucial for the generation of signals essential for the maintenance of ISCs and organoids (Sato et al., 2011). Even though these so-called mini-guts were found to be able to grow in vitro without a need of mesenchymal niche, the minimal growth conditions for these entities include factors and extracellular molecules that normally compose this niche. More precisely, Matrigel, a semi-viscous medium enriched with the extracellular matrix, provides the essential microenvironment for IEC self-renewal, differentiation, and cell–cell interactions (Figure 1A). In addition, a cocktail of biological enhancers such as the bone morphogenetic protein inhibitor Noggin, epidermal growth factor, R-spondin-1, and Wnt3a is required for IEC expansion and maintenance under the culture conditions (Ootani et al., 2009; Sato et al., 2009, 2011). An additional conserved feature of organoids compared with the in vivo system is that organoids exhibit the luminal region in which apoptotic enterocytes and metabolites are expelled. However, in contrast to the gut mucosa, where the external milieu is in contact with the enterocyte apical side, the polarized apical side of enterocytes faces to the inside of organoids, whereas the basolateral region is in contact with the growth medium (Sato et al., 2009). A recent strategy has been optimized to reverse this polarity by eversion of the apical side facing the culture medium (Co et al., 2019).

The use of the CreER/LoxP system, which allows conditional deletion of a targeted gene in an inducible manner, is a powerful tool for determining the role of various molecules in gut organoids that mimic intestinal pathologies (Bohin et al., 2018; Montenegro-Miranda et al., 2020). The recent establishment of organoids derived from the biopsies of patients with intestinal diseases provides the utmost advantage of exhibiting the similar characteristics of the primary diseased tissue from the perspective of developing personalized therapies. d’Aldebert et al. established and characterized colonoids from patients with IBD and compared them with those of healthy individuals. They concluded that despite these colonoids exhibit a similar cellular composition during the first 12 days of culture, IBD-derived organoids exhibit the characteristics of inflammation with increased cell death, reversal of cell polarization, and decreased expression of tight junction proteins (d’Aldebert et al., 2020). Noben et al. reported that IBD-derived colonoids are representative tools for assessing the molecular alterations responsible for the different types of inflammatory disorders in these patients. For example, the organoid cultures derived from patients with Crohn’s disease (CD) had a remarkable decrease in the expression of MUC2 gene transcripts as determined by qPCR and when compared with the organoids derived from healthy individuals or patients with UC. However, the inflammatory gene transcript signatures were not maintained compared with the original biopsies or the primary tissue sites of the corresponding patients (Noben et al., 2017).

Several reports have used gut organoids in studies pertaining to epithelial barrier functions and host–pathogen interactions. Using enteroids from wild-type C57BL/6 mice, Farin et al. (2014) observed a high and rapid sensitivity of Paneth cells toward degranulation and release of microbial peptides in the presence of interferon-γ, without generation of a similar response toward various molecular patterns associated with the microbes. The aforementioned apical-out enteroids not only retain their capacity for cell differentiation and native intestinal functions but also are suitable for barrier permeability assays including 4 kDa fluorescein isothiocyanate-dextrans (Co et al., 2019; Figure 2A). Other experimental protocols include dissociation of organoids, allowing the formation of polarized 2D monolayers directly on the pore membranes of Transwell systems (Kozuka et al., 2017; Tong et al., 2018; In et al., 2019; Figure 2B). Significant efforts are needed to obtain and reproduce the monolayers that mimic different properties of the tissue of origin, such as in vivo epithelial cell organization, epithelial cell absorption and transport mechanisms, and transepithelial electrical resistance (Fernando et al., 2017; Altay et al., 2019). Recently, Wang et al. showed that the fragmented colonic spheroids seeded as single cells on Transwells and exposed to an air–liquid interface for 21 days can recapitulate morphological changes and patterns of cell differentiation. This allowed goblet cells to produce a thick protective mucus layer covering the epithelium, opening the new possibilities for investigating the intestinal barrier under these conditions (Wang et al., 2019). On the other hand, Moon et al. (2014) used culture monolayers from colonoids of multiple genetic mouse strains to study the role of polymeric Ig receptor during immunoglobulin A transcytosis. Epithelial barrier dysfunction in the context of pathogen exposure has also been studied (Noel et al., 2017; Nakamura, 2019). A group of researchers established suitable enteroid monolayers derived from the human fetal small intestine to study viral replication of Enterovirus A71 and bacterial translocation of Listeria monocytogenes after apical infection (Roodsant et al., 2020). The colonoids generated from patients with CD could reproduce epithelial barrier properties, as observed in the native intestinal epithelium, based on the expression profile of key junctional components (ZO-1, OCLN, CLDN4, and CTNNB) and paracellular permeability assays using fluorescein isothiocyanate-dextran (Xu et al., 2018).

Gut organoid culture has provided important clues as to how IECs can integrate external signals from the environment and intrinsically affect cell fate determination during epithelial renewal. The high Wnt activity was found to be central to IEC determination. The high expression of Wnts favors ISC proliferative status and commitment of Paneth cells, whereas the low expression leads to the differentiation of enterocytes and goblet cells (Farin et al., 2014, 2016; Tian et al., 2015; Kim et al., 2020). Thus, pharmacological activators or inhibitors of Wnt, in addition to other pathways such as Notch and ROCK, can be used to manipulate epithelial cell differentiation in the small intestine toward a specific cellular fate (Yin et al., 2014; Beumer et al., 2018; Petersen et al., 2018). Similar strategies have been optimized recently for human and mouse colonoids (Wilson et al., 2021). Another study has reported the reprograming capacity of a specific cocktail of transcriptional factors (HNF4γ, GATA6, CDX2, and FOXA3) to produce gut-like organoids from mouse fibroblasts (Miura and Suzuki, 2017). HNF4γ was also proposed to act as a major driver of enterocyte differentiation by coupling the use of mouse enteroids with integrative systems biology analysis (Lindeboom et al., 2018). Interestingly, Cldn7 depletion in mouse enteroids revealed the crucial role for this tight junction membrane protein in the regulation of ISC survival as well as the differentiation of IEC cell lineage differentiation (Xing et al., 2020). Organoids offer thus exciting opportunities to elicit differentiation- related mechanisms in the context of developmental biology and medicine (Figure 2C).

The organoids derived from ESCs and iPSCs have emerged as an alternative approach when gut tissues are somewhat limited, or when invasive procedures are simply not possible for human patients. This method relies on specifying the progenitor cells from iPSCs with an optimized protocol sequence involving specific signals, factors, and culture conditions to ultimately stimulate their differentiation and subsequent formation of ex vivo organoids (Forbester et al., 2015; Dotti et al., 2017; Hibiya et al., 2017). Unlike other cellular systems, the organoids produced by this method simultaneously promote the presence of mesenchymal cells (Figure 1B), that surround the intestinal epithelium, forming a cellular microenvironment similar to that observed in the intestine (Spence et al., 2011; Gjorevski et al., 2016; Takahashi et al., 2018). Although many single organoids can regenerate from iPSCs, these structures do not recapitulate the mature differentiation process, and rather show characteristics similar to the embryonic development of the epithelium, which limits their use in the functional analysis of gut adult stages (Spence et al., 2011; Fordham et al., 2013). Furthermore, evidence supports that the organoids derived from PSCs exhibit a high tendency for tumor formation in the absence of specific, controlled growth conditions (He et al., 2020). Nevertheless, considerable efforts have been made to improve this method and to bring these models closer to the physiology related to the mature intestinal tissues. One study enhanced mini-gut maturation and cellular vascularization by generating enteroids from human ESCs or iPSCs that were subsequently engrafted in vivo into the kidney capsule of immunocompromised mice (Watson et al., 2014). Another group developed the first model of human intestinal organoids with a functional enteric nervous system using PSC-derived neural crest cells that allowed contractile activity (Workman et al., 2017). Similarly, Holloway et al. (2020) grew and co-differentiated endothelial cell populations in vitro within the culture of human PSC-derived intestinal organoids, with characteristics similar to those of the native endothelial cells, and thus ensuring vascularization of this system. Thus, ESCs and iPSCs are powerful tools for the study of normal intestinal development (McCauley and Wells, 2017; Perez-Lanzon et al., 2018; Sugimoto et al., 2018; Fowler et al., 2020) or for elucidating the mechanisms underlying the onset of diseases such as cancer (Crespo et al., 2017; Smith and Tabar, 2019). However, an important limitation to consider while designing such experimental strategies is the genetic heterogeneity of iPSCs, as opposed to crypt-derived IECs, that can further affect the phenotypes and gene expression of differentiated cells under these assays (DeBoever et al., 2017; Kilpinen et al., 2017).

Organoids are 3D structures, which have become the potential tools for the study of ex vivo intestinal physiology. While mouse organoid experiments can be functionally validated in vivo with available genetically modified mouse models, human organoids provide new perspectives into human biology. However, the expansion of progenitor cells that constitute the fundamental basis of this constantly-renewing system faces challenges in terms of reproducibility among different laboratories. The variations involved in this process range from individual manipulation in the laboratory; oxygen levels and the source and quality of growth medium factors used in the cultures; methodologies of stem cell isolation from the primary tissues; and genetic and epigenetic variability of the cellular source (Shuhendler et al., 2013; Lehmann et al., 2019). All these parameters represent important caveats for the successful establishment of gut organoid cultures. Another important aspect while studying the characteristics of these cultures is the absence of various cell types that normally interact with epithelial cells to maintain the homeostasis in intestinal epithelium. These include immune, mesenchymal, endothelial, muscle, and nerve cells, as well as microbes and their metabolites. Setting up mini-gut co-culture conditions with different cell types, such as isolated intraepithelial lymphocytes, monocytes/macrophages/neutrophils, or fibroblasts are attracting the possibility of reproducing the in vivo intestinal microenvironments under these conditions (Nozaki et al., 2016; Pastuła et al., 2016; Staab et al., 2020). In addition, the recent advances offer new possibilities for the genetic engineering of organoid culture systems. CRISPR/Cas9 technology is one of the most commonly used genomic editing techniques that combines Cas9 endonuclease action to short guide RNA sequences specifically binding to genomic target sequences (Drost et al., 2017; O’Rourke et al., 2017; Roper et al., 2018). The recent inclusion of bioengineering strategies to build biomimetic-based or microfluidic-based scaffolds, gut organoid-on-a-chip which are built as microfluidic devices allowing continuous perfusion as it is the case in a living tissue, and organoid-derived intestinal grafts (Rahmani et al., 2019) might enhance our understanding of the overall structural and cellular complexity of organoid culture.