# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 2 Technological Approaches of Intestinal Epithelium Models
### Cell Cultures Used in Epithelium Models
#### 2.3.4 Organoid Cultures  
Organoids are cell-based _in vitro_ models that mimic many characteristics of the corresponding _in vivo_ tissue [55]. During the generation of an intestinal organoid, tissues from  
Figure 4: Schematic of transepithelial electrical resistance (TEER) measurements [58]. the small or large intestine are fragmented. The fragments are treated with EDTA, which chelates the Ca\({}^{2+}\) and disrupts cell-cell adhesion. This way, primary intestinal crypts are obtained, which can be used for the generation of organoid cultures [55]. Cells are seeded into biologically derived matrices (e.g., Matrigel, collagen) or into synthetic hydrogels [55]. The cells of epithelial organoids are arranged in a single layer and form a sphere [8]. Besides the predominant absorptive enterocytes, other specialized cells are also present, like goblet, Paneth, Tuft and enteroendocrine cells [27]. Furthermore, the differentiation of M cells from the epithelial precursor cells can also be induced [27]. The proliferative regions, which are enriched with stem cells, form protrusions oriented in the outward direction of organoids [8]. Generally, in intestinal organoids, crypt andillus-like parts can be distinguished but the apical side of the epithelium is facing inward [17]. _In vivo_, the self-renewing capacity of the intestinal epithelium (turnover rate 4-5 days) may compensate for the extensive cell death caused by the digesta and the mechanical abrasion [14]. In organoids, new progenitor cells that differentiate into different intestinal cells types are continuously generated. It is possible that this regeneration could compensate for the toxicity of the digesta. By sustaining the self-renewing capacity of the stem cells, organoids imitate the structure and functions of the tissues, while keeping the cell-type diversity of the tissue [62]. Still, unpredictable growth can occur even in the case of the same stem cell lines [47]. This may diminish reproducibility [63]. The absorption and metabolism are higher in organoids than in 2D cell cultures [63]. Organoids are lacking some components of the whole intestine, for example, immune cells, neurons, and blood vessels covered with endothelium [28; 42; 45]. The morphology of the organoids significantly restricts the accomplishment of different experiments. In the case of organoids, the apical part of the epithelium faces the inside of the organoids. This hinders the investigation of the effect of bacteria and food components on organoids and the evaluation of transport processes [42; 62]. In contrast with the _in vivo_ organization, in this case, the regions that are similar to the villus do not form protrusions, while crypts extend outwards (Figure 5). In the case of normal gastrointestinal organization, the apical part of the epithelium is oriented in the direction of the lumen, where it comes into contact with the digesta and microbiota. It was proven that these \"mini-guts\" reproduce intestinal absorption effectively [17]. Due to the presence of goblet cells, a small amount of mucus is also secreted to the lumen together with the cell debris [8; 27; 28]. _In vivo_, the substances secreted by goblet, enteroendocrine, and Paneth cells, and the dead cells are removed by peristaltic motility and the flow of the digesta, while in the case of organoids, these accumulate in the central area [42]. The imitation of the _in vivo_ physical forces that affect the intestinal epithelium (shear stress generated by fluid flow and distortion caused by peristaltic movements) is impossible because the lumen of organoids is inaccessible [17; 28]. Figure 5: The structure of organoids compared to the intestinal epithelium [64]. The investigated food components and bacteria can be introduced to the luminal side of organoids by microinjection but this is a technically challenging and time-consuming task, which may cause damage to the organoid structure [17; 47]. Another approach to make the apical side more accessible is to reverse the epithelial polarity of organoids [41; 64].