# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 1 Introduction
### Challenges Regarding the Design of Intestinal Epithelium Models Used for Absorption Studies  
It is probably impossible to imitate all aspects of the small and large intestinal digestion phase in a manner that it reproduces the _in vivo_ state perfectly [24]. A major obstacle is the precise imitation of the intestinal epithelium. For example, it is difficult to reproduce the complex histological organization, the presence of the microbiota, and the effect gut motility in a single model [24].  
The gut bacteria participate in the differentiation of stem cells to enterocytes, the motility of the gastrointestinal tract, the metabolism of foodstuffs and drugs, the degradation of dietary toxins and carcinogens, the synthesis of essential vitamins (K, B12, folic acid, B1, B6) and short chain fatty acids, and bile acid circulation [25; 26]. They also synthesize enzymes, which can degrade dietary polysaccharides to simpler carbohydrates [26].  
The presence of stem cell niche, the simultaneous cell differentiation, migration, and the shedding of dead cells are prerequisites of tissue homeostasis in the intestine [27], which is also difficult to adjust for _in vitro_ analysis.  
Moreover, the gut motility should also be simulated but this also causes difficulties. The gut motility influences the mixture and propagation of gut content [28]. The distortion that can be noticed during peristaltic expansion and contraction, and the flow/shear stress caused by digesta movement influences the epithelium, by inducing the division and differentiation of epithelial cells [29]. Peristaltic movement also inhibits the overgrowth of bacteria _in vivo_[30]. According to [PERSON] et al. [31], it is difficult, if not impossible, to reproduce _in vitro_ the peristaltic movements of the gastrointestinal tract. The contractions of the small intestine are highly influenced by the time that has passed since the last meal, while large intestine motility is determined by circadian rhythm [8].  
During the investigation of the absorption of food components, several different approaches can be applied. In the case of some high-throughput experiments, non-cell-based transport models or brush border membrane vesicles (Section 2.2) can be used [1; 32].  
Still, the complex processes that determine the absorption rate of food components through the intestinal barrier [22], can only be precisely simulated with models that include mammalian cells. For example, the active transport of D-glucose, in the case of dialysis, is only reproduced as passive diffusion, which is only determined by the concentration gradient [33]. Due to the complexity of the intestinal tissue, numerous approaches (the application of different cell lines or stem cells, various cell culture techniques) were developed to imitate the epithelium. The simpler methods mainly focus on the establishment of proper epithelial barrier integrity and the development of a mucus layer on the surface of cells. The imitation of the physiological density of mucus is also important, since it may affect absorption [8]. Usually, the digesta cannot be added directly to the surface of mammalian cell cultures. It can diminish the viability of cells, due to the osmotic shock it may induce, or due to the presence of bile acids, digestive enzymes, and enzyme inhibitors [3].