# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 1 Introduction
### Description of Intestinal Epithelium  
The gut epithelium is made of numerous cell types (enterocytes/colonocytes, goblet cells, stem cells, enteroendocrine cells, Tuff cells, M cells, and Paneth cells) (Figure 1) [10]. While enterocytes are present everywhere, goblet cells are on the villi, and Paneth cells can be found in the crypts [11]. Junctional complexes (intercellular attachment structures) connect the epithelial cells that form the intestinal barrier. They limit the passive movement of food components across  
Figure 1: The small intestinal epithelium cells and their localization [6]. the epithelium. Out of these, the tight junctions regulate paracellular passive diffusion of ions and small molecules, and exclude large molecules [6; 12]. Enterocytes, with long, densely packed microvilli, are responsible for absorption of nutrients, like carbohydrates, peptides, amino acids, and lipids [6]. They also absorb ions and water, release antimicrobial proteins like \(\beta\)-defensins and cathelicidins, and express brush border enzymes [6]. Goblet cells secrete mucin fibers and antimicrobial peptides [6]. At the base of the crypts, quickly renewing epithelial stem cells can be found [8]. Paneth cells have a role in maintaining the proper stem cell niche, by presenting ligands and secreting growth factors, such as Wnt ligands [13; 14]. They also secrete antimicrobial peptides, which are synthesized by ribosomes [14; 15]. Enterroendocrine cells regulate appetite and gut motility by secretion of numerous hormones [8]. Tutf cells are chemosensory epithelial cells that secrete effector molecules with immunodulatory functions [16]. They have a role in the protection against protozoa and helminths [8]. M cells (\"microfold cells\") present the antigens from the lumen to the immune cells [8]. The proportion of M cells in the gastrointestinal tract is low (<1%). They can only be found in the small intestine, above the Peyer's patches [8]. All differentiated cells of the epithelium originate from stem cells [8]. The progenitors of stem cells migrate upwards from the crypts [17]. The migration and differentiation of cells on the crypt/villus axis are maintained by biochemical (e.g., growth factors) and biophysical gradients (e.g., extracellular matrix properties) [8; 18]. As digestive enzymes hydrolyze foodstuffs in the intestinal lumen, bioaccessible molecules are released and are transferred into the bloodstream by passive or active transport through the intestinal epithelium [19]. Passive transport is manifested in transcellular or paracellular diffusion, while active transport can be realized by influx/efflux transporter-facilitated transport, carrier-mediated transport, endocytosis, and transcytosis (Figure 2) [12]. All cell types are capable of these absorption processes but enterocytes express a selection of genes that maximize the absorption of food components [20]. The small intestine is covered by one mucus layer. The mucus is a hydrated, complex heterogeneous mixture of mucin fibers, lipids, proteins, carbohydrates, cell debris and bacteria [22]. The mucus layer covers and protects the surface of the intestine (e.g., from digestive enzymes, bacteria) [8]. As it influences the solubility on the surface of the cells, it  
Figure 2: Transport processes in the intestinal epithelium [21]. has a significant impact on the absorption of different compounds [12; 23]. In the mucus, the main type of mucin present is mucin 2 (Muc2) [8]. The large intestine differs from the small intestine in several aspects. It also contains crypts but no villi are present. The colonocytes of the large intestine are similar to enterocytes but they do not express brush border enzymes and have short, irregular microvilli [6]. They absorb water, electrolytes, and bacterial metabolites [6]. The large intestine has two mucus layers: a sterile, inner, strongly adherent layer and an outer, looser one, which forms a specific environmental condition for the growth of bacteria [8; 10].