# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 2 Technological Approaches of Intestinal Epithelium Models
### Cell Cultures Used in Epithelium Models
These can be the best choice, as they can be propagated in the long term, and they can differentiate into multiple cell types [5]. A disadvantage is that the acquirement and maintenance of stem cells is laborious [5]. The self-renewing 3D cultures originating from embryonic, induced pluripotent and multipotent stem cells are called organoids [47; 53]. The structure, metabolism, and phenotype of the organoids are similar to the characteristics of tissue of origin [27]. Enteroids are prepared from the small intestine, while colonoids from the colon [28]. Compared to 2D cell cultures, the arrangement of cell junctions and the extracellular matrix are more similar to the _in vivo_ situation, and more importantly, the expression of brush border proteins is better [9]. Another example of a 3D model is the Epi-IntestinalTM developed by MatTek Corporation [12]. Epi-IntestinalTM is a reconstructed 3D human gut tissue model derived from primary intestinal cells that can be very useful in the investigation of nutrient processing [12]. Unlike organoids, this organotypic microtissue has an accessible luminal surface [12]. To avoid microinjection, the organoids may be fragmented and cultured as a monolayer in 2D [28; 41]. These cultures also organize themselves in stem cell zones and differentiated cell areas, just as the original tissue [14]. When they are maintained on membrane insertsplates, the system is appropriate for studying nutrient absorption. However, in this case, only a thin mucus layer is formed [49]. According to [PERSON] et al. [23], cells originating from the intestinal organoids are also able to reproduce the physiological responses effectively. In contrast, according to [PERSON] et al. [54] and [PERSON] et al. [55], the generation of intestinal organoids from pluripotent stem cells may lead to the development of a tissue with fetal properties. In cases of nutrient absorption studies, the presence of endothelial cells can be important, as vasculature is fundamental in nutrient and waste transport [54]. The reproducibility of models that apply cell cultures can be diminished due to the utilization of animal serum during their maintenance [53]. It is very interesting that the digesta obtained by the Infogest protocol is toxic to Caco-2 cell monolayers [7]. The digestive enzymes may be toxic and may cause detachment of cell layers, and bile salts are not only toxic to cells but can also alter osmolality in a harmful manner [7]. Metabolites generated from _in vitro_ food digestion may also have deleterious effect on mammalian cells [7]. Although it was proven that the digesta imitates physiologically relevant osmolality and concentrations of bile acids and enzymes, a detoxification of digesta is necessary [7]. [PERSON] et al. [7] recommend different detoxification protocols, which should be chosen based on the purpose of the research. These will also alter the digesta, diminishing the precision of the imitation of the _in vivo_ digesta. Due to the above-mentioned setbacks, it is important to apply a negative control during the experiments, which contains all digestive juices in the digesta but the investigated food is not included [7].