# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 2 Technological Approaches of Intestinal Epithelium Models
### Cell Cultures Used in Epithelium Models  
The main categories of cell cultures that can be used in intestinal epithelium protocols are primary cells, cell lines, and stem cells [5]. Primary cells are directly obtained from the tissue of origin and they cannot be sub-cultured indefinitely. In contrast, cell lines that are immortalized cells can be cultured for long periods. The immortalized cells are either of cancerous origin or have been genetically modified to proliferate indefinitely [40]. The biochemical and physiological characteristics of primary cell cultures are closer to the conditions that can be noticed _in vivo_ since these cells do not incorporate genetic modifications [23,41]. The acquirement of these cells is more problematic than the maintenance of the immortalized cell lines [6]. Another disadvantage is that exogenous growth factors are necessary to maintain these cell cultures [23,42]. Besides this, it is still a challenge to develop a reproducible protocol for the long-term sustainment of primary cell culturesthat originate from the intestinal tissue, as primary cells, _in vitro_, are susceptible to apoptosis [5; 29]. Only a few studies describe treatment of primary cells with digested foods [7]. The main disadvantage of using cancerous cell lines is that their phenotype is different from the epithelial cells of the small or large intestine. Diverse culture conditions may also cause experimental variability [43]. This complicates the extrapolation of the results regarding the _in vivo_ situation [27]. The traditional and most used _in vitro_ model of the gastrointestinal microenvironment is the culture of Caco-2 and HT-29 cell lines in Transwell systems [28; 44; 45]. The Caco-2 cells are human colorectal adenocarcinoma cells, which differentiate into polarized epithelial cells, and are organized spontaneously in crypt and villi-like structures [23; 46; 47]. Besides its cancerous origin, this cell line has limitations. It is hard to control its differentiation, its transporters, and the diameter of its tight junctions are different than in the case of the intestinal epithelium [47]. The intestinal epithelium is covered with a mucus layer, which has not only a protective role but also affects the absorption rate and host-microbiota interaction. An important deficiency of Caco-2 cell cultures is that they do not secrete mucin glycoproteins that form the mucus layer [48]. In opposition, if Caco-2 cells are cultured under shear stress, in a gut-on-a-chip model (Section 2.3.5), the presence of MUC2 could be detected. The HT-29 culture also originates from human colorectal adenocarcinoma cells but in contrast to Caco-2 cells, it also contains mucin-secreting goblet cells [10]. If Caco-2 and HT29-MTX (HT-29 cells stimulated with methotrexate) are mixed, co-cultures secrete the gastric mucin called (MUC)5 AC instead of the typical intestinal MUC2 [6; 49]. Furthermore, the HT29-MTX cells divide significantly faster than Caco-2 cells [10; 30]. So, the absorptive: goblet cells ratio changes with the aging of the culture, in a manner that will not correspond to the _in vivo_ state [10; 30]. TC7, NCM460, T84, SK-CO15, and HCT-8 are other human cancerous cell lines that are often used as epithelium models [6; 7; 22; 50]. The latter four can be used as models of the large intestine epithelium [6; 7]. Models based on tumor cells do not incorporate all intestinal epithelial cell types. The expression of receptors, transporters, and enzymes is not the same as in case of _in vivo_ conditions [8]. Noncancerous cell lines of animal origin like, MDCK (Madin-Darby canine kidney), IEC-18 (established from rat small intestine), and IPEC-J2 (isolated from piglet small intestine) can be also utilized as _in vitro_ absorption models [22; 51; 52]. The phenotype of these is more similar to primary cells [9]. Out of the animal-derived cell lines, the IPEC-J2 culture is most similar to the human epithelium [1; 22]. Another approach to creating intestinal epithelium models is the utilization of stem cells.