# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 2 Technological Approaches of Intestinal Epithelium Models
### Ex Vivo Models  
_Ex vivo_ refers to experiments conducted in or on tissue from an organism in an external environment with minimal alteration of natural conditions. _Ex vivo_ models replicate the structure and physiology of the intestine more precisely since they apply tissues excised from animals or humans but unfortunately, the viability of these samples is very short (usually less than \(1\) day, in some cases, less than an hour) [22; 28]. The interaction of different cell types can be better evaluated with _ex vivo_ models than with microfluidic systems. In the case of microfluidic systems, besides epithelial cells, endothelial and/or immune cells [77] can be integrated in the chips but replicating the exact histological organization is difficult, if not impossible. In cases of tissues maintained _ex vivo_, the endothelial, immune cells, and neurons are already present in the right proportion. The _ex vivo_ absorption studies often use tissues of animal origin (e.g., rat, pig) to estimate the transport processes of the human intestine [22]. The human intestine tissue removed during surgeries provides a better representation of the human physiology. However, the inter-individual variability impedes the collection of systematic data regarding absorption in the intestine, while the utilization of animal tissues can be standardized [22]. It is hard to draw relevant, unequivocal conclusions regarding the human physiological processes from results that are obtained from different mammalian tissues. These experiments can be contradictory [41]. The application of _ex vivo_ models is also restricted by the limited accessibility of human tissue [47]. The application of _ex vivo_ models is quite easy and cheap [22]. The functioning of the Franz cell and the Using chamber (Figure 8) is based on the same principle: the intestinal tissue is mounted between two compartments, separating apical and basolateral sides [22; 47]. In the case of the Using chamber, the orientation of the intestinal tissue is vertical, while in the case of the Franz cells it is horizontal [22]. The Using chamber measures voltage, transepithelial resistance, and short-circuit current with electrodes [10; 22]. \"Short-circuit current reflects electrogenic ionic flux across the epithelium\" [22]. This allows the continuous surveillance of the integrity and viability of the investigated tissue/cell layer [22]. [PERSON] et al. [76] used the Using chamber  
Figure 8: Illustration of the Using chamber [47]. with mouse intestinal tissue and Caco-2 cell cultures to evaluate the absorption of dietary carotenoids. They concluded that the utilization of the Ussing chamber model with mouse intestinal tissue after _in vitro_ digestion with Infogest protocol can be an effective method to determine carotenoid bioavailability. In the case of Franz cells, the contents of the compartment that is in contact with the basal side of the epithelium (the compartment that is opposite to the chamber where the food/digesta is added) is continuously stirred, which may cause enhanced permeability compared to the Ussing chamber [22]. [PERSON] et al. [78] recently developed the gastrointestinal tissue robotic interface system (GI-TRIS), which enables long-term maintenance of the tissue [22]. In this case, the porcine intestinal tissue is incubated in a 96-well plate, enabling high-throughput screening, i.e., more samples and/or more parallel experiments can be performed [22; 78]. The GI-TRIS was developed in two different set-ups. The first design can be combined with commercially available multiwell plates, while the other one can be used for direct measurements in a spectrophotometer without disassembly and is also compatible with a robotic system [78]. In contrast to Caco-2 Transwell transport assays, where the digesta is toxic to cells, native intestinal fluid can be applied in GI-TRIS [78]. The InTESTine(r) system from TNO is also appropriate for high-throughput screening as it contains 24 or 96 chambers.