# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 2 Technological Approaches of Intestinal Epithelium Models
### Cell Cultures Used in Epithelium Models
#### 2.3.5 Microfluidic Systems That May Be Used in Absorption Studies  
Microfluidic systems (also called organ-on-a-chip models) are micrometer-sized chambers, where cells can be cultured in continuous contact with flowing culture media [14]. In gut-on-a-chips, the differentiation of epithelial cells is influenced by the reproduction of flow and peristaltic motility. The intestinal epithelium may be more robust when it is cultured under continuous flow [66]. The configuration of the microfluidic systems that reproduce the gut may be determined by the research goal, as the consideration of different factors is necessary in the case of absorption- or host-microbiota interaction studies. [PERSON] et al. [67] built a microfluidic system for the evaluation of absorption. They used a microchannel separated by a membrane on which Caco-2 cells were maintained. The medium was pumped to the lower and upper compartments at a flow rate of 1 uL/min. The validity of the system was proven by tests with cyclophosphamide (CP, a reagent used for chemotherapy) and Lucifer yellow (LY, a fluorescent dye). In control experiments without Caco-2 cells, both CP and LY could be transferred through the membrane. When Caco-2 cells formed a confluent layer, only CP could permeate from the upper compartment to the lower one. These results are in accordance with the _in vivo_ results, as CP can be absorbed _in vivo_ and the intestinal wall is impermeable for LY. Gut-on-a-chips usually contain two microchannels: the upper microchannel represents the lumen, while the lower one, where endothelial cells are cultured, simulates blood vessels (Figure 6) [58]. In the case of more complex microfluidic systems, more microchannels are applied, separated from each other by a porous semipermeable membrane [14]. Epithelial cell lines or fragmented organoids can be cultured on the membrane. Figure 6: The best-known method to simulate peristaltic motility in a gut-on-a-chip [42]. The membrane and the chambers can be covered with natural polymers, which can support the adhesion, migration, or specific function of the cells [23]. The development of intestinal villi in some microfluidic systems (e.g., [24]) may be facilitated by the presence of scaffolds (e.g., from collagen) [23]. Another approach is based on the fact that the Caco-2 cells and the organoids can form villi-like structures spontaneously due to mechanical signals [57]. On the side and the top of these villi-like structures, distinct types of differentiated epithelial cells (absorptive, goblet, enteroendocrine, Paneth cells) are present, while the proliferative cells are at the bottom of the crypts [57,68]. The integrity of the epithelial barrier, the tight junctions, and the mucus production are improved in the case of microfluidic systems, in comparison to static Caco-2 Transwell cultures [57]. The microfluidic systems can reproduce the chemical, hydrodynamic, and mechanical gradients that are characteristic of the investigated organ [69]. The flow can be established by different methods, like syringes, pumps, or gravity [43]. It removes dead cells and enhances access to nutrients [43]. It also induces the formation of villi-like structures and microvilli and mucin secretion [69]. Flow shear stress also induces a better reproduction of the _in vivo_ circumstances [9]. So, according to [PERSON] et al. [69], a proper model of the human intestine cannot be realized without reproduction of the fluid flow. In several cases, a pneumatic cyclic vacuum suction is applied in the chambers that surround the microchannels, which causes pulsatile flow and periodically stretches the membrane (Figure 6) [17]. The manipulation of the membrane induces cell distortion, which simulates the effect of peristalsis [8,47]. The high-throughput screening with these chips is hindered by the complexity of the system that is needed for its operation [5]. To imitate peristaltic motion, peristaltic liquid pumps can also be used instead of gas-actuated channels [69].