# 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
When fluid flow and cyclic mechanical force was applied together, the epithelial permeability measurements correlated better with _in vivo_ data, and mucin production was similar to _in vivo_ conditions [69]. It also optimized the formation of villi-like structures [69]. Gut-on-a-chip models usually simulate flow in one direction, while _in vivo_, besides peristalsis, segmentation is also present, which also moves flow in the opposite direction [58,61]. Microfluidic systems are more precise than organoids and 2D models due to the establishment of proper hydrodynamics, mechanical stimulation, and chemical (e.g., oxygen) gradients [5,69]. The oxygen gradient (hypoxic in the lumen, normoxic at the surface of the cells) is also important when integration of microbiota is necessary because of the research topic. The alterations in the simulated intestine can be continuously monitored in the case of many organ-on-a-chip models. These microfluidic systems can be made of transparent materials, and/or may contain sensors or real-time analytical instruments [23]. Through integrated sensors, for example, barrier integrity or oxygen concentration can be continuously monitored [70]. While in the case of 3D organoids, the inner side is not accessible for verification of barrier integrity, the culture of fragmented organoids on gut-on-a-chip models enables the evaluation of intestinal barrier effectiveness [42]. Induced pluripotent stem cell-derived organoids and also organoids derived from human biopsies have been cultured in gut-on-a-chip models [43]. The small scale of microfluidic systems entails several disadvantages [29]. For example, the utilization of gut-on-a-chip models requires experience and is also laborious and costly [12,41]. Several different approaches are applied to simulate and evaluate host-microbiota interaction. The \"HuMix\" device by [PERSON] et al. [71] and the \"HMI\" module by [PERSON] et al. [72] separate the microbiota from the Caco-2 cells by a membrane and an artificial mucus layer [65]. The so-called \"Intestine Chip\" [73] and the \"Anoxic-Oxic Interface-on-a-Chip\" [74] simulate the _in vivo_ circumstances more precisely because the mucus layer that separates the intestinal cells from the microbiota is secreted by the intestinal cells. The mucus acts as a lubricating barrier that protects the intestinal cells from the harmful components in the lumen [17]. It also influences absorption [11], so it can be a decisive part of an intestinal _in vitro_ absorption model. The \"Intestine Chip\" is presented in Figure 7, which is based on [65]. The sizes of the elements in Figure 7 are not proportional. [PERSON] et al. [73] designed the \"Intestine Chip\" both with Caco-2 cells and with intestinal epithelial cells isolated from organoids. In the case of Caco-2 cell cultures, oxygen sensors were also included in the system. When culturing Caco-2 cells, immortalized endothelial cells were also cultured in the bottom channel. [PERSON] et al. [74] cultured Caco-2 cells in the \"Anoxic-Oxic Interface-on-a-Chip\". The main difference between the \"Intestine Chip\" and the \"Anoxic-Oxic Interface-on-a-Chip\" is the strategy applied for oxygen modulation [65]. In both cases, aerobic cell medium is perfused through the lower channel. In the case of the \"Intestine Chip\", the chip is placed in an anaerobic box, while in the top channel of \"Anoxic-Oxic Interface-on-a-Chip\" anoxic medium (obtained by mixing the medium with 6.5 mM sodium sulfide) is perfused [65]. In the case of the \"Intestine Chip\", endothelial cells can also be included in the bottom channel. As the gut bacteria may produce metabolites from some food components (e.g., polyphenols), it also may be an advantage that these gut-on-a-chip models (Figures 6 and 7) also include the microbiota. Microbiota and their metabolites affect each cell type differently [64]. [PERSON] et al. [66] prepared a modular microfluidic-based platform to imitate the gastrointestinal tract.