# Upgrading _In Vitro_ Digestion Protocols with Absorption Models
## 3 Conclusions  
A proper absorption model should be chosen according to the precision and feasibility that is necessary for obtaining relevant results [65]. The control and interpretation of  
\begin{table}
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**Method** & **Advantage** & **Disadvantage** \\ \hline Centrifugation & & \\ \hline Filtration & Easy to use; cheap; reproducible; high-throughput screening. & It simplifies the _in vivo_ conditions too much; active transport is not simulated \\ \hline Biomimetic membranes & Easy to use; cheap; reproducible; high-throughput screening [22] & Only passive transcellular transport is measured [22] \\ \hline Brush border membrane & & It contains only one side of the intestinal epithelial cell (apical or basolateral part); the isolation of completely pure vesicles is not possible; transporters, enzymes associated with the membrane can be damaged during isolation; small sample volume for analytical analysis [22] \\ \hline Primary cells & Phenotype similar to _in vivo_ conditions & Laborious; low reproducibility \\ \hline Cell lines & Reproducible & Usually, they have the phenotype of cancerous cells, which is different than the _in vivo_ status; In cases of monocultures (e.g., Caco-2 cells), it may lack the mucus layer that influences absorption \\ \hline Organoids & Imitates the 3D organization of intestinal epithelial cells & Laborious; the apical side is not accessible (with the exception of fragmented organoids and organoids with reversed polarity) \\ \hline Transwell systems\({}^{\text{\textcircled{\textcircled{\textcircled{\textcircled{\textcircled{\textcircled{\text  
simpler systems (non-cell-based transport-, 2D, _ex vivo_ models) is easier but they may lack details that can be essential in the absorptive process. Organoids sustain the self-renewing capacity of stem cells, maintain the cell-type diversity of the tissue, and imitate the structure and function of tissues [62]. However, experiments with 3D organoids are hindered by the inaccessibility of the inner side of the organoid. According to our knowledge, absorption experiments with digested food on gut-on-a-chip models are rare. Still, studies about drug absorption [12; 22] already recommend the utilization of gut-on-a-chip models. The application of fragmented organoids in gut-on-a-chips would make possible a more precise estimation of the absorption of food components from digested food. This review describes the most relevant methods that can be applied for the simulation of the absorption of food in the intestinal tract. Until now, there are no standardized methods to imitate intestinal absorption of food components. It was proven that the standardized COST Infogest protocol is an effective and cheap method to evaluate food digestion in the upper gastrointestinal tract. As it lacks brush border enzymes and the intestinal tissue that regulates absorption, it should be complemented with a standardized protocol for the imitation of the intestinal barrier. It is possible that it is unachievable to apply a single model for each research question and several standardized protocols have to be developed. For example, several foodstuffs can be metabolized by the intestinal microbiota. In these cases, more complex models are needed that enable the cultivation of anaerobic intestinal microbiota in the presence of aerobic intestinal cells. This review not only could be a starting point in the standardization of absorption protocols, it also can promote the reduction of _in vivo_ experiments necessary for the evaluation of foodstuffs. We presented numerous _in vitro_ methods that do not need animal tissue at all for the investigation of absorption. _Ex vivo_ models need animal sacrifice but in numerous cases, the tissue of one animal can be used for multiple experiments. Choosing the proper _in vitro_ or _ex vivo_ absorption model allows reproducible transport, nutrition, and toxicity studies with the replacement or reduction of animal experiments. Conceptualization, O.A.