Three-stage enzymatic digestive system for a gut-on-a-chip

In this work, we present the development of a three-stage microfluidic system as a cell-free model for digestion in the human gastrointestinal (GI) tract. Larger-scale digestion models are currently being used for pharmacological, toxicological and nutritional studies to determine the possible effects of enzymatic digestion on samples containing drugs, toxicants and nutrients. We describe a miniaturized, continuously flowing digestive system, which will be coupled to a gut-on-a-chip [1] containing living human intestinal epithelium. This will allow pharmacokinetic and toxicokinetic studies in vitro in a system that resembles the in vivo situation well. This miniaturized digestive system consists of three stages of identical chaotic micromixers (Figure 1A and B) [2,3], representing the mouth, stomach and small intestine. These micromixers each contain herringbone-shaped grooves in the channel surface, causing a perturbation of the flow patterns in the channel which greatly enhances mixing. Artificial digestive juices containing minerals and enzymes were prepared according to a modified literature procedure [4]. pH values of 6.8, 3.0 and 7.3 in the respective stages of digestion were achieved after optimization to mimic the in vivo situation and provide an optimal environment for enzyme function [5]. A sample solution of fluorescein (20 µM in water) was mixed with artificial saliva in the first stage (Figure 1C), after which the mixture flowed to the second micromixer. Here, mixing with gastric juice caused the pH to drop to 3.0. In the final stage, the liquid was further mixed with duodenal juice and bile, with recovery of the pH to a value of 7.3 to mimic intestinal conditions. This miniaturized digestive system will be used for both short-term and long-term cell exposure studies, allowing both single dose and continuous exposure of cells to the compounds of interest and their metabolites in a fully integrated gut-on-a-chip