Speaker
Description
Introduction: The assessment of bioaccessibility and bioavailability of orally-ingested compounds is key to determine their efficacy and safety. However, there is a lack of toxicological studies that combine simulated gastrointestinal (GI) digestion with subsequent intestinal absorption. Many studies expose cells directly to pristine bioactive substances, failing to consider the series of biochemical transformations that occur throughout the GI tract. Intestinal cell lines have been widely explored to build different gut-epithelial models. However, one-fits-all approaches fall short of recreating physiologically-relevant responses. Patient-derived stem-like cells are emerging as sophisticated and refined models that ultimately surpass the potential of immortalised cells.1 Organ-on-chip offers a disruptive technology to predict in vitro toxicology, presenting a reliable approach with significant advantages when compared to in vivo and cell-based models. Here we describe a two-module microfluidic device comprising the complete GI tract, where digestive and absorptive functions are combined.
Methodology: Microfluidic digestive devices were produced from rapid prototyping (CNC micromachining) of acrylic sheets, while the cell-based module was fabricated from polydimethylsiloxane using a replica moulding technique. On-chip digestion was validated using a fluorescently-labelled casein derivative and compared to the static digestion INFOGEST protocol.2 The Gut-Chip consisted of two channels separated by an in-house fabricated membrane, coated with collagen/Matrigel. Caco-2 and HT29-MTX cells were seeded on-chip at a 9:1 ratio and cultured under continuous flow (120 µL∙h-1) for 7 days. Cell morphology and the epithelial barrier formation were assessed by immunocytochemistry of occludin tight junctions and by measuring the paracellular transport of Lucifer Yellow. Intestinal crypts were isolated from human colon samples of patients undergoing tumour resection surgeries, with full patient consent and approval by Ethics Committee.3 Lgr5+ cells will be expanded as organoids to be used in human primary cell organ-on-chips.
Results: Automated on-chip digestion was in agreement with the current gold standard protocol and was able to replicate typical Michaelis-Menten kinetics. Critically, our device offers enhanced time-resolution over static methods in both gastric and intestinal digestion phases, with on-line pH and temperature sensing and actuation. On-chip Caco-2/HT29-MTX co-cultures displayed 3D villi-like structures after 7 days in culture, a significantly more relevant architecture than the one obtained with Transwell inserts. Furthermore, the on-chip intestinal barrier showed a higher permeability (3.4x10-6 cm/s) when compared to the insert culture (3.0x10-7 cm/s), closely resembling the ex vivo (4.0x10-6 cm/s). Intestinal crypts were successfully isolated from patient samples and their integration on-chip is the subject of our current goals aiming translational applications.
Conclusion: The modular microfluidic device described here shows great promise to be used as a robust tool for pharmacokinetics studies of orally-ingested compounds. The use of patient-derived organoid cultures will allow the execution of personalised studies with significant implications in both food and health applications.
Acknowledgements: We thank Joana Reis (2CA-Braga – Centro Clínico Académico de Braga) for her help with project management regarding clinical samples.
References:
1. Dutton, J. S. et al, Trends Biotechnol. 37, 744-760 (2018)
2. Brodkorb, A. et al, Nat Protoc. 14, 991-1014 (2019)
3. Sato, T. et al, Nature. 459, 262-265 (2011)
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