Speaker
Description
INTRODUCTION
The role of gut microbiota in neurodegeneration is becoming a very interesting topic nowadays, and innovative in vitro and in vivo tools are becoming increasingly a need to help in dissecting the biochemical pathways involved in microbiota-brain interaction. An innovative engineered multiorgan-on-a-chip platform able not only to mimic microbiota-gut-brain connection but also to reproduce in vitro biological and mechanical stimuli to represent the so-called microbiota-gut-brain axis (MGBA) is the aim of our ERC project “MINERVA”. MINERVA platform is based on a 3D printed device compatible with commercially available tissue culture inserts and characterized by optical accessibility and capability to be connected to other devices. In the present work, we present our preliminary results related to the development of the gut epithelium modelling unit of our MGA engineered platform.
METHODOLOGY
Computational fluid dynamic simulations were performed with the software COMSOL Multiphysics® testing different flow rate values to obtain suitable oxygenation and shear stress values.
Biological validation was performed using human Caco2 cells seeded on collagen-coated inserts. After 7 days in static conditions, we assembled the seeded inserts into MINERVA devices under perfusion for other 7 days. As control we used seeded inserts maintained in static culture for 14 days. Cellular viability was tested by MTS test, FITC-dextran permeability and Trans-epithelial electrical resistance (TEER) evaluation. To assess cell layer morphology and maturation, immunofluorescence tests were performed with anti-mucin, anti-occludin, FITC-phalloidin and Hoechst dyes.
RESULTS AND DISCUSSION
From computational simulations we set flow rate at 30µL/min to guarantee oxygen supply and suitable shear stress value.
While MTS test showed no difference in terms of viability between static and dynamic condition, TEER values of static samples showed significant differences in line with apparent permeability (Papp) value, that resulted enhanced in dynamic condition.
Occludin and mucin reactivity are comparable between static and dynamic samples and their expression changes along the villus vertical axis. Morphological analysis confirmed mature cell layer formation in both static and dynamic samples with significantly higher villi in dynamic condition.
CONCLUSION
The selected flow rate set to mimic optimal physiological condition, maintained cell viability, promoted villi height and induced TEER value comparable to those of in vivo human intestinal epithelium [1]. Lower TEER values and higher permeability in dynamic condition are coherent with immunofluorescent data that confirmed mature cell layer formation characterized by lower cellular differentiation near to the villus base, likely required to guarantee villi turnover [2].
Overall our results confirm MINERVA device suitability for gut epithelium modelling in MINERVA MGA multi-organ platform.
ACKNOWLEDGMENT
MINERVA project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement N° 724734).
REFERENCES
[1] Srinivasan B, et al. (2015) J Lab Autom. 2015;20(2):107-126.
[2] Gommers, L. et al. (2019). Acta biomaterialia, 99, 110–120.
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