Human gut epithelium features recapitulated in MINERVA 2.0 millifluidic organ-on-a-chip device.

We developed an innovative millifluidic organ-on-a-chip device, named MINERVA 2.0, that is optically accessible and suitable to serial connection. In the present work, we evaluated MINERVA 2.0 as millifluidic gut epithelium-on-a-chip by using computational modeling and biological assessment. We also tested MINERVA 2.0 in a serially connected configuration prodromal to address the complexity of multiorgan interaction. Once cultured under perfusion in our device, human gut immortalized Caco-2 epithelial cells were able to survive at least up to 7 days and form a three-dimensional layer with detectable tight junctions (occludin and zonulin-1 positive). Functional layer development was supported by measurable trans-epithelial resistance and FITC-dextran permeability regulation, together with mucin-2 expression. The dynamic culturing led to a specific transcriptomic profile, assessed by RNASeq, with a total of 524 dysregulated transcripts (191 upregulated and 333 downregulated) between static and dynamic condition. Overall, the collected results suggest that our gut-on-a-chip millifluidic model displays key gut epithelium features and, thanks to its modular design, may be the basis to build a customizable multiorgan-on-a-chip platform.

Development of an Induced Pluripotent Stem Cell-Based Liver-on-a-Chip Assessed with an Alzheimer's Disease Drug.

Liver-related drug metabolism is a key aspect of pharmacokinetics and possible toxicity. From this perspective, the availability of advanced in vitro models for drug testing is still an open need, also to the end of reducing the burden of in vivo experiments. In this scenario, organ-on-a-chip is gaining attention as it couples a state-of-the art in vitro approach to the recapitulation of key in vivo physiological features such as fluidodynamics and a tri-dimensional cytoarchitecture. We implemented a novel liver-on-a-chip (LoC) device based on an innovative dynamic device (MINERVA 2.0) where functional hepatocytes (iHep) have been encapsulated into a 3D hydrogel matrix interfaced through a porous membrane with endothelial cells (iEndo)]. Both lines were derived from human-induced pluripotent stem cells (iPSCs), and the LoC was functionally assessed with donepezil, a drug approved for Alzheimer's disease therapy. The presence of iEndo and a 3D microenvironment enhanced the expression of liver-specific physiologic functions as in iHep, after 7 day perfusion, we noticed an increase of albumin, urea production, and cytochrome CYP3A4 expression compared to the iHep static culture. In particular, for donepezil kinetics, a computational fluid dynamic study conducted to assess the amount of donepezil diffused into the LoC indicated that the molecule should be able to pass through the iEndo and reach the target iHep construct. Then, we performed experiments of donepezil kinetics that confirmed the numerical simulations. Overall, our iPSC-based LoC reproduced the in vivo physiological microenvironment of the liver and was suitable for potential hepatotoxic screening studies.

The microbiota-gut-brain axis and epilepsy from a multidisciplinary perspective: Clinical evidence and technological solutions for improvement of in vitro preclinical models.

Epilepsy is a common neurological disease characterized by the enduring predisposition of the brain to generate seizures. Among the recognized causes, a role played by the gut microbiota in epilepsy has been hypothesized and supported by new investigative approaches. To dissect the microbiota-gut-brain (MGB) axis involvement in epilepsy, in vitro modeling approaches arouse interest among researchers in the field. This review summarizes, first of all, the evidence of a role of the MGB axis in epilepsy by providing an overview of the recent clinical and preclinical studies and showing how dietary modification, microbiome supplementations, and hence, microbiota alterations may have an impact on seizures. Subsequently, the currently available strategies to study epilepsy on animal and in vitro models are described, focusing attention on these latter and the technological challenges for integration with already existing MGB axis models. Finally, the implementation of existing epilepsy in vitro systems is discussed, offering a complete overview of the available technological tools which may improve reliability and clinical translation of the results towards the development of innovative therapeutic approaches, taking advantage of complementary technologies.

Technological tools and strategies for culturing human gut microbiota in engineered in vitro models.

The gut microbiota directly impacts the pathophysiology of different human body districts. Consequently, microbiota investigation is an hot topic of research and its in vitro culture has gained extreme interest in different fields. However, the high sensitivity of microbiota to external stimuli, such as sampling procedure, and the physicochemical complexity of the gut environment make its in vitro culture a challenging task. New engineered microfluidic gut-on-a-chip devices have the potential to model some important features of the intestinal structure, but they are usually unable to sustain culture of microbiota over an extended period of time. The integration of gut-on-a-chip devices with bioreactors for continuous bacterial culture would lead to fast advances in the study of microbiota-host crosstalk. In this review, we summarize the main technologies for the continuous culture of microbiota as upstream systems to be coupled with microfluidic devices to study bacteria-host cells communication. The engineering of integrated microfluidic platforms, capable of sustaining both anaerobic and aerobic cultures, would be the starting point to unveil complex biological phenomena proper of the microbiota-host crosstalks, paving to way to multiple research and technological applications.

Microbiota-Host Immunity Communication in Neurodegenerative Disorders: Bioengineering Challenges for In Vitro Modeling.

Human microbiota communicates with its host by secreting signaling metabolites, enzymes, or structural components. Its homeostasis strongly influences the modulation of human tissue barriers and immune system. Dysbiosis-induced peripheral immunity response can propagate bacterial and pro-inflammatory signals to the whole body, including the brain. This immune-mediated communication may contribute to several neurodegenerative disorders, as Alzheimer's disease. In fact, neurodegeneration is associated with dysbiosis and neuroinflammation. The interplay between the microbial communities and the brain is complex and bidirectional, and a great deal of interest is emerging to define the exact mechanisms. This review focuses on microbiota-immunity-central nervous system (CNS) communication and shows how gut and oral microbiota populations trigger immune cells, propagating inflammation from the periphery to the cerebral parenchyma, thus contributing to the onset and progression of neurodegeneration. Moreover, an overview of the technological challenges with in vitro modeling of the microbiota-immunity-CNS axis, offering interesting technological hints about the most advanced solutions and current technologies is provided.

Intracellular label-free detection of mesenchymal stem cell metabolism within a perivascular niche-on-a-chip.

The stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overall fate of stem cells within it. Mesenchymal stem cells (MSCs) in particular, experience influential microenvironmental conditions, which induce specific metabolic profiles that affect processes of cell differentiation and dysregulation of the immunomodulatory function. Reports focusing specifically on the metabolic status of MSCs under the effect of pathophysiological stimuli - in terms of flow velocities, shear stresses or oxygen tension - do not model heterogeneous gradients, highlighting the need for more advanced models reproducing the metabolic niche. Organ-on-a-chip technology offers the most advanced tools for stem cell niche modelling thus allowing for controlled dynamic culture conditions while profiling tuneable oxygen tension gradients. However, current systems for live cell detection of metabolic activity inside microfluidic devices require the integration of microsensors. The presence of such microsensors poses the potential to alter microfluidics and their resolution does not enable intracellular measurements but rather a global representation concerning cellular metabolism. Here, we present a metabolic toolbox coupling a miniaturised in vitro system for human-MSCs dynamic culture, which mimics microenvironmental conditions of the perivascular niche, with high-resolution imaging of cell metabolism. Using fluorescence lifetime imaging microscopy (FLIM) we monitor the spatial metabolic machinery and correlate it with experimentally validated intracellular oxygen concentration after designing the oxygen tension decay along the fluidic chamber by in silico models prediction. Our platform allows the metabolic regulation of MSCs, mimicking the physiological niche in space and time, and its real-time monitoring representing a functional tool for modelling perivascular niches, relevant diseases and metabolic-related uptake of pharmaceuticals.