Simone Perottoni

Biological barriers in our body are dedicated to maintaining systemic homeostasis by enabling selective transport, the dysregulation of which plays a pathogenic role in several diseases. Transendothelial/epithelial electrical resistance (TEER) analysis is conventionally used to assess the barrier permeability. Engineering TEER setups within advanced in vitro models has become essential for acquiring real-time information on barrier integrity inside organ-on-a-chip (OOC) devices. However, current approaches often involve multiple assembly steps, require external access to the culture chamber, rely on cumbersome systems, or provide highly localized measurements that do not represent the entire surface of the tissue under examination. Specifically, in Transwell-based OOC systems, continuous and nondisruptive monitoring is often precluded by the need to disassemble the system for TEER measurement. To address these limitations, we combined conventional technology for patterning thin conductive films with low-cost, benchtop 3D printing to integrate planar electrodes within a Transwell-based hybrid OOC system. Our integrated TEER electrode device, ITE-MINERVA, can be easily employed with commercial measurement instruments, representing a useful tool for real-time, in situ detection of barrier functionality, potentially altered by drug toxicity and absorption processes, on Transwell-based OOCs, both in single- and multiorgan configurations, noninvasively and without stopping the experiment. In this work, we present the design and fabrication strategy of our innovative setup and its assessment in recording real-time TEER modulation on a Caco-2 cell-based in vitro gut epithelial barrier model.

Current drug development pipelines are time-consuming and prone to a significant percentage of failure, partially due to the limited availability of advanced human preclinical models able to better replicate the in vivo complexity of our body.

A dermal perivascular microenvironment has been reproduced on a chip within the MOAB's chambers by combining 3D microscaffolds, and controlled fluid dynamics and mass transport conditions, predicted by computational modelling of in vivo angiogenesis.

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.

Epilepsy is a common neurological disease characterized by the enduring predisposition of the brain to generate seizures. Among the recognised 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 modelling 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.

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.

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.