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.

Induced pluripotent stem cell-based organ-on-a-chip as personalized drug screening tools: A focus on neurodegenerative disorders.

The Organ-on-a-Chip (OoC) technology shows great potential to revolutionize the drugs development pipeline by mimicking the physiological environment and functions of human organs. The translational value of OoC is further enhanced when combined with patient-specific induced pluripotent stem cells (iPSCs) to develop more realistic disease models, paving the way for the development of a new generation of patient-on-a-chip devices. iPSCs differentiation capacity leads to invaluable improvements in personalized medicine. Moreover, the connection of single-OoC into multi-OoC or body-on-a-chip allows to investigate drug pharmacodynamic and pharmacokinetics through the study of multi-organs cross-talks. The need of a breakthrough thanks to this technology is particularly relevant within the field of neurodegenerative diseases, where the number of patients is increasing and the successful rate in drug discovery is worryingly low. In this review we discuss current iPSC-based OoC as drug screening models and their implication in development of new therapies for neurodegenerative disorders.

Using integrated meta-omics to appreciate the role of the gut microbiota in epilepsy.

The way the human microbiota may modulate neurological pathologies is a fascinating matter of research. Epilepsy is a common neurological disorder, which has been largely investigated in correlation with microbiota health and function. However, the mechanisms that regulate this apparent connection are scarcely defined, and extensive effort has been conducted to understand the role of microbiota in preventing and reducing epileptic seizures. Intestinal bacteria seem to modulate the seizure frequency mainly by releasing neurotransmitters and inflammatory mediators. In order to elucidate the complex microbial contribution to epilepsy pathophysiology, integrated meta-omics could be pivotal. In fact, the combination of two or more meta-omics approaches allows a multifactorial study of microbial activity within the frame of disease or drug treatments. In this review, we provide information depicting and supporting the use of multi-omics to study the microbiota-epilepsy connection. We described different meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics), focusing on current technical challenges in stool collection procedures, sample extraction methods and data processing. We further discussed the current advantages and limitations of using the integrative approach of multi-omics in epilepsy investigations.

Microbiological-Chemical Sourced Chondroitin Sulfates Protect Neuroblastoma SH-SY5Y Cells against Oxidative Stress and Are Suitable for Hydrogel-Based Controlled Release.

Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer's disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H2O2-induced oxidative stress. After assessing that our CS (1-100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.

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.

3D brain tissue physiological model with co-cultured primary neurons and glial cells in hydrogels.

Recently, researchers have focused on the role of gut microbiota on human health and reported the existence of a bidirectional relationship between intestinal microbiota and the brain, referred to as microbiota-gut-brain axis (MGBA). In this context, the development of an organ-on-a-chip platform recapitulating the main players of the MGBA would help in the investigations of the biochemical mechanisms involved. In this work, we focused on the development of a new, hydrogel-based, 3D brain-like tissue model to be hosted in the brain compartment of the aforementioned platform. We previously cultured primary mouse microglial cells, cortical neurons and astrocytes independently, once embedded or covered by a millimeter layer of two selected collagen-based hydrogels. We evaluated cell metabolic activity up to 21 days, cell morphology, spatial distribution and synapse formation. Then, we exploited the best performing culturing condition and developed a more complex brain-like tissue model based on the co-culture of cortical neurons and glial cells in physiological conditions. The obtained results indicate that our 3D hydrogel-based brain tissue model is suitable to recapitulate in vitro the key biochemical parameters of brain tissue.

A miniaturized hydrogel-based in vitro model for dynamic culturing of human cells overexpressing beta-amyloid precursor protein.

Recent findings have highlighted an interconnection between intestinal microbiota and the brain, referred to as microbiota-gut-brain axis, and suggested that alterations in microbiota composition might affect brain functioning, also in Alzheimer's disease. To investigate microbiota-gut-brain axis biochemical pathways, in this work we developed an innovative device to be used as modular unit in an engineered multi-organ-on-a-chip platform recapitulating in vitro the main players of the microbiota-gut-brain axis, and an innovative three-dimensional model of brain cells based on collagen/hyaluronic acid or collagen/poly(ethylene glycol) semi-interpenetrating polymer networks and ß-amyloid precursor protein-Swedish mutant-expressing H4 cells, to simulate the pathological scenario of Alzheimer's disease. We set up the culturing conditions, assessed cell response, scaled down the three-dimensional models to be hosted in the organ-on-a-chip device, and cultured them both in static and in dynamic conditions. The results suggest that the device and three-dimensional models are exploitable for advanced engineered models representing brain features also in Alzheimer's disease scenario.

Human Gut-Microbiota Interaction in Neurodegenerative Disorders and Current Engineered Tools for Its Modeling.

The steady increase in life-expectancy of world population, coupled to many genetic and environmental factors (for instance, pre- and post-natal exposures to environmental neurotoxins), predispose to the onset of neurodegenerative diseases, whose prevalence is expected to increase dramatically in the next years. Recent studies have proposed links between the gut microbiota and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Human body is a complex structure where bacterial and human cells are almost equal in numbers, and most microbes are metabolically active in the gut, where they potentially influence other target organs, including the brain. The role of gut microbiota in the development and pathophysiology of the human brain is an area of growing interest for the scientific community. Several microbial-derived neurochemicals involved in the gut-microbiota-brain crosstalk seem implicated in the biological and physiological basis of neurodevelopment and neurodegeneration. Evidence supporting these connections has come from model systems, but there are still unsolved issues due to several limitations of available research tools. New technologies are recently born to help understanding the causative role of gut microbes in neurodegeneration. This review aims to make an overview of recent advances in the study of the microbiota-gut-brain axis in the field of neurodegenerative disorders by: (a) identifying specific microbial pathological signaling pathways; (b) characterizing new, advanced engineered tools to study the interactions between human cells and gut bacteria.

Advanced Organ-on-a-Chip Devices to Investigate Liver Multi-Organ Communication: Focus on Gut, Microbiota and Brain.

The liver is a key organ that can communicate with many other districts of the human body. In the last few decades, much interest has focused on the interaction between the liver and the gut microbiota, with their reciprocal influence on biosynthesis pathways and the integrity the intestinal epithelial barrier. Dysbiosis or liver disorders lead to0 epithelial barrier dysfunction, altering membrane permeability to toxins. Clinical and experimental evidence shows that the permeability hence the delivery of neurotoxins such as LPS, ammonia and salsolinol contribute to neurological disorders. These findings suggested multi-organ communication between the gut microbiota, the liver and the brain. With a view to in vitro modeling this liver-based multi-organ communication, we describe the latest advanced liver-on-a-chip devices and discuss the need for new organ-on-a-chip platforms for in vitro modeling the in vivo multi-organ connection pathways in physiological and pathological situations.

An Organ-On-A-Chip Engineered Platform to Study the Microbiota-Gut-Brain Axis in Neurodegeneration.

After decades of research, the etiology of neurodegenerative disorders such as Alzheimer's or Parkinson's disease is still mostly unknown. Recent findings indicate that the microorganisms in the human gut might be involved in neurodegenerative pathways. Here, we discuss an innovative groundbreaking bioengineering approach that could make a difference in this intriguing scenario.

Microbiota-gut brain axis involvement in neuropsychiatric disorders.

Introduction: The microbiota-gut brain (MGB) axis is the bidirectional communication between the intestinal microbiota and the brain. An increasing body of preclinical and clinical evidence has revealed that the gut microbial ecosystem can affect neuropsychiatric health. However, there is still a need of further studies to elucidate the complex gene-environment interactions and the role of the MGB axis in neuropsychiatric diseases, with the aim of identifying biomarkers and new therapeutic targets, to allow early diagnosis and improving treatments. Areas covered: To review the role of MGB axis in neuropsychiatric disorders, prediction and prevention of disease through exploitation, integration, and combination of data from existing gut microbiome/microbiota projects and appropriate other International '-Omics' studies. The authors also evaluated the new technological advances to investigate and modulate, through nutritional and other interventions, the gut microbiota. Expert opinion: The clinical studies have documented an association between alterations in gut microbiota composition and/or function, whereas the preclinical studies support a role for the gut microbiota in impacting behaviors which are of relevance to psychiatry and other central nervous system (CNS) disorders. Targeting MGB axis could be an additional approach for treating CNS disorders and all conditions in which alterations of the gut microbiota are involved.

Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture.

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.

Towards bioinspired in vitro models of intestinal mucus.

Intestinal mucus is a biological structure that acts as a barrier between the external environment and the epithelium. It actively selects nutrient and drug intake, regulates the symbiosis with the intestinal microbiota and keeps the epithelium protected from the attack of pathogens. All these functions are closely connected to the chemical and structural complexity of this biological material, on which its viscoelastic and diffusive properties depend. Many models have been proposed to replicate these characteristics using glycoproteins in solution and possibly the addition of other mucus components, such as lipids and other proteins. In the field of mucus modelling, an overall view of the mucus as a material, having its own viscous, rheological and diffusive characteristics, has been undersized with respect to a pure biological-functional analysis. In this review, we propose a description of the mucus as a biomaterial, including a presentation of its chemical and structural complexity, and of its main viscoelastic-diffusive properties, in order to provide a synthesis of the characteristics necessary for the engineering of more advanced mucus models.

Organ-On-A-Chip in vitro Models of the Brain and the Blood-Brain Barrier and Their Value to Study the Microbiota-Gut-Brain Axis in Neurodegeneration.

We are accumulating evidence that intestinal microflora, collectively named gut microbiota, can alter brain pathophysiology, but researchers have just begun to discover the mechanisms of this bidirectional connection (often referred to as microbiota-gut-brain axis, MGBA). The most noticeable hypothesis for a pathological action of gut microbiota on the brain is based on microbial release of soluble neurotransmitters, hormones, immune molecules and neuroactive metabolites, but this complex scenario requires reliable and controllable tools for its causal demonstration. Thanks to three-dimensional (3D) cultures and microfluidics, engineered in vitro models could improve the scientific knowledge in this field, also from a therapeutic perspective. This review briefly retraces the main discoveries linking the activity of gut microbiota to prevalent brain neurodegenerative disorders, and then provides a deep insight into the state-of-the-art for in vitro modeling of the brain and the blood-brain barrier (BBB), two key players of the MGBA. Several brain and BBB microfluidic devices have already been developed to implement organ-on-a-chip solutions, but some limitations still exist. Future developments of organ-on-a-chip tools to model the MGBA will require an interdisciplinary approach and the synergy with cutting-edge technologies (for instance, bioprinting) to achieve multi-organ platforms and support basic research, also for the development of new therapies against neurodegenerative diseases.

Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson's disease related toxin 6-hydroxydopamine.

Neurodegenerative diseases, as Parkinson's disease (PD), involve irreversible neural cell damage and impairment. In PD, there is a selective degeneration of the dopaminergic neurons leading to motor symptoms. A common finding in PD neurodegeneration is the increase of reactive oxygen species (ROS), leading to oxidative stress. To date there are only interventions to relieve PD symptoms, however progress has been made in the development of therapies that target the immune system or use its components as therapeutic agents; among these, mesenchymal stem cells (MSCs), which are able to express neuroprotective factors as cytokines, chemokines and angiogenic molecules, collectively named secretome, that accumulate in MSC culture medium. However, lasting cell-free administration of secretome in vitro or in vivo is challenging. We used the conditioned media from rat adipose tissue-derived MSCs (RAA-MSCs) to check for neuroprotective activity towards pro-oxidizing agents such as hydrogen peroxide (H2O2) or the dopaminergic selective toxin 6-hydroxydopamine (6-OHDA) that is commonly used to model PD neurodegeneration. When neuroblastoma SH-SY5Y cells were pre-conditioned with 100% RAA-MSC media, then treated with H2O2 and 6-OHDA, mortality and ROS generation were reduced. We implemented the controlled release of RAA-MSC secretome from injectable biodegradable hydrogels that offer a possible in situ implant with mini-invasive techniques. The hydrogels were composed of type I bovine collagen (COLL) and low-molecular-weight hyaluronic acid (LMWHA) or COLL and polyethylene glycol (PEG). Hydrogels were suitable for RAA-MSC embedding up to 48h and secretome from these RAA-MSCs was active and counteracted 6-OHDA toxicity, with upregulation of the antioxidant enzyme sirtuin 3 (SIRT3). These results support a biomaterials-based approach for controlled delivery of MSC-produced neuroprotective factors in a PD-relevant experimental context.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

The effect of scaffold pore size in cartilage tissue engineering.

INTRODUCTION: The effect of scaffold pore size and interconnectivity is undoubtedly a crucial factor for most tissue engineering applications. The aim of this study was to examine the effect of pore size and porosity on cartilage construct development in different scaffolds seeded with articular chondrocytes. METHODS: We fabricated poly-L-lactide-co-trimethylene carbonate scaffolds with different pore sizes, using a solvent-casting/particulate-leaching technique. We seeded primary bovine articular chondrocytes on these scaffolds, cultured the constructs for 2 weeks and examined cell proliferation, viability and cell-specific production of cartilaginous extracellular matrix proteins, including GAG and collagen. RESULTS: Cell density significantly increased up to 50% with scaffold pore size and porosity, likely facilitated by cell spreading on the internal surface of bigger pores, and by increased mass transport of gases and nutrients to cells, and catabolite removal from cells, allowed by lower diffusion barriers in scaffolds with a higher porosity. However, both the cell metabolic activity and the synthesis of cartilaginous matrix proteins significantly decreased by up to 40% with pore size. We propose that the association of smaller pore diameters, causing 3-dimensional cell aggregation, to a lower oxygenation caused by a lower porosity, could have been the condition that increased the cell-specific synthesis of cartilaginous matrix proteins in the scaffold with the smallest pores and the lowest porosity among those tested. CONCLUSIONS: In the initial steps of in vitro cartilage engineering, the combination of small scaffold pores and low porosity is an effective strategy with regard to the promotion of chondrogenesis.