Proof-of-Concept Human Organ-on-Chip Study: First Step of Platform to Assess Neuro-Immunological Communication Involved in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) are complex chronic inflammatory disorders of the gastrointestinal (GI) tract. Recent evidence suggests that the gut-brain axis may be pivotal in gastrointestinal and neurological diseases, especially IBD. Here, we present the first proof of concept for a microfluidic technology to model bilateral neuro-immunological communication. We designed a device composed of three compartments with an asymmetric channel that allows the isolation of soma and neurites thanks to microchannels and creates an in vitro synaptic compartment. Human-induced pluripotent stem cell-derived cortical glutamatergic neurons were maintained in soma compartments for up to 21 days. We performed a localized addition of dendritic cells (MoDCs) to either the soma or synaptic compartment. The microfluidic device was coupled with microelectrode arrays (MEAs) to assess the impact on the electrophysiological activity of neurons while adding dendritic cells. Our data highlight that an electrophysiologic signal is transmitted between two compartments of glutamatergic neurons linked by synapses in a bottom-up way when soma is exposed to primed dendritic cells. In conclusion, our study authenticates communication between dendritic cells and neurons in inflammatory conditions such as IBD. This platform opens the way to complexification with gut components to reach a device for pharmacological compound screening by blocking the gut-brain axis at a mucosal level and may help patients.

Human Brain Organoids-on-Chip: Advances, Challenges, and Perspectives for Preclinical Applications.

There is an urgent need for predictive in vitro models to improve disease modeling and drug target identification and validation, especially for neurological disorders. Cerebral organoids, as alternative methods to in vivo studies, appear now as powerful tools to decipher complex biological processes thanks to their ability to recapitulate many features of the human brain. Combining these innovative models with microfluidic technologies, referred to as brain organoids-on-chips, allows us to model the microenvironment of several neuronal cell types in 3D. Thus, this platform opens new avenues to create a relevant in vitro approach for preclinical applications in neuroscience. The transfer to the pharmaceutical industry in drug discovery stages and the adoption of this approach by the scientific community requires the proposition of innovative microphysiological systems allowing the generation of reproducible cerebral organoids of high quality in terms of structural and functional maturation, and compatibility with automation processes and high-throughput screening. In this review, we will focus on the promising advantages of cerebral organoids for disease modeling and how their combination with microfluidic systems can enhance the reproducibility and quality of these in vitro models. Then, we will finish by explaining why brain organoids-on-chips could be considered promising platforms for pharmacological applications.

Modeling Neurodegenerative Diseases Using In Vitro Compartmentalized Microfluidic Devices.

The human brain is a complex organ composed of many different types of cells interconnected to create an organized system able to efficiently process information. Dysregulation of this delicately balanced system can lead to the development of neurological disorders, such as neurodegenerative diseases (NDD). To investigate the functionality of human brain physiology and pathophysiology, the scientific community has been generated various research models, from genetically modified animals to two- and three-dimensional cell culture for several decades. These models have, however, certain limitations that impede the precise study of pathophysiological features of neurodegeneration, thus hindering therapeutical research and drug development. Compartmentalized microfluidic devices provide in vitro minimalistic environments to accurately reproduce neural circuits allowing the characterization of the human central nervous system. Brain-on-chip (BoC) is allowing our capability to improve neurodegeneration models on the molecular and cellular mechanism aspects behind the progression of these troubles. This review aims to summarize and discuss the latest advancements of microfluidic models for the investigations of common neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Label-Free Electric Monitoring of Human Cancer Cells as a Potential Diagnostic Tool.

Dielectrophoresis is widely used for cell characterization, and the exerted force on cells depends on the difference of polarizability between the latter and the surrounding medium. This physical phenomenon is translated by the real part of the Clausius-Mossotti factor. It is mostly modeled from the imaginary part, measured by electrorotation. The method described here measures experimentally the real part of the Clausius-Mossotti factor. It relies on the cell velocity when submitted to pure dielectrophoresis, and it was conducted on several human cell lines, at different times. A variety of cell lines was evaluated, from different organs or representative of different stages of cancer, with promising findings for early cancer detection.

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks.

The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for in vitro CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create in vitro neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basic brain motifs that have been previously observed in vivo and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce in vitro the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.

Trajectory Control of Self-Propelled Micromotors Using AC Electrokinetics.

3D control of the motion of self-powered micromotors is demonstrated using AC electrokinetics by applying an AC electric field on indium tin oxide transparent electrodes.

Involvement of membrane proteins and ion channels on the self-rotation of human cells in a non-rotating AC electric field.

Dielectrophoresis is a force that has been exploited in microsystems for label-free characterization and separation of cells, when their electrical signature is known. However, the polarization effect of cells at the transmembrane protein level is not well established. In this work, we have use the self-rotation effect of cells in a non-rotating field, known as the "Quincke effect," in order to measure the maximum rotation frequency (frotmax ) of different cell populations when modifying the composition of their membrane. We investigated the influence of active ionic transportation of membrane protein concentration on frotmax of HEK cells. Our results show that ionic transportation is responsible for the reduction of conductivity within the cytoplasm, which results in higher frotmax . However, the influence of the concentration of proteins in the membrane, achieved by silencing gene expression in cancer cells, changes significantly frotmax , which is not explained by the changes of ionic conductivity within the cell.

Design of a 2D no-flow chamber to monitor hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are the most commonly used cell type in cell-based therapy. However, the investigation of their behavior in vitro has been limited by the difficulty of monitoring these non-adherent cells under classical culture conditions. Indeed, fluid flow moves cells away from the video-recording position and prevents single cell tracking over long periods of time. Here we describe a large array of 2D no-flow chambers allowing the monitoring of single HSCs for several days. The chamber design has been optimized to facilitate manufacturing and routine use. The chip contains a single inlet and 800 chambers. The chamber medium can be renewed by diffusion within a few minutes. This allowed us to stain live human HSCs with fluorescent primary antibodies in order to reveal their stage in the hematopoiesis differentiation pathway. Thus we were able to correlate human HSCs' growth rate, polarization and migration to their differentiation stage.

Comprehensive analysis of human cells motion under an irrotational AC electric field in an electro-microfluidic chip.

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.

Moving pulsed dielectrophoresis.

In this paper, we introduce a dielectrophoresis (DEP)-based handling method that allows fine 3D manipulation of beads in suspension using a lab on a chip device. The device consists of two layers of linear electrodes on the top and bottom of a microfluidic channel. Each electrode layer has a 53 × 53 donut trap matrix, with traps that are linearly connected into rows along the top, and columns along the bottom of the channel. To address in this matrix a single particle in suspension, we introduce pulsed dielectrophoresis (puDEP) where the AC signal used to induce dielectrophoresis is chopped, such that only beads that are at the intersection of two perpendicular electrodes are constantly polarized. Finally, by combining puDEP and moving dielectrophoresis (mDEP), we introduce a generic application of dielectrophoresis namely moving pulsed dielectrophoresis (mpuDEP) that allows the contactless, micron accuracy, addressable displacement in a 2D array of a single bead in suspension.

Electrokinetic confinement of axonal growth for dynamically configurable neural networks.

Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 10(5) Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode 'gates' that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca(2+) imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks.