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Modeling of the human blood-brain barrier (BBB) in stroke
Isabelle Matthiesen, PhD Student, KTH Royal Institute of Technology
We report on a novel human blood-brain barrier (BBB) ischemic stroke model, evaluating trans-endothelial electrical resistance (TEER) in a dynamic microfluidic chip system with integrated real time sensors compared to conventional static culture in transwell plates. The microengineered chip was made using off-stoichiometry thiol-ene-epoxy (OSTE+), a polymer with multiple advantages compared to the prevalent polymethylsiloxane (PDMS). The chip consists of two layers with molded channels, separated by a porous polycarbonate membrane, with integrated gold electrodes for TEER measurements. Induced pluripotent stem cells (iPS)-derived microvascular brain endothelial cells and astrocytes were cultured in these channels under a continuous flow of medium.
Our studies evaluated a healthy model compared to the diseased state where an excess of glutamate was introduced to the system. Both TEER and tight junction biomarkers such as ZO-1 and VECAD, characterized with immunofluorescence to evaluate barrier properties, were compared between the BBB chip and cells grown in conventional transwell plates. Our study demonstrates how the fluidic BBB model serves as a more biologically relevant in vitro platform for studies of cellular response in neurovascular diseases.
Modeling of the human blood-brain barrier (BBB) in stroke
Isabelle Matthiesen, PhD Student, KTH Royal Institute of Technology
We report on a novel human blood-brain barrier (BBB) ischemic stroke model, evaluating trans-endothelial electrical resistance (TEER) in a dynamic microfluidic chip system with integrated real time sensors compared to conventional static culture in transwell plates. The microengineered chip was made using off-stoichiometry thiol-ene-epoxy (OSTE+), a polymer with multiple advantages compared to the prevalent polymethylsiloxane (PDMS). The chip consists of two layers with molded channels, separated by a porous polycarbonate membrane, with integrated gold electrodes for TEER measurements. Induced pluripotent stem cells (iPS)-derived microvascular brain endothelial cells and astrocytes were cultured in these channels under a continuous flow of medium.
Our studies evaluated a healthy model compared to the diseased state where an excess of glutamate was introduced to the system. Both TEER and tight junction biomarkers such as ZO-1 and VECAD, characterized with immunofluorescence to evaluate barrier properties, were compared between the BBB chip and cells grown in conventional transwell plates. Our study demonstrates how the fluidic BBB model serves as a more biologically relevant in vitro platform for studies of cellular response in neurovascular diseases.
In vitro development of microvascular networks with customized structure using biodegradable fibrin cylinders
Yen-Ting Tung, PostDoc, National Chung-Hsing University
A biomimetic “scaffold-wrapping” method was developed for the fabrication of microvascular networks with customized pattern in vitro. Unlike current methods that the microvascular networks were generated by seeding endothelial cells inside the channels made within extra-cellular-matrix (ECM) like gels, the cylindrical fibrin scaffolds with desired structure were fabricated for culturing human umbilical vein endothelial cells (HUVECs) onto the scaffold and could be degraded via plasmin for the formation of microvascular lumen after the scaffold was fully covered by cells. The fibrin cylinders with the diameter as small as 30 µm were produced by integration of soft-lithography and thermal reflow technique. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin (VE-cadherin) on cultured HUVECs that formed the microvascular network have demonstrated the feasibility of fabricating a microvascular network with a degradation-controllable fibrin scaffolds. In addition, seeding cells on a solid scaffold enables monitoring cell conditions in a much direct manner and removes the requirement of medium circulation system during the early stage of vascular development. These cylindrical fibrin scaffolds with the characteristic of controllable degradation can be applied to develop microvascular chips with uniform vascular structure or serve as the framework for building large tissues.
Microengineered crisscross grooves for multiple tissue-tissue interfaces in Organ-on-chips
Jose Yeste Lozano, PhD student, Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC)
Introduction
Single tissue-tissue interfaces are largely recreated in organ-on-chips by culturing different cells on opposite sides of a porous membrane or by connecting the cells of two adjacent compartments through microchannels. Unfortunately, these strategies are not suitable to address multiple tissue tissue interfaces. We have developed a microfluidic chip where cells can be arranged in parallel compartments and highly interconnected through a network of crisscross microgrooves [1].
Results
The compartmentalized device comprises a PDMS slab containing 7 compartments (500µm in width, 230µm in height, and spaced by 50 µm) and a glass substrate with etched microgrooves (2µm in width, 4µm in depth, and spaced by 10µm). In addition, the device includes electrodes on the substrate for quantification of barrier functions and the recording of neuronal electrical activity. With this planar strategy, it is possible the formation of multiple tissues enabling heterotypic cell-cell interaction across the microgrooves. The system has been validated with the co-culture of primary human retinal endothelial cells (HREC), a human neuroblastoma cell line (SH-SY5Y), and a human retinal pigment epithelial cell line (ARPE-19).
Conclusion
The interconnection of different tissue-tissue interfaces may extend organ-on-chips to a new generation of sophisticated models capable of recapitulating more complex organ-level functions.
The impact of surface topography on 3D cell alignment
Fatemeh Navaee, PhD student , EPFL
3D culturing and the organization of the cardiomyocytes and fibroblasts is important to mimic the structural and functional properties of native myocardium. The context of this project is “heart-on-chip”. Our aim is to develop new devices and protocols for providing better in-vitro models of cardiac tissues. For this purpose, two specific aspects have been studied:1) The protocol of the differentiation of H9c2 cells in co-culture with fibroblasts has been defined in combination to the use of a new hydrogel composed of decellularized extracellular matrix (dECM) mixed with fibrin which has structural and functional properties similar to native ones.2) The impact of the surface topography on the alignment of the cells has been evaluated. We present a simple and direct method to control the cellular organization in 3D, using grooves of different dimensions. We showed that cells that are aligned to the wall propagate their alignment to cells that are further inside the 3D space up to more than 300micrometers and therefore have a bulk 3D alignment of the cells in the middle of the hydrogel. The effect of groove dimensions on cell organization has been studied. This system could provide a better in-vitro model for investigating cell and tissue morphogenesis.
Paving the way towards an in-vitro 3D mechanosensory-motor circuit on a chip
Maider Badiola, PhD student, IBEC
Neuromuscular diseases (NMD) are neurological disorders affecting muscles and their control through nervous system. The effects might be reflected in the mechanosensory-motor circuit at different cellular levels (including sensory and motor neurons[1], glia[2] and muscle dysfunctions[3]), and in the connexion among them (neuromuscular junction[4] and muscle spindle[5], as well as the intraspinal circuits).
The aim of this research is to create an in-vitro model to mimic the 3D microenvironment of a healthy neural circuit for locomotion to understand and find treatments for NMDs. To that end, organ-on-a-chip technologies are used for the integration of sensorial and motor neural components together with a functional muscular unit.
First, a compartmentalised microfluidic device is fabricated in PDMS using soft lithography techniques. Then, primary spinal motor- or dorsal root ganglia sensory- neurons are cultured in different compartments together with optogenetically sensitive myocytes (a channelrhodopsin-2 positive cell line), embedded in a 3D matrix.
This study presents the first steps for an innovative motor and sensorial neuronal pathways integration, with the muscle effector in a 3D compartmentalised microfluidic device. Therefore, it provides the basis for future steps towards NMD in-vitro study models, that would improve understanding their mechanisms and would help finding putative treatments.
Skin-on-Chip Model a Novel Approach to Study Wound Healing
Sahar Biglari, Postdoctoral researcher, University of Sydney
Organ-on-chips are an emergent technology that enables the high throughput screening of bioactive molecules and drugs. Our goal was to develop a skin-on-chip model for wound modelling and testing of transdermal drugs on wound healing mechanism. A 3D in vitro skin model was manufactured by integrating microfluidics and human cells with pivotal roles in wound healing. Human umbilical vein endothelial cells (HUVECs), fibroblasts, and macrophages were co-cultured within the microfluidic device that generated a natural microenvironment that facilitated cell growth and the production of cytokines and growth factors. The inflammatory cytokines produced by macrophages were quantified by flow cytometer while vascularization was visualized by immunostaining. M1 macrophages produced IL-6 and IL-1ß as an inflammatory cytokine at the initiation of wound healing whereas IL-8 was released by M2 macrophages at the later stages as a pro-inflammatory chemokine. Our results indicate that this human skin-on-chip model is a promising tool to bridge the gap between 2D cell culture methods and preclinical animal studies/human trials.
Designing of a Gut-on-a-Chip to Model Intestinal Inflammation
Chiara Fois, PhD Student, The University of Sydney
The cost of new drug development is raising and there are many concerns using animal studies as a model of the human physiology. Organs-on-a-chip is an alternative approach that may address these issues for drug discovery[1]. Gut inflammation is a critical disorder of high social impact, and it is important to design an in vitro model that represents this condition. The aim of this study was to develop a cost-effective gut-on-a-chip system as an in vitro model to mimic the inflammatory conditions. To this end, ANSYS Fluent simulation model was used primarily to determine the effect of fluid dynamic conditions, shear stress and geometry of the microfluidic channels on the cells viability[2]. To construct the gut epithelium and simulating inflammatory response, Caco-2 cells and macrophages were grown in two separate channels that are connected by a porous membrane for the diffusion of media between these two compartments. The modelling facilitated the upfront selection of the best geometry and most compatible components (e.g. membrane pores size) that can translate into an in vivo case. Collectively, the in silico findings led to the selection of the optimal parameters for improving epithelium cell functions including the 3D villi-like structures and mucin production.
Developing an Ovarian Cancer Model-on-a-Chip
Matthew Dibble, PhD Student, Queen Mary University of London
Microfluidics and tissue-on-a-chip technology is a rapidly expanding field, their success led to the development of various ‘cancer-on-a-chip’ models, with breast cancer [1], glioblastoma [2] and fibrosarcoma [3] all being effectively imitated. High-Grade Serous Ovarian Cancer (HGSOC), which was responsible for 4,271 deaths in the UK in 2012, is the deadliest gynaecological malignancy. A microfluidic model of HGSOC offers an opportunity to further understand disease progression and discover novel chemotherapeutic targets. One of the hallmarks of cancer is the development of a tumour promoting vasculature, this is characterised by vessel hyper-permeability, vessel immaturity and lack of blood vessel hierarchy. Tumorigenic blood vessels promote the development of cancer through a number of mechanisms, i.e. reducing chemotherapeutic delivery. Investigating cancer-vasculature interaction is therefore imperative in understanding cancer progression and treatment. This project proposes an interconnected, four channelled microfluidic device to model HGSOC, featuring adjacent tumour and vascular compartments. This system enables analysis of cancer-vasculature interaction using live-imaging platforms and confocal microscopy.
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