MIRA University of Twente

Poster Session

Poster session IOOCS16 abstracts

Microfluidics for Complex Tissue Engineering

T. Kamperman, S. Henke, J. Leijten and M. Karperien

Multiscale hierarchy is found throughout nature and is essential for tissue function. Incorporating hierarchies into biomaterials is expected to provide engineered tissues with the multifunctionality that drives the behavior of native tissues. To this end, the inclusion of nano/micromaterials into the design of biomaterials is explored. Microfluidic technologies are uniquely suited to produce such micromaterials. We have developed a variety of microfluidic chips for the controlled production of single and multi-cell-laden microgels and microfibers. We also exploited microfluidics to control the biofunctionalization and micromechanical properties of these micromaterials. The resulting cell-laden modified micromaterials can be incorporated into distinct biomaterials to create unique spatially defined constructs. Moreover, this approach is compatible with biofabrication techniques such as injection molding, photolithography, and 3D printing. Currently, we are exploring novel microfluidic technologies that allow production upscaling and clinical translation of multiscale hierarchical biomaterials. Here, we present our microfluidics-based concept of engineering complex tissue constructs.

Non-invasive multiplexed phenotyping of human stem cell-derived cardiomyocytes for microfluidic chips

B.J. van Meer; M.C. Ribeiro; L.G.J. Tertoolen; P.C.J.J. Passier; C.L. Mummery

Organ-on-Chip devices could recapitulate human physiology if substrate elasticity, topology, mechanical stimuli and fluid dynamics mimic that of each organ. It is thought that biophysical stimuli might even enhance maturation of differentiated human pluripotent stem cells (hPSC). This is currently one of the major limitations of hPSC based models. In excitable cells dynamics of and correlation between membrane potential and ion fluxes are among the most important readouts of maturation and drug or disease response.
Here, we demonstrate a non-invasive optical measurement system with high spatial (~200 nm) and temporal (~333 Hz) resolution for simultaneous readout of action potential, calcium flux and force of contraction in hPSC derived cardiomyocytes (hPSC-CMs). We show these biophysical dynamics can be used to assess hPSC-CM maturation and reveal disease phenotypes when the stem cells have been derived by reprogramming from patients.

In vitro cardiac tissue model reaveals fibroblast threshold for synchronized beating

ACC van Spreeuwel, NAM Bax, CVC Bouten

In cardiac pathologies, synchronized beating of the heart is often hampered by the formation of fibrosis. Gaining insight into the mechanisms that are responsible for the decreased cardiac function could help to improve currently used therapies for cardiac disease. Therefore, in this study an in vitro cardiac tissue model was used to unravel the effect of fibroblast density and collagen content on cardiomyocyte contractility. Cardiac microtissues were created by seeding mouse neonatal cardiomyocytes and cardiac fibroblasts in a collagen/matrigel hydrogel in arrays of microwells containing flexible posts. Upon spontaneous beating, deflection of the posts was used to measure the beating frequency and dynamic contraction force of the microtissues. Interestingly, the results revealed that only a fibroblast density higher than 50% inhibited spontaneous beating of the microtissues. Furthermore, the developed cardiac tissue model holds great promise as a tool for predictive safety pharmacology and toxicology screenings.

SkinonChip: Integrating SkinTissue and Microsystems Engineering

L. Bergers, T. Waaijman, T. De Gruijl, A. Van De Stolpe, R. Dekker, S. Gibbs

All skin disease has an underlying immune component e.g. skincancer, fibrosis, allergy. Current animal and in vitro models are inadequate for studying these complex human immune interactions. Microsystems engineering offers dynamic control of the tissue microenvironment, enabling better in vitro models. Our goal is to create a microsystem suitable for studying interactions between immune cells migrating into and out of cultured skin tissue. We present results of 1) a microfluidics device consisting of two channels separated by a microporous membrane 2) integration of tissue engineering full thickness skin equivalents in the device showing first steps towards skin-on-chip.

A microfluidic platform to study the blood-brain barrier

Marinke van der Helm, Jean-Philippe Frimat, Mathieu Odijk, Albert van den Berg, Jan Eijkel & Loes Segerink

A realistic model of the blood-brain barrier (BBB) is valuable to perform drug screening experiments and to improve the understanding of the barrier’s physiology in normal and pathological conditions. Conventional in vitro systems (e.g. Transwell systems) lack reproducibility and have a static environment. To overcome these disadvantages, we have recently developed a “BBB-on-a-chip”, which uses microfluidics and (human) cells to mimic organ function.
hCMEC/D3 cells were cultured inside this microfluidic chip for up to 15 days. With four integrated electrodes reliable transendothelial electrical resistance measurements were carried out. Additionally, using immunohistochemistry it was shown that the endothelium expressed tight junction proteins, which is an essential characteristic of the BBB.
To further improve the physiological relevance of this promising platform, the cells inside the channels will be cultured under fluid flow. As application, this platform will be used to study the transport of nanocarriers with Alzheimer medication through the BBB.

Functional assays for the assessment of contractile behavior in hPSC-derived vascular smooth muscle cells (vSMCs)

Oleh Halaidych, Marcelo Ribeiro, Robert Passier, Christine Mummery and Valeria Orlova

vSMCs are essential for the regulation of vascular tone through coordinated constriction/vasodilation. The constriction of vSMCs is regulated by intracellular Ca2+. Different vasoconstrictors, such as endothelin-1 (ET1), carbachol, phenylephrine etc. are known to increase intracellular Ca2+ and to induce vasoconstriction. Here we established functional assays for the assessment of contractile behaviour of hPSC-derived vSMCs: (1) intracellular Ca2+ imaging upon controlled addition of the vasoconstrictors using a highly precise multichannel microfluidic pump and (2) traction force measurement on micropatterned acrylamide gels (Rape et al., 2011). We next tested neural crest (NC)-derived vSMCs differentiated from hPSCs. This was of particular interests since vSMCs in the central nervous system (CNS) originate from NC. Vascular contribution to the development of different neurodegenerative conditions became apparent recently. Therefore, having a renewable source of vSMCs, as well as robust functional assays will be beneficial for the understanding of different vascular neurodegenerative conditions. Stimulation with vasoconstrictors induced robust responses in hPSC-derived NC-vSMCs. In the future, we aim to apply this protocol for neurovascular disease modelling, as well as comparative biophysical studies of contraction and intracellular Ca2+ imaging in NC and mesoderm-derived vSMCs.

Emulating the Human Vasculature in a Multi-Organ-Chip Platform

Tobias Hasenberg, Katharina Schimek, Severin Mühleder, Andrea Dotzler, Sophie Bauer, Krystyna Labuda, Wolfgang Holnthoner, Heinz Redl, Roland Lauster, Uwe Marx

Our Multi-Organ-Chip (MOC) platform contributes to the ongoing efforts to initiate a paradigm change in substance development away from the reliance on animal models toward predictions via in vitro microphysiological systems. One major aspect is the vascularization of the platform’s inhabited organoids to overcome limitations in size and complexity. The object of this work is to recreate a continuous perfusable barrier of human endothelial cells towards a subjacent tissue. To recreate the human vasculature three major aspects had to be addressed: (1) Provide a near-physiological, pulsatile flow that recreates an in vivo-like shear environment. (2) Create an endothelial lumen in the chip’s microfluidic system. And (3) establish a microvascular network in the cultivation cavities for organoid integration. We will present an overview of our ongoing approaches towards the emulation of the human vasculature.

Design of a microfluidic system for measurement of oxygen consumption by tissue slices

Pieter E. Oomen, Maciej D. Skolimowski, Geny M.M. Groothuis and Elisabeth Verpoorte

A gas-impermeable, microfluidic perfusion system has been developed that will allow local changes in medium oxygenation to be monitored in the presence of tissue slices. The micromilled, polycarbonate (PC) device can be equipped with microelectrodes to measure oxygen in medium flowing towards and from the culture chamber. Moreover, the device can be rapidly opened and closed to facilitate the inserting of slices. An oxygenation module based on gas-permeable silicone tubing ensures sufficient oxygenation of the medium before it enters the incubation device.

High-end analytical detection coupled to a gut-on-a-chip

Pim de Haan, Milou Santbergen, Michel WF Nielen, Hans Bouwmeester and Elisabeth Verpoorte

Current in vitro models lack predictive validity for efficacy and safety assessment of chemicals, nutrients and drugs. We are developing an integrated miniaturized gastrointestinal system, mimicking the human gut in function and structure. Three digestive modules, representing the mouth, stomach and small intestine, are employed for a microfluidic digestion using artificial digestive juices. A subsequent epithelial module is used to study the gastrointestinal absorption of compounds. The latter module is coupled to microfluidic sample preparation modules designed for each class of analytes, and state-of-the-art detection systems using ESI-ICP/MALDI-MS. This orthogonal total analysis system was designed to study the behavior of small molecules, nanoparticles and protein samples in the gastrointestinal tract, including possible effects of enzymatic digestion. This system is ideally positioned to replace animal trials in the testing of nutrients and chemicals or in pharmacological lead finding.

Vascular Healing of Coronary Arteries studied in a human disease model

Jeroen Leuven, Volkert van Steijn, Johan W. van Neck, Heleen M. M. van Beusekom

Cardiovascular disease, the leading cause of death in the world, is often treated by drug eluting stents (DES) to re-open blocked arteries. Atherosclerosis and arterial damage following DES - leading to scar formation and delayed healing - is often studied using experimental animal models. These models, however, do not enable to study the complex healing process with high spatial and temporal resolution.

In this work, we present a human disease model of a coronary arterial wall, which comprises two parallel microchannels separated by a thin porous polymeric membrane. Endothelium is grown on one side until confluent, and smooth muscle cells contralateral until the smooth muscle cell layer resembles a thin fibrous cap. We demonstrate how to controllably damage the endothelium and follow the healing dynamics using confocal time-lapse microscopy. This data is instrumental in understanding healing dynamics and the development of new treatments.

Early Health Technology Assessment for the development of Thrombosis-on-a-chip

Heleen H.T. Middelkamp; J. Marjan Hummel; Maarten J. IJzerman; Christine L. Mummery; Robert Passier; Andries D. van der Meer

Organ-on-chip technology can contribute to drug development and may replace animal testing. To maximize future impact, it is essential to make informed decisions regarding features of organs-on-chips even though the technology is still in early development. It is likely that different stakeholders in organ-on-chip development have different perspectives on aspects like cost, materials, cell source, read-out technology, types of data and compatibility with existing technology and how this will maximize the future impact. Early health technology assessment (HTA) is needed to integrate potentially conflicting views in technology development. We suggest using multi-criteria decision analysis (MCDA) as a tool in early HTA of organs-on-chips. It is crucial to design and perform a comprehensive MCDA for organ-on-chip development, maximizing the future impact of organ-on-a-chip technology. As a case study we plan to use HTA in the development of thrombosis-on-a-chip. What do end-users want implemented in our model to maximize its impact in terms of decreasing animal testing?

Unravelling Macular Degeneration: An Organ-on-a-Chip Model of the Human Retina

Yusuf Bilgehan Arik, Andries van der Meer, Albert van den Berg, Robert Passier

Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 in the world. Exudative, or ‘wet’ AMD, the most aggressive form, is characterized by the growth of leaky blood vessels in retina, which leads to the rapid degeneration of retinal tissue and loss of central vision. Current treatment consists of expensive and invasive, intra-ocular injection of antibodies that inhibit the growth of new blood vessels. Anecdotal evidence in patients suggests two over-the-counter drugs (i.e. aescin and cetirizine) that can halt the progression of wet AMD, by normalizing the leaky state of blood vessels in the eye. Current animal models of AMD suffer from specific issues such as physiological differences between species, and the time course of the disease. The goal of the current project is to develop an organ-on-a-chip model mimicking the microenvironment of the human retina, which will be used to study effects of the experimental treatment.

Hypertrophic Cardiomyopathy - Sarcomeric and contractile dysfunction caused by MYBPC3 deficiency in hPSC-derived cardiomyocytes

Marcelo C. Ribeiro, Matthew J. Birket, Leon Tertoolen, Jantine Monshouwer-Kloots, Christine L. Mummery and Robert Passier

Hypertrophic Cardiomyopathy (HCM) is a cardiac disease, morphologically characterized by cardiac hypertrophy, fibrosis and impaired heart function and is primarily associated with mutations in sarcomeric proteins. Mutations in sarcomeric protein Myosin Binding Protein C (MYBPC3) account for approximately 25% of all HCM. The majority of mutations in MYBPC3 are nonsense mutations, predicted to produce truncated proteins. However, instead of truncated proteins, lower levels of full-length protein of MYPBC3 are frequently found in heart samples of patients with mutations in MYBPC3, strongly suggesting that haploinsufficiency is responsible for the cardiac disease phenotype. Induced pluripotent stem cell-derived cardiomyocytes (PSC-CMs) generated from HCM patients carrying this MYBPC3 mutation showed a lower force of contraction. Similarly, knock-down of MYBPC3 by lentiviral-based shRNA in control hPSC-CMs induced contractile dysfunction. Additionally, we observed a decrease in diastolic sarcomere length together with a decrease in shortening and elongation amplitude of the sarcomeres of MYBPC3 deficient hPSC-CM. Knock-down of MYBPC3 did not affect calcium handling of hPSC-CMs, but reduced the ATP consumption per contraction cycle. In conclusion, we successfully developed a human in vitro model for functional analysis of MYBPC3 haploinsufficiency in hPSC-CMs using advanced biophysical and imaging techniques, which may further enable us to comprehend the molecular mechanisms responsible for the development of HCM.

Magnetic hyperthermia: Application of magnetic nanoparticles in cancer medicine

Matthias Bischoff , Maarten van Rossum, Peter Schön and Martin Bennink

Magnetic nanoparticles (MNP) have gained tremendous interest in recent years due to their potential use in applications such as medicine and novel materials. In the bio-medical field, the particles are maily used as contrast agents for imaging, target for specific (cancer) cells, carriers for drug delivery, and sources for in vivo heating. The latter firming under the term magnetic hyperthermia, i.e. the local elevation of body temperature between 40-44°C by means of MNP reacting to a rapidly alternating magnetic field. Here, the MNPs are used in combination with chemotherapy reducing thereby the side effects drastically, because hyperthermia makes the heterogeneous tumor tissue more susceptible to the drug. In this project we fabricate and modify MNPs based on iron oxide and study their magneto thermal coupling and performance in hyperthermia application. We investigate the particles in suspension as well in a more biomimetic environment, for instance a blood-brain-barrier on a chip.