Heart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other.... Show moreHeart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other. Although independent human in vitro models for heart and kidney exist, they do not capture their dynamic crosstalk. We have developed a microfluidic system which can be used to study heart and kidney interaction in vitro. Cardiac microtissues (cMTs) and kidney organoids (kOs) derived from human induced pluripotent stem cells (hiPSCs) were generated and loaded into two separated communicating chambers of a perfusion chip. Static culture conditions were compared with dynamic culture under unidirectional flow. Tissue viability was maintained for minimally 72 h under both conditions, as indicated by the presence of sarcomeric structures coupled with beating activity in cMTs and the presence of nephron structures and albumin uptake in kOs. We concluded that this system enables the study of human cardiac and kidney organoid interaction in vitro while controlling parameters like fluidic flow speed and direction. Together, this "cardiorenal-unit" provides a new in vitro model to study the cardiorenal axis and it may be further developed to investigate diseases involving both two organs and their potential treatments. Show less
To improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These... Show moreTo improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These microphysiological systems (MPS) or “Organs-on-Chip” (OoC) are being developed to include all aspects of the human physiology to improve the in vitro cellular response. OoCs combined with differentiated human induced pluripotent stem cells (hiPSC) allow the use of cells with patient specific genotypes and aid the development of personalized and precision medicine.In this thesis, the development of tractable models of the vasculature is described. These models allow for the combination of hiPSC-derived vascular and tissue specific cells with haemodynamics to recapitulate essential stimuli of blood vessels. Show less
Pharmaceutical companies, governments and the general public have become increasingly aware that animal models used in drug testing lack vital aspects to serve as an accurate representation of... Show morePharmaceutical companies, governments and the general public have become increasingly aware that animal models used in drug testing lack vital aspects to serve as an accurate representation of human biology. As models of the human body should become more physiologically relevant, animal models no longer suffice because responses of animal cells often differ from human cells. Instead, in vitro cell culture models with 3D architecture, microfluidics and high throughput capabilities are a promising technology. These 3D models can be developed in ways that they will likely surpass animal models on important aspects like resemblance to a human body, predicting compound safety and efficacy, high throughput testing capabilities, ethical aspects, and costs.To demonstrate the feasibility of an advanced 3D in vitro model, we used a microfluidic in vitro platform to develop a kidney-on-a-chip platform which possess the ability to reproduce the tubular response to known and unknown nephrotoxicants and compounds as seen in in vitro and in clinical studies. Furthermore, we assessed the response of the model to renal ischemia/reperfusion injury and could measure the prevention of tubular damage when adding protective compounds.These findings show that 3D tissue models can compete with alternatives like animal models and 2D models. Show less
Vascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow. The high degree of... Show moreVascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow. The high degree of heterogeneity among these cells is dictated by (1) differences in their developmental origin and (2) their location in the vascular bed. Phenotype switching contributes to this heterogeneity especially following in vitro culture. In the absence of distinguishing molecular markers, functional assays that capture their heterogeneity in vitro are needed. Spatiotemporal changes in intracellular Ca2+ levels and contraction in response to vasoconstrictors reflect the differences between vascular pericyte and smooth muscle cell. In order to capture this heterogeneity in vitro, large ensembles of cells need to be analyzed. Here we developed an automated image processing method to measure intracellular Ca2+ and contraction in large cell groups which in combination with a computational approach for integrative analysis allowed vascular pericytes and smooth muscle cells to be distinguished without knowledge of their anatomical origin. (C) 2019 The Authors. Published by Elsevier Inc. Show less
Duinen, V. van; Zhu, D.; Ramakers, C.; Zonneveld, A.J. van; Vulto, P.; Hankemeier, T. 2019
The vascular system is one of the first to develop during embryogenesis and is essential for all organs and tissues in our body to develop and function. It has many essential roles including... Show moreThe vascular system is one of the first to develop during embryogenesis and is essential for all organs and tissues in our body to develop and function. It has many essential roles including controlling the absorption, distribution and excretion of compounds and therefore determines the pharmacokinetics of drugs and therapeutics. Vascular homeostasis is under tight physiological control which is essential for maintaining tissues in a healthy state. Consequently, disruption of vascular homeostasis plays an integral role in many disease processes, making cells of the vessel wall attractive targets for therapeutic intervention. Experimental models of blood vessels can therefore contribute significantly to drug development and aid in predicting the biological effects of new drug entities. The increasing availability of human induced pluripotent stem cells (hiPSC) derived from healthy individuals and patients have accelerated advances in developing experimental in vitro models of the vasculature: human endothelial cells (ECs), pericytes and vascular smooth muscle cells (VSMCs), can now be generated with high efficiency from hiPSC and used in 'microfluidic chips' (also known as 'organ-on-chip' technology) as a basis for in vitro models of blood vessels. These near physiological scaffolds allow the controlled integration of fluid flow and three-dimensional (3D) co-cultures with perivascular cells to mimic tissue- or organ-level physiology and dysfunction in vitro. Here, we review recent multidisciplinary developments in these advanced experimental models of blood vessels that combine hiPSC with microfluidic organ-on-chip technology. We provide examples of their utility in various research areas and discuss steps necessary for further integration in biomedical applications so that they can be contribute effectively to the evaluation and development of new drugs and other therapeutics as well as personalized (patient-specific) treatments. (C) 2018 The Authors. Published by Elsevier B.V. Show less
The human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and... Show moreThe human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and validation of these metabolomic biomarkers requires large-scale cohort studies, typically involving thousands of samples. This analytical challenge drives novel technological developments to enable faster, cheaper, and more comprehensive metabolomic analysis: more for less.This review summarises recent (2012–2018) developments towards this goal in all aspects of the analytical workflow, in relation to NMR but primarily to mass spectrometry (MS). Recent trends include miniaturisation and automation of extraction techniques, online coupling to fast analysis methods including direct infusion ion mobility MS, integrated microfluidic devices, and sharing and standardizing metabolomics software and data.The technological advances in metabolomics support its widespread application, integration with other -omics fields, and ultimately disease prediction and precision medicine. Show less
There is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the... Show moreThere is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the development of the OrganoPlate, a microfluidic platform that enables enhanced physiology in cell culture models by combining 3D cell culture, co-culture and perfusion flow, whilst maintaining ease of use, compatibility and throughput. Phaseguides are capillary pressure barriers that enable microfluidic liquid routing and patterning without the use of membrane or other physical barriers. This technology was further developed to enable complex liquid routing using only a standard pipette Phaseguide technology was implemented for gel patterning in a dedicated 3D cell culture device embedded in a standard 384 wells plate. Each plate contains up to 96 microfluidic networks that enable perfusion culture of extracellular matrix embedded tissues and perfused epithelial or endothelial tubules. The standard dimensions and high quality optical readout allows interrogation of these tissues using high content readers as well as other standard readout equipment. The platform has been used for the culture of a variety of tissue types and disease models by the authors, but has also been adopted by expert and non-expert users across the field. Show less
The ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of... Show moreThe ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of organoids, and other 3D culture systems, is the lack of a vascular network. Protocols developed for establishing in vitro vascular networks typically use human or rodent cells. A major technical challenge is the culture of functional (perfused) networks. In this rapidly advancing field, some microfluidic devices are now getting close to the goal of an artificially perfused vascular network. Another development is the emergence of the zebrafish as a complementary model to mammals. In this review, we discuss the culture of endothelial cells and vascular networks from mammalian cells, and examine the prospects for using zebrafish cells for this objective. We also look into the future and consider how vascular networks in vitro might be successfully perfused using microfluidic technology. Show less
One of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem.... Show moreOne of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem. The zebrafish is a promising model for in vitro vasculogenesis and angiogenesis studies, as a replacement for currently used mammalian models. However, the culture of endothelial cells from this species is not well characterized. Here, we test different culture strategies, medium supplementations and culture substrates for their effect on the generation of putative endothelial (fli:GFP+ and kdrl:GFP+) cells and vascular morphogenesis in zebrafish blastocyst cell derived embryoid body culture. we have also developed a perfused culture model, using microfluidic technology, to culture zebrafish vascular networks. This study is a step forward to the development of zebrafish vascular networks in vitro. Show less
A major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such... Show moreA major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such studies. Unfortunately, methods of culturing endothelial cells from this species are not well characterised. Here, we tried two methods (embryoid body culture and organ explants from transgenic zebrafish kdrl:GFP embryos) to develop in vitro vascular networks. In the kdrl:GFP line, endothelial cells expresses green fluorescent protein, which allows to track the vascular development in live cultures. We found that embryoid bodies showed significantly longer and wider branches of connected endothelial cells when grown in a microfluidic system than in static culture. Similarly, sprouting of kdrl:GFP+ cells from the tissue explants was observed in a 3D hydrogel matrix. This study is a step towards the development of zebrafish vascular networks in vitro. Show less
Meer, B.J. van; Vries, H. de; Firth, K.S.A.; Weerd, J. van; Tertoolen, L.G.J.; Karperien, H.B.J.; ... ; Mummery, C.L. 2017
The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb... Show moreThe polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like "Organs-on-Chip", designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license. Show less
Ent, W. van der; Veneman, W.J.; Groenewoud, A.; Chen, L.P.; Tulotta, C.; Hogendoorn, P.C.W.; ... ; Langenau, D.M. 2016
Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies... Show moreZebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines. To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research. Show less
The unification of isotachophoresis (ITP) and electric field gradient focusing (EFGF) is proposed, based on theoretical considerations and experimental evidence from previous literature. This... Show moreThe unification of isotachophoresis (ITP) and electric field gradient focusing (EFGF) is proposed, based on theoretical considerations and experimental evidence from previous literature. This unification increases the versatility of each EFGF method. A novel isotachophoretic separation method is developed which is based on one of the EFGF techniques, namely micro/nanofluidic concentration polarization devices, This novel method, coined depletion zone isotachophoresis (dzITP), requires a single electrolyte only, which is a simplification over conventional isotachophoresis. Moreover, positioning of focused analyte zones, selective release of individual analyte zones and continuous filtering based on ionic mobility differences is possible. Furthermore, it is shown that microvalves in PDMS chip devices can act as nanochannels upon closure, giving rise to a wide range of concentration polarization phenomena. Compounds can be concentrated over 1000-fold and subsequently be released. These techniques give novel possibilities for bioassays and sample preparation. Show less
In this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed... Show moreIn this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed the focusing and separation of analytes in a 0.4 picoliter volume, which is a volume in the order of a sample from a single cell. Depletion zone isotachophoresis (dzITP) is demonstrated which uses a nanofluidic phenomenon, concentration polarization, to enable isotachophoresis in a microchannel while using only a single electrolyte. A concept for surface enhanced Raman spectroscopy (SERS) sensor (SERSOR) is explored; a coating protecting the SERS surface from irreversible adsorption may enable dynamic measurements of biomolecules in solution in minute volumes. Working with nanochannels has led to the discovery of new unexpected fundamental phenomena: the very high surface to volume ratio in nanochannels causes acidification of solutions introduced in them, despite the presence of up to 1 mol/L of buffer. Also, extreme pressures of more than a 1000 bar may be induced in a nanochannel by what we named electrocavitation, an effects shown to impose a limit on further downscaling of ITP. Show less
