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
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
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
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
