NeuroExaminer: an all-glass microfluidic device for whole-brain in vivo imaging in zebrafish

While microfluidics enables chemical stimuli application with high spatio-temporal precision, light-sheet microscopy allows rapid imaging of entire zebrafish brains with cellular resolution. Both techniques, however, have not been combined to monitor whole-brain neural activity yet. Unlike conventional microfluidics, we report here an all-glass device (NeuroExaminer) that is compatible with whole-brain in vivo imaging using light-sheet microscopy and can thus provide insights into brain function in health and disease

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets.

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization

Mikrofluidische Vorrichtungen für das Screening von pharmazeutischen Produkten

This thesis focuses on the development, production and testing of four novel microfluidic systems in which different forms of living matter ranging from cells to organisms can be cultivated and investigated. Such systems are referred to in the literature as organ-on-chip systems. They typically find applications in research and development in the fields of pharmacy and biology. The order of the chapters in this work is derived from the order of growing complexity of the life forms that shall be cultivated in the systems. At first, a user-friendly new development of a fully dynamic fluidic system for cell layer cultivation with online monitoring of cell integrity for drug absorption studies is described This system was realized by methods of precision engineering. An artificial human hemicornea tissue model could be successfully transferred into the device. With the implemented electrodes and commercially available measurement electronics a successful long term validation of the cell culture barrier was possible. Next, a system for with three-dimensional capillary structures for mimicking neuronal structures in the human brain is described. This system is fabricated by so called soft-lithographic approaches. Endothelia cells were successfully seeded into a fibrin matrix and injected into the device to form artificial blood vessels. Thirdly, a system for cultivation of Pancreatic cell islands forming fully functional mini organs represents a further increase in complexity. This system is completely made of transparent glass structured by femtosecond laser ablation. Combined with a special post-treatment an excellent optical quality could be achieved. This enabled successful multi parametric examinations of the cell islets. The last system presented in this work allows to image the neuronal activities in the full brain of living zebrafish larva (Danio rerio). The required high optical quality could be realized with glass micro structuring processes already developed for the pancreas system. With a successful hydrodynamic immobilization, a real-time 3D-light-sheet-imaging with single cell resolution was achieved.Diese Arbeit konzentriert sich auf die Entwicklung, Herstellung und Erprobung von vier neuartigen mikrofluidischen Systemen, in denen verschiedene Formen von Lebewesen von Zellen bis hin zu Organismen kultiviert und untersucht werden können. Solche Systeme werden in der Literatur als Organ-on-Chip-Systeme bezeichnet. Typischerweise finden sie Anwendungen in der Forschung und Entwicklung in den Fachbereichen Pharmazie und Biologie. Die Reihenfolge der Kapitel in dieser Arbeit ergibt sich aus der Reihenfolge der wachsenden Komplexität der Lebensformen, die in den Systemen kultiviert werden sollen. Zunächst wird eine anwenderfreundliche Neuentwicklung eines volldynamischen Strömungssystems für die Zellschichtkultivierung mit Online-Überwachung der Zellintegrität für Arzneimittelabsorptionsstudien beschrieben. Dieses System wurde mit Methoden der Feinmechanik realisiert. Ein künstliches menschliches Hämicornea-Gewebemodell konnte erfolgreich in das Gerät übertragen werden. Mit den eingesetzten Elektroden und der marktüblichen Messelektronik war eine erfolgreiche Langzeitvalidierung der Zellkulturbarriere möglich. Als nächstes wird ein System zur Bildung von dreidimensionalen Kapillarstrukturen beschrieben. Dieses System wird durch so genannte soft-lithographische Ansätze hergestellt. Endothelzellen wurden erfolgreich in eine Fibrinmatrix gesät und in das Gerät injiziert, um künstliche Blutgefäße zu bilden. Drittens wird ein System zur Untersuchung von Pankreaszellinseln vorgestellt, bei diesen Mini-Organen handelt es sich um eine weitere Steigerung der Komplexität. Das System besteht vollständig aus transparentem Glas, das durch Femtosekunden-Laserablation strukturiert wurde. In Kombination mit einer speziellen Nachbehandlung konnte eine hervorragende optische Qualität erreicht werden. Dies ermöglichte erfolgreiche multiparametrische Untersuchungen der Zellinseln. Das letzte System, das in dieser Arbeit vorgestellt wird, ermöglicht es, die neuronalen Aktivitäten im gesamten Gehirn von lebenden Zebrafischlarve (Danio rerio) darzustellen. Die erforderliche hohe optische Qualität konnte mit bereits für das Pankreas-System entwickelten Glasmikrostrukturierungsverfahren realisiert werden. Mit einer erfolgreichen hydrodynamischen Immobilisierung wurde ein Echtzeit-3D-Lichtblattaufnahme mit Einzelzellenauflösung erzielt

DynaMiTES - A dynamic cell culture platform for in vitro drug testing PART 2 - Ocular DynaMiTES for drug absorption studies of the anterior eye

In the present study, a formerly designed Dynamic Micro Tissue Engineering System (DynaMiTES) was applied with our prevalidated human hemicornea (HC) construct to obtain a test platform for improved absorption studies of the anterior eye (Ocular DynaMiTES). First, the cultivation procedure of the classic HC was slightly adapted to the novel DynaMiTES design. The obtained inverted HC was then compared to classic HC regarding cell morphology using light and scanning electron microscopy, cell viability using MTT dye reaction and epithelial barrier properties observing transepithelial electrical resistance and apparent permeation coefficient of sodium fluorescein. These tested cell criteria were similar. In addition, the effects of four different flow rates on the same cell characteristics were investigated using the DynaMiTES. Because no harmful potential of flow was found, dynamic absorption studies of sodium fluorescein with and without 0.005 %, 0.01 % and 0.02 % benzalkonium chloride were performed compared to the common static test procedure. In this proof-of-concept study, the dynamic test conditions showed different results than the static test conditions with a better prediction of in vivo data. Thus, we propose that our DynaMiTES platform provides great opportunities for the improvement of common in vitro drug testing procedures

LED-Based Tomographic Imaging for Live-Cell Monitoring of Pancreatic Islets in Microfluidic Channels

A portable lensless imaging device combining light-emitting diodes (LEDs) and a CMOS image sensor was developed and its suitability for non-invasive live-cell in vitro monitoring of pancreatic islets was demonstrated. A microfluidic lab-on-a-chip platform containing micro wells with various depths was also fabricated and integrated into the optical sensor system, which allows for immobilization of the single islets and continuous recording of their behavior. This promising technique may provide further insight into the structure and function of pancreatic islets and their deficiencies in type 2 diabetes

Continuous Live-Cell Culture Monitoring by Compact Lensless LED Microscopes

A compact lensless microscope comprising a custom-made LED engine and a CMOS imaging sensor has been developed for live-cell culture imaging inside a cell incubator environment. The imaging technique is based on digital inline-holographic microscopy, while the image reconstruction is carried out by angular spectrum approach with a custom written software. The system was tested with various biological samples including immortalized mouse astrocyte cells inside a petri dish. Besides the imaging possibility, the capability of automated cell counting and tracking could be demonstrated. By using image sensors capable of video frame rate, time series of cell movement can be captured

Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage