A minimalistic co-culture platform for alpha-synuclein spreading in human dopaminergic neurons

Parkinson’s disease (PD) is the second major neurodegenerative disease and the most common movement disorder. Due to age being a critical risk factor, the rapid ageing of the world population further increases the prevalence of PD. So far no treatment is available and therapies mainly focus on motor symptoms by pharmacologically substituting striatal dopamine, caused by the loss of dopaminergic neurons in the substantia nigra. This neuronal loss and intracellular protein aggregates, termed Lewy bodies (LBs), are pathological characteristics of PD. With disease progression, a spread of LBs through the brain can be observed which mainly follows axonal projections. Understanding the mechanisms of this progressive spread could be central to discovering the underlying molecular pathogenesis of the disease. As LBs mainly consist of alpha-synuclein (-syn), a prion-like spreading of -syn was suggested and is now widely accepted as a component in the PD pathogenesis. New dopaminergic model systems to study the exact mechanisms underlying -syn spread are urgently needed. As PD is a human disease, in vitro models should be derived from humans. Lund human mesencephalic (LUHMES) cells are a suitable alternative to other, mostly non-human, dopaminergic cell lines. However, difficulties cultivating them in microfluidics devices has made them thus far inaccessible for co-cultivation studies in the field of PD spreading. In the first part of this thesis, a human dopaminergic cell model system for studying the spreading of -syn fibrils is presented. First, the well-characterized LUHMES cell line was tested for suitability of PD research on prion-like spreading, as no data is currently available on this matter. For the analysis, immunofluorescence light microscopy was employed. An extended period of differentiation aimed for a high degree of neuronal maturity and long neurites to facilitate the connectivity of spatially-separated cell populations. Seeding experiments with -syn fibrils revealed a weak toxicity against these assemblies, even at prolonged differentiation. Second, to study the transmission of -syn fibrils via neuronal projections, we developed a light microscopy-compatible microfluidic co-culturing device, to maintain two LUHMES cell populations in separate cell compartments for up to two weeks of differentiation. During this time, a neurite network is formed which connects the fluidically isolated cell growth compartments. The ability to cultivate cells with neurites and soma in an isolated environment enabled seeding and transmission experiments in anterograde and retrograde directions. In the second part of this thesis, implementation strategies of the microfluidic co-culturing chip for alternative analysis methods are discussed. Firstly, the accessibility of the cells in the co-culturing device using a single-cell lysis instrument is evaluated. The tool allows for targeted lysis of individual adherent cells. Preliminary tests point in a promising direction, while LUHMES single cell lysate was successfully transferred to different analysis techniques. However, direct access to the channels of the microfluidic co-culturing chip was problematic and needs further modifications. Secondly, an implementation of the microfluidic device aiming for co-cultivation of LUHMES cells on electron microscopy grids to study neurite architecture was pursued. Thereby, microfluidic devices harbor only cell soma, but neurites can grow onto an electron microscopy grid, as only they are thin enough to be visualized by cryo-electron microscopy. Proof-of-concept experiments demonstrate the direct visualization of LUHMES cell neurites in a near-native, frozen-hydrated state

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics

Nanomimics of host cell membranes block invasion and expose invasive malaria parasites

The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics—polymersomes presenting receptors required for parasite attachment to host cells—were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM

Differential Visual Proteomics: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force

Formation of lipid and polymer based gold nanohybrids using a nanoreactor approach

Nanocarriers encapsulating gold nanoparticles hold tremendous promise for biomedical applications. The nanoreactor approach offers a versatile, efficient, and highly reproducible preparation technology.</p

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 320 nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and realtime monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics. (C) 2016 The Author(s). Published by Elsevier Inc

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible. A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM. The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-andplunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples

openBEB: open biological experiment browser for correlative measurements

Background: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. Results: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. Conclusions: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated