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Cells are the fundamental functional units in all forms of life. Even though largelyall cells in a human body carry the same genetic information, we still knowsurprisingly little about their detailed composition. This is due to the vastamount of variation in a cell's gene expression. Specifically, each cell at a certainmoment in time has a certain set of genes that are switched on or off, resultingin different biomolecules which are being expressed, in their turn regulatingcellular processes. In order to understand the biological driving forces of healthand disease, a detailed map of the trillions of different cells with their specificbiomolecules and within their natural environment is needed. However, asbiomolecules are often much smaller than 200 nm, direct visualization in largequantities, e.g. through light microscopy imaging, is challenging. Recently, amethod named expansion microscopy addresses this issue in an elegant andcost-effective way. Compared to other, more classical super-resolutionfluorescence microscopy techniques, expansion microscopy embeds biologicalsamples in a water-absorbing polymer which can be physically expanded, layingbare their subcellular structures with nanometer precision on conventional lightmicroscopes. Following this approach, expansion microscopy has several advantages overother super-resolution fluorescence microscopy methods. Yielding a perfectlytransparent matrix, it is most suitable for nanoscale imaging of volumetricsamples such as pieces of tissue. For this reason, the field is now rapidly evolvingin a direction where it is combined with state-of-the-art fluorescent methods forsingle cell genome, transcriptome and proteome mapping in multicellularsamples. Nevertheless, expansion microscopy is still in its infancy, and certainhurdles remain to be tackled. In this thesis, we aim to contribute to thisendeavor, evaluating whether ExM is a suitable platform used to multiplexdifferent omics, by studying if expansion is isotropic, and if heterogeneities insample composition influence the expansion factor. We found that theexpansion hydrogel indeed has the potential to ensure isotropic expansion in alldimensions, but care should be taken during sample handling. Furthermore, ascell function is determined by different DNA, RNA and protein markers, the nextstep will be to study a combination of biomarkers in their spatial context viaintegrative techniques. For this reason, we seek for improved labeling strategies viii which better retain fluorescent signal in the expanded sample, and whichfacilitate simultaneous crosslinking of different biomolecules. These efforts haveresulted in an optimized expansion microscopy workflow, which was adopted toperform the first tests for implementing a protein multiplexing readout-schemein expansion microscopy, referred to as immuno-SABER. Finally, we exploredwhich resolution can be achieved when imaging expanded samples on superresolutionmicroscopy setups, with possible applications in resolving proteinultrastructures with nanoscale precision. Overall, this work brought us one stepcloser to using expansion microscopy as a multi-modal fluorescent detectionplatform with higher efficiency and reliability.

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Skeletal muscle tissue is one of the three types of muscle tissue in the human body, along with cardiac and smooth muscle. Skeletal muscle tissue is composed of long, multinucleated cells called myofibers, which are arranged in bundles called fascicles. Myoblasts, which are muscle progenitor cells, differentiate into myocytes which then fuse together to form multinucleated myotubes that further mature into myofibers. Skeletal muscle has some self-repair and renewal properties, but when there is considerable muscle loss or damage, this proves to be insufficient. Tissue engineering of skeletal muscle tissue is a promising future treatment for such medical conditions. Tissue engineering involves the development of biological tissue substitutes through a combination of cells, growth factors and scaffolds to mimic and recreate the 3D structure of native tissue. Skeletal muscle tissue constructs from tissue engineering could also be used to complement or replace animal models in research, for example in disease modelling or drug testing. Fibrin is a commonly used hydrogel for tissue engineering due to its biocompatibility but has rather poor characteristics when used for 3D bioprinting. By creating a hybrid hydrogel from fibrin and gelatin, the properties of fibrin could be improved upon for 3D bioprinting. A big challenge for tissue engineering is providing nutrients and oxygen to cells in the tissue engineered constructs through vascularization. Spheroids, which are multicellular aggregates of cells, have shown potential as an answer to the problem of vascularization. Sprouting of capillary-like structures has been observed in heterocellular spheroids containing endothelial cells. Therefore, incorporating spheroids in tissue engineered constructs could be a potential strategy for vascularization of these constructs. The aim of this thesis was to create skeletal muscle tissue constructs in the form of bioartificial muscles or manually extruded constructs using human myoblasts encapsulated in a fibrin-gelatin hydrogel to assess the effect of fibrin concentration on cell viability, formation and alignment of myotubes. Additionally, homo- and heterocellular spheroids were cultured in different conditions to further determine the characteristics of these spheroids for future applications in the biofabrication of skeletal muscle tissue constructs. Results showed that myotube formation increased as fibrin concentration decreased. Myotube alignment was also more prominent in the 1 mg/ml fibrin constructs compared to constructs of higher concentration. Furthermore, sprouting of capillary-like structures from triculture spheroid was dependent on the type of culture medium which highlights the need for novel formulations of culture medium for coculture spheroids. Finally, one day old spheroids doublets proved to fuse together at a faster rate than three or ten day old spheroids. Triculture spheroid doublets also fused at a quicker rate than monoculture spheroid doublets. The use of triculture myogenic spheroids containing endothelial cells and stem cells could therefore prove to be a viable strategy for biofabrication of muscle tissue constructs, especially in combination with a suitable hydrogel such as fibrin-gelatin hydrogel. However, further work needs to be done including, formulating the right culture medium and conditions while also adapting the hydrogel for the specific biofabrication application, e.g. 3D bioprinting.

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In this project the effect of the extracellular matrix (ECM) on cancer associated fibroblasts (CAF) and cancer interplay is being investigated. Different aspects of cancer associated fibroblast and the extracellular matrix are being studied on a colorectal isogenic cell line model, namely KM12C and KM12SM which are non-metastatic and metastatic cell lines respectively. The influence of cancer associated fibroblast on cancer was studied in two dimensions as well as in three dimensions using different experimental setups. Likewise, the role of the extracellular matrix was investigated in three dimensions. The influence of secreted components by the CAF was studied in a wound healing assay using conditioned media and induced an increment in migration speed for both KM12 cell lines in non-diluted and diluted media. In the co-culture wound healing assays we observed a pulling effect of CAF on KM12 cells. This was not observed with the non-cancerous counterpart BJ-hTERT cells. Likewise, in a three-dimensional model, which was realized by making spheroids, it could be observed that the CAF exert pulling forces on the KM12 cells. In the three-dimensional models it was also found that the spheroids degrade back to a monolayer after several days. Also, migrating individual cells at the outer edge of the spheroids were observed. Fluorescent analysis of different regions of the spheroids (top, bottom and middle region) indicates an increment in all those regions over a period of 72 hours on different surfaces (glass, Matrigel and PIC). However, it seems that KM12SM spheroids increase faster in Matrigel compared to the mixed spheroids (composed of KM12SM and CAF cells) which seems to increase faster on glass.

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It is often said that good communication would solve 90% of the world’s problems. This is no different for every cell in the human body. In order to survive in their dynamic environment, these cells require communication with other cells and with the matrix in which they are embedded. Communication can come in many forms. We often think of how certain chemicals can influence cells, but mechanical stimulations through poking, friction and pulling also have a great effect on cell functions. Inside the cell thousands and thousands of chemical (and mechanical) reactions regulate how a cell behaves itself. Since the cell’s interior is separated from the exterior by the plasma membrane, the cell needs to translate the mechanical signal into another language that can be analyzed by the cell and induce reactions. This process is called mechanotransduction. There are many molecules embedded in the membrane that can act as translators, one of which being mechanosensitive ion channels (MSCs) which gate the passage of small charged molecules (ions) through the membrane. Among these MSCs, Piezo channels gate calcium ions and are solely activated through mechanical stimulation. These calcium ions are one of the most important messengers to regulate processes inside the cell. Moreover, the structure of Piezo channels resembles that of a lever where small changes on one side can be multiplied and elicit bigger ones on the other side. This makes Piezo channels very sensitive to many physical forces such as friction applied by fluids to the membrane in which the channel resides. Since your body is primarily consisting of liquids, Piezo-channels have an important function all over the body. Consequently, when something goes wrong in the careful equilibrium of mechanisms your body is maintaining, e.g. during cancer, it can have a disastrous effect and has been proven that Piezo-channels enhance the spreading of cancer cells throughout the body, making this an interesting target for research. By using fluorescent calcium sensors, we can record how the activity of Piezo-channels and as a consequence, calcium ion concentration changes over time. These sensors can be brought inside the cell and can be monitored through light detection. The mechanical friction (shear stress) is applied to cells in a controlled environment by pushing fluids over a layer of cells within a chamber at a fixed speed. When we compare the gathered light data with systems that are more defined, we can optimize the experiment to make it represent the processes taking place in your body more accurately, which helps us to gather more mechanical details from the calcium sensors in the future.

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Kanker is wereldwijd een belangrijke ziekte, die zorgt voor een aanzienlijke last op de globale gezondheidszorg. Maar liefst negentig procent van alle kanker-gerelateerde overlijdens wordt veroorzaakt door uitzaaiing van de kanker naar een andere plaats in het lichaam, wat metastase genoemd wordt. Wij leggen de focus op dikkedarmkanker, momenteel de derde meest voorkomende en tweede meest dodelijke kanker wereldwijd. Eén van de belangrijkste uitdagingen bij dikkedarmkanker is dat symptomen vaak pas opgemerkt worden in een vergevorderd stadium wanneer de kanker reeds is uitgezaaid, terwijl vroege detectie belangrijk is voor het starten van een behandeling en voorkomen van metastase. Wij bestudeerden daarom eiwitten in de cel die mogelijks betrokken zijn bij metastase in dikkedarmkanker. We focussen op integrine eiwitten, receptoren die op de buitenkant van de cel staan en signalen naar de binnenkant van de cel kunnen doorgeven. Integrines kunnen dus voelen wat er zich buiten de cel bevindt; namelijk andere cellen, en de extracellulaire matrix (ECM), een 3-dimensionaal netwerk van onder andere collageen, fibronectine en vitronectine dat de cel ondersteunt. Niet alleen kunnen cellen daarom reageren op hun omgeving, ook kunnen ze zo door de ECM voortbewegen. Integrines worden niet voor niets de “handen en voeten” van de cel genoemd. Tijdens metastase is de ECM stijver en bewegen cellen sneller, en daarom onderzoeken wij hoe integrines betrokken zijn bij metastase. In het algemeen bestaan er meerdere soorten integrine complexen, elk met hun specifieke eigenschappen, en hier ligt de focus op twee soorten structuren: focale adhesies en reticulaire adhesies. We bekeken 2 componenten van integrine complexen, namelijk vinculine en integrine αVβ5, in een standaard kankermodel (HeLa), alsook in gemetastaseerde en niet-gemetastaseerde dikkedarmkankercellen. We gebruikten hiervoor een gewone fluorescentiemicroscoop (confocaal) en een microscoop die ons meer details kan laten zien (superresolutie). Vervolgens probeerden we een onderscheid te maken tussen focale en reticulaire adhesies op basis van hun samenstelling. Structuren die zowel vinculine als integrine αVβ5 bevatten, werden beschouwd als focale adhesies, terwijl structuren die enkel integrine αVβ5 bevatten, werden beschouwd als reticulaire adhesies. Daarnaast keken we naar de invloed van de extracellulaire omgeving door middel van coatings, bestaande uit verschillende componenten van de ECM, op integrine complexen. We ontdekten dat reticulaire adhesies het best gevisualiseerd konden worden op collageen en vitronectine, en dat deze structuren veel minder te zien waren op een glazen oppervlak of een fibronectine coating. Bovendien probeerden we focale en reticulaire adhesies te onderscheiden door te kijken waar vinculine overlapt met integrine αVβ5, en zo mogelijke verschillen tussen ECM componenten te ontrafelen, maar een nauwkeurige kwantificatie bleek uitdagend. Samengevat zijn deze resultaten een belangrijke eerste stap in het begrijpen van de moleculaire processen die bijdragen aan de uitzaaiing van colorectale kanker. Verder onderzoek is nodig voor de ontwikkeling van nieuwe vroegtijdige detectiemethoden en therapieën om deze ziekte effectief te bestrijden.