| 1. |
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| 2. |
- André, Oscar
(författare)
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Data-driven microscopy: placing high-fidelity data in a population-wide context
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mikroskopi är idag ett fundamentalt verktyg inom forskning, där det tillåter oss att skåda in och utforska våra prover i hög detalj. Mycket utav utvecklingen av nya mikroskopimetoder har strävat efter att öka den detaljnivå vi kan uppnå. Samtidigt har utvecklingen inom hårdvara, med tillgång till bättre och mer kraftfulla instrument, lett till utveckligen av metoder där fokuset är att studera en hel population av celler. Till skillnad från när vi studerar ett fåtal celler i hög detalj, tillåter det oss att sätta perspektiv på det vi ser. Det ger oss en förmåga att säga vad det normala beteendet som man kan förvänta sig är, och vilka celler som sticker ut i en population. Med andra ord, vad som är intressant.Samtidigt finns det ett stort intresse av att veta hur varje individuell cell beter sig. Varje cell är, precis som oss människor, unik. De har olika historia, olika ålder och befinner sig i olika tillstånd. Precis som våra celler i kroppen är unika, är även de cellerna som kan orsaka sjukdom unika. För att förstå varför vissa personer är mer känsliga mot sjukdom, och hur en infektion svarar på våra behandlingar behövs en förståelse och an förmåga att studera celler på individuell nivå, samtidigt som vi bibehåller ett perspektiv utifrån populations-nivå.Denna brist på perspektiv har länge varit ett problem inom mikroskopi. Den vanliga lösningen på detta problem är att vi, som människor, kan tolka en bild och peka på vad det är som är intressant eller inte. Vi är, trots allt, extremt duktiga på att tolka visuell information. Men detta är inte en helt felfri lösning. Som människor kan vi vara relativt okonsekventa, vi tolkar oftast utifrån hur vi vill att datan ser ut. Med andra ord, vi saknar förmågan att vara objektiva i vår metodik för att samla in bilder i hög detalj.Min avhandling har till stor del handlat om att utveckla ett verktyg som tillåter oss att sätta perspektiv på det vi studerar med mikroskopi. Detta har lett till Arbete 1, där vi presenterar en allmän strategi (data-styrd mikroskopi) för hur vi kan arbeta med mikroskopi för att samla in data på en hel population, samtidigt som vi kan samla in data med hög detalj på relevanta fynd i populationen. Vi presenterar även här en teknisk lösning, och utför metoden i tre olika scenarion: ett för att studera en population av celler mer allmänt, ett för att fånga det ögonblick som bakterier infekterar mänskliga celler, och ett där vi studerar och fångar in data på relevanta (från ett populations-kontext) cancerceller och följer dem över tid. Denna metod tillåter oss att samla in data i hög detalj på ett objektivt sätt, och att sätta perspektiv på det vi studerar.I Arbete 2 har vi vidare utvecklat på vår metod, där vi försöker lösa problemet att hitta en och samma cell i flera olika mikroskop. Eftersom vi, genom mikroskopi, jobbar på en så ofantligt liten skala, är det oftast väldigt svårt att orientera sig och hitta rätt inom ett prov. Det är lite som att spela På spåret och gissa vart man är, fast utan alla ledtrådar man får på varje nivå. Eftersom vi har tillgång till data på en hel population, så utgick vi från att det borde finnas samband mellan celler och deras grannar i ett prov som är unika för just dem. Genom att använda sig av dessa unika samband kom vi fram med en lösning där vi snabbt kan kalibrera ett prov på ett nytt mikroskop. Det öppnar dörrarna för oss forskare att återanvända prov, att lättare justera provet med nya markörer (för det vi vill visualisera inom cellerna), och att kunna tolka ett prov med data insamlat från flera system.COVID-19 pandemin var en stor omställning för samhället och vården. Likväl var det en stor omställning för många forskningslabb, där en kapplöpning startade för att så snabbt som möjligt förstå sig på hur viruset fungerar och hur vårt immunförsvar svarar på dess infektion. Det var i detta kontext som mitt tredje arbete utfördes. Genom den erfarenhet jag samlat på mig inom mikroskopi och att analysera bilder på stora dataset, bidrog jag med hjälp för att studera hur framtagna antikroppar kan förhindra bindningen av virus-lika partiklar till celler. Antikroppar är ett protein som immunförsvaret producerar i respons mot en patogen. En bättre förståelse kring hur antikroppar verkar, och vad skillnaden mellan en bra och en dålig antikropp är kan leda till framtagningen av bättre vaccin-program och behandlingar inom sjukvården.I Arbete 4 medverkade jag i ett arbete där bakterien Streptococcus pyogenes var i fokus. S. pyogenes enda värd är människor, och ansvarar för över 600 miljoner infektionsfall per år globalt. På bakteriens yta dominerar ett protein, M-proteinet, ett multi-funktionellt protein som bakterien (bland annat) använder sig för att binda till ytor och förhindra immunförsvarets förmåga att göra sig av med bakterien. I arbetet upptäckte vi att fibronektin binder till bakterien (specifikt M-proteinet) olika mycket beroende på mängden antikroppar som finns i miljön. Fibronektin är ett protein som vi människor producerar, och bidrar (bland annat) till att skapa den miljön som celler befinner sig i. Mängden fibronektin varierar beroende på var i kroppen man kollar. Till exempel, i saliv har du en relativt låg mängd fibronektin jämfört med i blodet. Detta ledde till hypotesen att bakterien är special-anpassad för olika miljöer i dess förmåga att undkomma immunförsvaret. En bättre förståelse kring hur bakterien är anpassad till våra olika miljöer och dess infektionsförlopp kan leda till bättre och mer anpassade behandlingar inom sjukvården.
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| 3. |
- Cavallaro, Sara, 1992-
(författare)
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Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology.
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| 4. |
- Clausson, Carl-Magnus, 1985-
(författare)
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Making Visible the Proximity Between Proteins
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment. In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.
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| 5. |
- Cros, Olivier
(författare)
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Structural properties of the mastoid using image analysis and visualization
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The mastoid, located in the temporal bone, houses an air cell system whose cells have a variation in size that can go far below current conventional clinical CT scanner resolution. Therefore, the mastoid air cell system is only partially represented in a CT scan. Where the conventional clinical CT scanner lacks level of minute details, micro-CT scanning provides an overwhelming amount of ne details. The temporal bone being one of the most complex in the human body, visualization of micro-CT scanning of this boneawakens the curiosity of the experimenter, especially with the correct visualization settings.This thesis first presents a statistical analysis determining the surface area to volume ratio of the mastoid air cell system of human temporal bone, from micro-CT scanning using methods previously applied for conventional clinical CT scans. The study compared current results with previous studies, with successive downsampling the data down to a resolution found in conventional clinical CT scanning. The results from the statistical analysis showed that all the small mastoid air cells, that cannot be detected in conventional clinical CT scans, do heavily contribute to the estimation of the surface area, and in consequence to the estimation of the surface area to volume ratio by a factor of about 2.6. Such a result further strengthens the idea of the mastoid to play an active role in pressure regulation and gas exchange.Discovery of micro-channels through specific use of a non-traditional transfer function was then reported, where a qualitative and a quantitative pre-analysis were performed and reported. To gain more knowledge about these micro-channels, a local structure tensor analysis was applied where structures are described in terms of planar, tubular, or isotropic structures. The results from this structural tensor analysis suggest these microchannels to potentially be part of a more complex framework, which hypothetically would provide a separate blood supply for the mucosa lining the mastoid air cell system.The knowledge gained from analysing the micro-channels as locally providing blood to the mucosa, led to the consideration of how inflammation of the mucosa could impact the pneumatization of the mastoid air cell system. Though very primitive, a 3D shape analysis of the mastoid air cell system was carried out. The mastoid air cell system was first represented in a compact form through a medial axis, from which medial balls could be used. The medial balls, representative of how large the mastoid air cells can be locally, were used in two complementary clustering methods, one based on the size diameter of the medial balls and one based on their location within the mastoid air cell system. From both quantitative and qualitative statistics, it was possible to map the clusters based on pre-defined regions already described in the literature, which opened the door for new hypotheses concerning the effect of mucosal inflammation on the mastoid pneumatization.Last but not least, discovery of other structures, previously unreported in the literature, were also visually observed and briefly discussed in this thesis. Further analysis of these unknown structures is needed.
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| 6. |
- Grannas, Karin, 1983-
(författare)
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Improvements and Applications of in situ Proximity Ligation Assays
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.
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| 7. |
- Gustafsson, Johan, 1976, et al.
(författare)
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Cellular limitation of enzymatic capacity explains glutamine addiction in cancers
- 2022
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Metabolism within the tumor microenvironment, where a complex mixture of different cell types resides in a nutrient-deprived surrounding, is not fully understood due to difficulties in measuring metabolic fluxes and exchange of metabolites between different cell types in vivo. Genome-scale metabolic modeling enables estimation of such exchange fluxes as well as an opportunity to gain insight into the metabolic behavior of individual cell types. Here, we estimated the availability of nutrients and oxygen within the tumor microenvironment using concentration measurements from blood together with a metabolite diffusion model. In addition, we developed an approach to efficiently apply enzyme usage constraints in a comprehensive metabolic model of human cells. The combined modeling reproduced severe hypoxic conditions and the Warburg effect, and we found that limitations in enzymatic capacity contribute to cancer cells’ preferential use of glutamine as a substrate to the citric acid cycle. Furthermore, we investigated the common belief that some stromal cells are exploited by cancer cells to produce metabolites useful for the cancer cells. We identified a total of 233 potential metabolites that could support collaboration between cancer cells and cancer associated fibroblasts, but when limiting to metabolites previously identified to participate in such collaboration, no growth advantage was observed. Our work highlights the importance of enzymatic capacity limitations for cell behaviors and exemplifies the utility of enzyme constrained models for accurate prediction of metabolism in cells and tumor microenvironments.
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| 8. |
- Hahn, Max, 1993-
(författare)
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Characterizing the pancreatic "isletome" : 3D optical imaging to study diabetes
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pancreas is a specialised multipurpose organ, that can be separated into two major compartments: endocrine and exocrine. The exocrine part makes up the majority of the organ volume and functions to secrete digestive enzymes into the small intestine. Notably, endocrine islets of Langerhans are embedded and scattered in vast numbers throughout the exocrine space. These miniature functional units are composed of different cell types that secrete hormones into the blood stream. The most abundant islet-cell is the insulin-producing β-cell. Highly coordinated, the endocrine cells are the primary regulators of energy homeostasis in the body. Together, the collective islet volume constitutes the pancreatic “isletome”, a synchronised, complex and size-equilibrated system that is able to respond to various metabolic conditions. Indeed, environmental and/or genetic conditions often lead to impaired islet function and/or β-cell destruction leading to elevated blood glucose levels over time and eventually diabetes. Diabetes mellitus is a disease that currently affects more than 400 million individuals worldwide. As such, understanding pancreatic disease-related mechanisms is pivotal to the development of new and more effective therapeutic, or even curative, regimens. The deep location of the pancreas in the abdomen and the relatively low resolution of current clinical imaging approaches, however, render the pancreatic islets difficult to study when visually assessing endocrine function. Although non-invasive imaging techniques have yet to reach their full potential, post-mortem studies of the pancreas and rodent disease models offer unique insights into the process of diabetes disease dynamics.Diabetes induced by streptozotocin (STZ) is a widely used model system in pre-clinical research, where it is generally believed that the b-cells are depleted upon the administration of the drug. Yet, quantification of β-cell volume dynamics and underlying disease mechanisms have not been extensively described. Using optical projection tomography (OPT), light sheet fluorescence microscopy (LSFM) and advanced protocols for ex vivo whole organ three-dimensional (3D) imaging, this study demonstrated that STZ-induced β-cell depletion is modest, primarily affecting large islets, and is not the primary cause for the development of diabetes in STZ-diabetic mice. Combined with islet gene expression studies, the remaining β-cell volume in STZ-diabetic mice displayed a downregulation of glucose transporter type 2 (GLUT2), a transmembrane carrier vital for sensing blood glucose levels. Islet transplantation into the anterior chamber of the eye (ACE) reversed the STZ-induced hyperglycaemia and partially restored islet function, including GLUT2, but did not restore β-cell volume loss. Extensive 3D image datasets were generated as a resource to the research community. The combined results of this study indicated that STZ-induced hyperglycaemia is not caused by β-cell loss, but rather by dysfunctional β-cells and that recovery of islet function is restrained by continuous hyperglycaemia.3D imaging using OPT has proven to be a reliable technique in quantifying cellular/anatomical features of the mouse pancreas. However, the technique has rarely been applied to patient-derived tissues. Here, a label-free and non-destructive method was developed to assess clinical biopsies within hours of collection. Specifically, this study showed that autofluorescence-based imaging can be used to delineate tumours of the pancreas (pancreatic ductal adenocarcinoma, PDAC) in 3D, which may aid in identifying tumour margins in conjunction with resective surgery. Importantly, the protocol included a reversal pipeline so that other histological workflows could be applied to the same specimen. Furthermore, this study demonstrated that natural fluorescent substances in the endocrine cells provide sufficient contrast when quantifying both the volume and number of islets of Langerhans in the healthy pancreas. Altogether, the developed technique may provide a novel tool for the rapid 3D analysis of pancreatic biopsies that may complement and improve traditional pathological assessments.With the emergence of islet transplantation networks worldwide, access to fixed pancreatic tissues from diseased donors has dramatically improved. Hereby, the near instant autolysis of the pancreas post-mortem can generally be avoided, which provides the opportunity to quantitatively study the entire gland ex vivo within a conserved spatial context. Yet, mesoscopic 3D imaging of the pancreas (by OPT and/or LSFM) has been limited predominantly due to the obstacle of labelling larger tissue volumes. As such, a simple approach to antibody labelling and cellular imaging was developed in cubic centimetre-sized tissue cuboids that were mapped to the whole organ. By stitching the resultant datasets back into 3D space, this approach demonstrated how essentially any human organ may be analysed in full with high resolution. This technique was applied to pancreata from non-diabetic and type 2 diabetic (T2D) donors, analysing over 200 thousand islets, revealing features of the human pancreas that were not analysed in 3D previously, including high islet dense regions and intra-islet haemorrhaging. Crucially, this new technique may contribute to unveil a wealth of new insights into the complex pathophysiology of the “diabetic pancreas”.By applying the above method to the entire volume of the human pancreas, the absolute distribution and volume of insulin-positive cells in a pancreas from a donor with longstanding type 1 diabetes (T1D) was demonstrated for the first time. By dividing the 19 cm long organ into smaller pieces, followed by insulin labelling, OPT imaging and reconstruction in 3D space, approximately 173,000 insulin-positive objects were identified. By utilising tissue autofluorescence, the entire organ was reconstructed in 3D, together with blood vessels and ducts. These data indicated several important regional differences in β-cell mass, such as the uncinate process showing the highest density, which potentially reflects key aspects of disease dynamics. Furthermore, regions with a “punctated distribution” of single β-cells in close proximity to each other were identified. Although the significance of these observations needs to be elucidated, we speculate that these regions could be associated with pancreatic regeneration, which might permit the development of new interventions for clinical regenerative processes in the future. Altogether, this study represents the first whole organ account of β-cell distribution at the current level of resolution in an entire organ. As such, it may serve as an important advancement towards detailed whole organ analyses of endocrine cell identity/function, via a wide range of markers, in the study of normal anatomy and pathophysiology of the human pancreas.
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| 9. |
- Langer, Krzysztof, et al.
(författare)
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Rapid production and recovery of cell spheroids by automated droplet microfluidics
- 2019
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.
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| 10. |
- Senkowski, Wojciech
(författare)
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High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS.In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines.In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.
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