Fysik // Physics

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Explores and uses physics to understand the world, and provide applications, new materials and innovations that meet today's needs, and the challenges of the future.
Stimulated by major needs and challenges in society and industry, our ambition is to foster a creative environment for academic research, learning, innovations and utilisation. We provide a competitive advantage by linking top-level international and interdisciplinary academic performance in the areas of material science, nanotechnology, life science engineering and energy research with world-leading industrial R&D&I projects.

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Antimicrobial Effect on Bacteria in Biofilm and Planktonic state
(2018) Linse, Julia; Larsson, Anna; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
The elimination of contaminating bacteria is essential in order to achieve proper wound healing. Bacteria can occur in two different states, as free-living (planktonic) cells or in aggregates adhered to a surface (biofilm). The bacteria behaves differently depending on in which state they occur and their differences and their sensitivity against Chlorhexidine digluconate have been of key interest during this thesis work. To investigate this, time-kill studies have been performed on the two common wound bacteria P. aeruginosa and S. aureus under static and dynamic conditions. The studies were performed on bacteria in both biofilm and in planktonic state. The minimum inhibitory concentration (MIC), the minimum biocidal concentration (MBC) and the minimum biofilm eradication concentration (MBEC) for the antimicrobial compound Chlorhexidine digluconate against the bacteria were used in time-kill studies under static and dynamic conditions. In the static time-kill studies the bacterial were treated with different concentrations of Chlorhexidine digluconate in the range from below the MIC to above the MBC. In the dynamic time-kill studies Chlorhexidine digluconate was added continuously to the bacteria to reach the determined MBC in different times. Results from the experiments showed that P. aeruginosa was less sensitive against treatment of Chlorhexidine digluconate than S. aureus was. There seemed to be persister cells within the P. aeruginosa population during all experiments since they managed to recover from antimicrobial treatment even if the bacterial concentration at a point was below the limit of detection. The experiments also showed that killing of P. aeruginosa seemed to be Cmax-driven, which means that a high concentration Chlorhexidine was needed to be delivered fast to the bacteria to achieve the best rate of killing. For S. aureus the killing seemed to be AUC-driven which means that a lower amount of the antimicrobial could be used but the bacteria needed to be exposed for a longer time. When the two bacterial species were in biofilm, both were less sensitive to Chlorhexidine digluconate and a higher concentration was needed in order to achieve the same rate of killing as for bacteria in planktonic state.
De- and Recellularization of Kidneys Humanized 3D disease models for the pharmaceutical industry
(2018) Kullberg, Linn; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
Detection and quantification of COMP neoepitope in equine saliva: A biomarker for detection of early stages of Osteoarthritis
(2021) Lord, Moa; Chalmers tekniska högskola / Institutionen för fysik; Karlsteen, Magnus; Nyström, Susanne; Skiöldebrand, Eva
Osteoarthritis (OA) is a chronic in ammatory joint disease, with painful destructive processes locally in the joint. It is not possible to diagnose the early destructive biochemical events within the joint that leads to pain and subsequent lameness. Many horses with local joint pain due to early OA with a low-grade in ammation are used today in the equestrian industry. This disease develops over time and is usually not diagnosed until the end stages, where irreversible damage and clinical symptoms can be noticed. A biomarker for early matrix degradation of articular cartilage is needed as a diagnostic tool for early diagnosis of OA. A speci c neoepitope of cartilage oligomeric matrix protein (COMP) have previously been identi ed and shown to increase in synovial uid and serum from horses with acute lameness. The aim of this thesis was to detect and quantify the speci c COMP neoepitope in saliva sampled from healthy horses and horses with OA. This aim included evaluation of sample collection and preparation, sample collection throughout the day and validation of the capillary western blot Wes™by inhibition ELISA. Salivette®, a speci c collection device developed for salivary collection, was used to collect the saliva. Saliva sampled on the horse tongue, no food intake within one hour, obtained lower background noise, showed higher chemiluminescence signal and more de ned peaks compared to saliva sampled close to food intake, when analysed with Wes™. In Wes™, the COMP neoepitope could be detected at the apparent molecular weight of 58 kDa. The concentration of the COMP neoepitope was determined with inhibition ELISA developed for quanti cation of COMP neoepitope in synovial uid and serum from horses. The highest concentration of COMP neoepitope was quanti ed in a horse recently diagnosed with OA, compared to saliva from healthy horses. This indicates that saliva from horses can be used for detection and quanti cation of COMP neoepitope. However, this thesis included a limited amount of horses and they were not clinically examined by a veterinarian prior to saliva sampling. A larger group of well-de ned healthy horses and horses with OA is required to draw statistical conclusions. Quanti cation of COMP neoepitope in saliva is a step toward the future goal of developing a point of care diagnostics, which can be an accessible tool for diagnosis of OA, monitoring the horse during rehabilitation and also be used in everyday training of athletic horses.
Development and characterization of human liver spheroid cultures for drug transporter studies and microphysiological systems
(2017) Johansson, Linnea; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
A current problem in the drug development process today is the lack of predictable in vitro liver models for pre-clinical studies. Current in vitro methods and animal trials often fail to predict the effect of potential drugs in humans. In addition, drug-drug interactions which could lead to drug induced liver injury is often a reason for drugs not reaching the market, and it can be related to changes in efflux of metabolites from hepatocytes in liver. In this study, a two-organ-chip has been developed with aggregates formed of HepaRG cells co-cultured with stellate cells as well as pancreatic islets, and evaluated as an in vitro model. The focus of the present master thesis project was to evaluate functionality of liver aggregates used in the two-organ-chip system. As a comparison, liver aggregates from chip-system with liver aggregates only has been evaluated. The results showed a significant increase in albumin production from liver aggregates that were in the two-organ-chip system together with pancreatic islets. The liver aggregates showed no significant difference of glycogen production when cultured with or without pancreatic islets. As a conclusion, the chip-system with two organs used showed increased liver specific functions in liver aggregates. Another part of the study included evaluation of activity of the canalicular transporter multidrug resistance protein 2 (MRP2) in hepatic spheroids formed of primary human hepatocytes or hepatocytes co-cultured with stellate cells. The activity was evaluated using a substrate, 5-chloromethylfluorescein diacetate (CMFDA), which is converted to a fluorescent substrate inside cells and transported by MRP2 into bile canaliculi of spheroids. When using medium lacking of calcium it was possible to measure the amount of fluorescent substrate that was transported into bile canaliculi. The result indicated that MRP2 was active in spheroids, but the method used needs further development to reduce the large variation and the challenging experimental procedure.
Development and evaluation of an in vitro cardiovascular model for determining impact of genetic variability in calcium handling on drug-induced cardiotoxicity
(2019) Kha, Michelle; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
Cardiotoxicity is evaluated throughout the process of drug discovery and development and is a leading cause of drug attrition during clinical trials. One of the major challenges includes the understanding of differential pharmacology responses in healthy and disease states. This can be improved by the development of human in vitro models and consideration of genetic variability in patients. In this thesis, a human induced pluripotent stem cell (hiPSC) line was developed with a heterozygous single nucleotide polymorphism (SNP) rs3766871 (G1886S) within a calcium handling gene, RYR2. This SNP is the second most common heterozygous SNP within RYR2 and has been associated with cardiac diseases. The SNP line was obtained by reverse transfection of hiPSCs using CRISPR/Cas9 and was validated by Sanger sequencing. Furthermore, an existing hiPSC line with a heterozygous knock-out within RYR2 was evaluated in this thesis. The heterozygous knock-out hiPSC line mimics the partial reduction of RYR2 expression observed in aging and heart disease. This line was differentiated into cardiomyocytes and characterized by FACS, ICC and Western blotting. In addition, impedance-based measurements were conducted as a surrogate of contractility to obtain functional data of amplitude and beat rate of the cardiomyocytes. Differentiation of these hiPSC lines into cardiomyocytes, followed by drug treatment, will allow the hiPSC-derived cardiomyocytes to act as in vitro cardiovascular safety models of genetic variability. This can help understanding of the impact of genetic variability and its role in drug-induced cardiotoxicity.
Development of a Hepatocyte Spheroid Culture Model for Drug Uptake, Metabolism and Transporter Studies
(2015) Chouhan, Bhavik; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
The liver is a key organ in drug bioavailability including uptake, metabolism, and excretion as well as drug toxicity. Thus, a robust liver in vitro model that resembles in vivo micro environment with improved predictive capabilities is highly warranted. The focus in this thesis has been to develop and characterize a 3D liver spheroid model for the study of long-term drug uptake, metabolism, and transport. The bipotent progenitor cell line HepaRG has been used as it is known to express various liver specific markers and function in its differentiated state similar to primary human hepatocytes. Culturing these cells onto ultra-low attachment plates derived spheroid formation after 3 days independent of cellular concentration. Various staining techniques were used to investigate long-term sustainability of 2000 and 4000 cell spheroid culture. The gene expression analysis of the drug metabolizing cytochrome P450 (CYP) enzymes illustrated higher expression level in spheroid formation compared to 2D control while drug transporter, MRP2, hepatocyte markers Ki67, albumin, and CK19 all showed moderate to low expression. The HepaRG spheroid model showed promising results. The enzyme activity of CYP2C9, CYP2D6, and CYP3A4 was measurable throughout the whole cultivation period with a peak activity at day 7 while CYP1A2 showed no activity.
Development of a Non-enzymatic, Non-invasive Sensor for Detection of Lactate in Sweat
(2018) ljungh, annie; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
An athlete’s goal is to perform the best way possible and in many cases the level of lactate in blood is an indication of performance possibilities. Blood lactate measurement is conducted to obtain knowledge of the lactate threshold (LT) which is the intensity at which blood lactate starts to increase. Lactate can also be found in sweat and some studies have reported a correlation between lactate in sweat and blood, implying for the possible usage of sweat instead for these types of measurements. Using sweat result in non-invasive measurement, both suitable for the discomfort of the athlete tested and to not affect the training while testing. Most non-invasive sweat sensor are based on degrading of lactate with enzymes and detection with amperometric sensors. For endurance sport, such as marathon’s, an enzyme-based sensor can’t measure for the total performance time without losing effect. The aim of the thesis was to produce a sensory device for evaluating the lactate concentration in sweat. Molecular imprinted polymers (MIP) made of overoxidized polypyrrole (oPPy) using ammonium persulfate ((NH4)2S2O8) as an oxidative agent was successfully drop coated onto screen printed carbon electrodes (SPCE) modified with gold nanoparticles (AuNP). By evaluating the peak current observed by cyclic voltammogram between -0.5 V to -1.5 V at a scan rate 0.05 V/s a decreasing peak could be observed at -1 V to -1.5 V with a higher lactate concentration. By data analysis both with modified and unmodified SPCE-AuNP, showed a linear relationship between lactate concentration and peak current. The electrodes were used to evaluate the lactate level in a real sweat sample. The unmodified electrodes overestimated the lactate level with approximately 10 percent while the modified underestimated by approximately 30 percent.
Development of proximal tubule-on-a-chip assays for predicting kidney toxicity
(2024) Johansson, Emma; Chalmers tekniska högskola / Institutionen för fysik; Chalmers University of Technology / Department of Physics; Adiels, Caroline
Differentiation and characterization of MafA-GFP reporter iPS cells into beta cell lineage
(2020) Avijgan, Mahtab; Chalmers tekniska högskola / Institutionen för fysik; Gold, Julie; Gupta, Shailesh
Diabetes is a chronic disease characterized by increased blood sugar because of dysfunctional glucose homeostasis by the beta cells in the pancreas. Functional beta cells can be generated by primary cells or stem cells. Primary cells are isolated directly from tissues (blood or bone marrow) and retain the morphological and functional characteristics of their tissue of origin. However, they have a finite period of cell culture, a limited potential for self-renewal and differentiation and are more sensitive than stem cells, they often require additional nutrients and growth factors. In contrast, stem cells allow to investigate basic biological processes, manipulate cellular functions, establish new methods, or perform preliminary screenings. Considering the limitations of the primary cells, stem cells can be an alternative source. Stem cells are at the forefront of research in cell therapy, drug discovery, and disease-modelling. Generation of pancreatic beta cells is possible by differentiating human induced pluripotent stem cells (hiPS cells), although in stem cell research, efficiency of mature beta cells generation is a key problem (optimal efficiency at 80%, current efficiency at 20%). This project first aims to improve efficiency of pancreatic beta cell generation from hiPS cells by performing six differentiation processes following the same protocol and later screening for beta cell production at different stages of the differentiation process. It uses a wild type and two MafA-GFP reporter iPS cell lines, where MafA as a critical beta-cell-specific transcription factor is tagged with GFP by using CRISPR/Cas9 technology. Second, flow cytometry and immunofluorescence analysis are used at different stages of the differentiation process to characterize the differentiated cells to confirm faithful expression of MafA. Expression of GFP corresponds to expression of MafA in adult beta cells, since MafA is only present in mature beta cells, which this confirms complete differentiation and maturation of iPS cells into beta cells. The results obtained from the differentiation processes showed low level of reproducibility, although this project was successful in showing the GFP and MafA expression of MafA reporter lines. The MafA reporter lines successfully expressed GFP signals (~11% efficiency) at stage 7 of beta cell differentiation.
Differentiation of human pluripotent stem cell-derived mesenchymal progenitors into osteogenic, chondrogenic and adipogenic lineages
(2012) Bergman, Alexandra; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Abstract Differentiated cell types are of great interest in applications such as tissue engineering and drug screening. As harvesting of fully differentiated, adult cells neither is a safe nor scalable alternative, in vitro differentiated cells have become an attractive option. hES-MP002.5 (mesenchymal progenitors) is a cell type established by and at Cellectis Stem Cells from human embryonic stem cells (hESCs), that carries adult mesenchymal stem cell like characteristics. An examination of hES-MP002.5’s mesenchymal differentiation potential was performed in this project, in particular into the osteogenic, chondrogenic and adipogenic lineage. A second objective was to establish a mesenchymal progenitor cell line from a human induced pluripotent stem cell line (ChiPSC4) and compare its characteristics to those of hES-MP002.5. Results were obtained primarily through the use of differentiation in cell culture, histological stains, immunocytochemistry and quantitative PCR. An osteogenic differentiation study concluded that hES-MP002.5 show vast osteogenic potential when a previously established protocol was used. However, hES-MP002.5 showed near to no responsiveness to variations of commonly used chondrogenic or adipogenic treatments in terms of visible chondrogenic extracellular matrix deposition or intracellular lipid accumulation, respectively. Yet, hypoxic culture conditions ought to be considered if chondrogenic differentiation is attempted in future studies. Gene expression studies of adipogenically induced cells implied that a gene essential for adipogenic differentiation was down- regulated for both MP-lines; a fact that could help to explain the low differential response. With an established protocol for hESCs, a cell population resembling hES-MP002.5 was developed from ChiPSC4 (ChiPSC4-MP), but the cell population seemed less homogenous and had lower proliferative potential. hES-MP002.5 and ChiPSC4-MP seemed to respond similarly to mesenchymal differentiation inductions – however ChiPSC4-MP consistently showed a weaker response. This might be due to heterogeneity in the cellular population obtained. With the broad laboratory investigations in mind, it is not recommended that hES-MP002.5 is marketed as a mesenchymal progenitor with the ability of differentiating into chondrogenic and adipogenic lineages. A recommendation is that the protocol for mesenchymal progenitor establishment is reviewed and further developed, since the MP-cells established from both embryonic and induced pluripotent stem cells share the same strengths and weaknesses.
Effect of nanotopography on bacterial adhesion and EPS production.
(2011) Najafinobar, Neda; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Bacterial infection after surgeries is a serious problem that cannot be treated by traditional antibiotics because of antimicrobial resistance and biofilm formation. Recently, a new coating (Bactiguard coating) has been developed that is able to reduce the infection significantly without compromising biocompatibility. This coating is comprised of nanosized deposits of Ag, Pd, and Au. It is not clear how this coating reduces infection. Therefore, this study aims to investigate how bacterial adhesion and production of extra cellular substances (EPS) are affected by surface nanotopography. However, the exact mechanism of action is not yet fully understood. The studied topographies are a flat and nanostructured gold surfaces. The fabrication method used was self-assembly with 20 to 30 nm gold nanoparticles. For the cell study staphylococcus epidermidis, was used. Bacterial cell adhesion and the formation of EPS were investigated. FilmTracer TM SYPRO Ruby biofilm matrix stain was used in order to stain EPS. In order to characterize the surfaces contact angle and scanning electron microscopy (SEM) were used. Dynamic light scattering and spectrophotometry was applied to analyze the size of the particles. SEM analyzed the number of cells attached on both surfaces. No ifference was observed on the number of cells attached on both surfaces. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) was used to study the adhesion of bacteria and the production of EPS in real time. The results show that dissipation is lower on nanostructured surface that can be due to firmly attachment of bacterial cells on the surface and more production of EPS. Therefore, bacteria seem to behave differently when adsorbing on a flat or nanostructured surface.
Effects of different pH and oxygen levels on proliferation and chondrogenic differentiation of human mesenchymal stem cells cultured in hydrogels
(2014) SINGH, SUKHDEEP; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Low back pain is a very common health problem and 70-85% of the world’s population suffers from it at some point in their life. One possible cause for this pain is intervertebral disc (IVD) degeneration. Current treatments for IVD degeneration include surgical treatments which are very invasive. Thus there is a need for a strategy which is minimally invasive and for this cell therapy could be a solution. The IVD degeneration often starts in the center of the IVD, in the nucleus pulposus (NP), where chondrocyte-like cells reside. As human mesenchymal stem cells (hMSC) can undergo chondrogenic differentiation, these cells are a good candidate for the use in cell therapy to treat IVD degeneration. Transplantation of these cells together with a hydrogel into a degenerated IVD is thought to arrest the degeneration as hMSC could undergo chondrogenic differention while the hydrogel acts as a temporary matrix. The environment inside a degenerated IVD is hypoxic and acidic and thus the effects of these factors on the hMSC are important to study. The aim of this study was to evaluate the effects of different pH and oxygen levels on the proliferation and chondrogenic differentiation of human bone marrow-derived MSC cultured in the hydrogels HydroMatrix™ and PuraMatrix™. In the oxygen experiment, cells were cultured in the hydrogels for 14 days at either 10% O2 or 21% O2 (standard cell culturing condition). In the pH experiment, cells were cultured in a hydrogel for 6 days either at 10% O2 or 21% O2 and the pH levels tested were pH 7.4 (standard cell culturing condition), 7.1, 6.8 and 6.5 representing pH levels in healthy and degenerated IVDs. FACS was used to characterize the hMSC population and it could be concluded that the vast majority of the cells were in fact MSC. The results from the oxygen experiment showed gene and protein expressions of SOX9 and gene expression of collagen IIA1 in MSC cultured in HydroMatrix™ at both oxygen levels indicating chondrogenic differentiation. In addition, sulfated glycosaminoglycans were present in samples of hMSC cultured in HydroMatrix™ at 10% O2 indicating that a decreased oxygen level of 10% O2 could induce chondrogenic differentiation of hMSC. Cells were also shown to proliferate in HydroMatrix™ at both oxygen levels as cells positive for PCNA were observed in IHC studies. In the PuraMatrix™ samples, SOX9 and collagen IIA1 gene expressions were observed in some samples showing indications of chondrogenic differentiation. Deviating trends of HIF-1α gene expression were observed which suggested that 10% O2 might not be low enough to induce the HIF-1α gene. In the pH experiment, the results showed that the presence of viable hMSC decreased with decreasing pH, indicating that a decreased pH affects the survival of hMSC by resulting in a lower cell viability. However, some viable cells could be observed when cultured in HydroMatrix™ at pH 6.8 and 10% O2. This indicated that some hMSC could survive in the pH found in mildly degenerated IVDs at a decreased oxygen level of 10% O2. When comparing the results of culturing hMSC at different pH at 10% O2 with 21% O2, no clear difference in the presence of viable or dead cells could be observed however, quantification was not performed. Thus it cannot be said whether a combination of decreased pH and an oxygen level of 10% has a stronger negative effect on the survival of hMSC than a decreased pH alone.
Effects on Chondrocyte Derived Induced Pluripotent Stem Cells Adhered to Nano Gradients Functionalized with Transforming Growth Factor Beta-1, -3 and Growth Differentiation Factor 5
(2018) Andreasson, Linnea; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
Osteoarthritis (OA) is the most common joint disease, causing pain for the patient due to cartilage degeneration and a lot of costs to the society. Due to lack of knowledge regarding disease mechanisms, there is no existing drug based modifying therapy. In this project, the impact of TGFβ-1, TGFβ-3 and GDF5 signaling on the expression of aggrecan in chondrocytederived iPSCs, were elucidated by using a concentration gradient to gain information regarding OA disease mechanisms. The overall aim of the project was to use gold nanoparticle gradients and surfaces to optimize formation of cartilage producing cells from iPSCs where the focus was to induce a high aggrecan expression in the cells. Also, cells were stimulated on gradients and surfaces and then removed from the surfaces and formed to a 3D structure for further differentiation and visualization of the components. Particle gradients were produced by attaching gold nanoparticles to a glass surface with different spacing between particles and the gradients were then functionalized with signaling molecules and seeded with c-iPS cells, differentiated for 5 days and thereafter visualized using fluorescence imaging. Due to the protein concentration gradients, cells were stimulated differently over the gradient and by visualization of the cell behavior at the gradient, an area of interest was observed and new uniform surfaces were produced. No specific aggrecan expression was observed on surfaces differentiated for 5 days, especially not for GDF5 and TGFβ-1. TGFβ-3 indicated an elevated expression at high concentrations. Despite this result, 5 days of differentiation was found to be insufficient time and this result was also confirmed by the study were iPSCs were further differentiated for 6 weeks after removal from surfaces where there was a significant elevation of the aggrecan expression after 3 weeks of differentiation and it is suggested that the time should be prolonged in order to yield high aggrecan expressions. Even though the aim of the project to stimulate the cells to express a high expression of aggrecan was not fully fulfilled, other reactions of interest were observed on the surfaces. GDF5 stimulate cells tended to form a condensed structure where cells clustered together. In low concentrations of TGFβ-1, the effect in cells was indicated to be knocked out by laminin and higher concentrations were indicated to be sufficient for a cellular response of TGFβ-1. For cells on TGFβ-3 surfaces, aggrecan expression was indicated to be elevated in high TGFβ-3 concentrations. It can be concluded that the nanoparticle coated surfaces are effective substrates to use to study the effect of signaling molecules on cells and to optimize the differentiation process in stem cells.
Evaluating and Optimizing a Protocol for Mesodermal Differentiation of Pluripotent Stem Cells towards Cardiomyocytes and Adipocytes
(2013) Lundin, Anders; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
The fat epidemic is an increasing issue in today’s society, and with it follows obesity-related diseases, such as type 2 diabetes and coronary heart disease. Many of today’s drug candidates are withdrawn from the market due to negative cardiac effect, which result in huge setbacks for drug companies if detected at late stage trials Therefore, there is a necessity of appropriate in vitro models, which can detect these toxic effects at early stages in drug development. Consequently, there is an increasing demand in providing cells with high biological relevance. A large portion of the study models used today are performed with in vitro cells lines of cancerous origin, and often from species other than human. In addition, usage of different in vivo models also add to the complexity with the matter of accurate translability. This issue can be addressed by using primary cells. However, this raises the problem of accessibility, which can almost be impracticable when it comes to accessing and isolating cells from neuronal or heart tissue. Fatty tissue is more accessible, but the amount of cells that can be isolated from a subject are not sufficient to run large drug screens. Using cells from different subjects can be a solution to the problem of poor accessibility, but creates a large complexity of donor to donor variations. Another model, using pluripotent stem cells (PSC), provides beneficial properties of indefinite growth with sustained pluripotency, an immense source of cells, and the theoretical possibility of differentiation into any cell type in the body. In addition, the cells can be of human origin, providing a good biological relevance. The discovery and development of the induced pluripotent stem cell (iPSC) technique, meaning the possibility of creating a PSC from a somatic cell, also circumvented the major ethical hurdles of isolating embryonic stem cells. To be able to take advantage of the beneficial properties of the iPSC technology there is a need for establishing robust differentiation protocols, which can transform iPSCs into mature functional somatic cells. This thesis focused on evaluating and optimizing differentiation protocols to derived cardiomyocytes and adipocytes from iPSCs. A robust protocol for providing stem cell derived adipocytes was not achieved. Evaluation of the differentiation process mainly focused on functional assays, such as; glucose uptake of radioactive isotopes, lipolysis and western blot. These assays did not supply data supporting derivation of functional adipocytes. The observed poor functionality was due to the low percentage of derived adipocytes, a feature correlating with recent publications. Differentiation of cardiomyocytes was achieved, both through embryo body formation and by direct differentiation in a monolayer format. Characterization of the cardiomyocytes was conducted by immunocytochemistry (ICC), fluorescent activated cell sorting (FACS), quantitative PCR and high content imaging (ImageXpress). Moreover, the cardiomyocytes exhibited the characteristics of spontaneous contractions and expressed the typical cardiac markers, as Nkx2.5 and cTnT. Finally, the outcome of the cardiomyocyte differentiation protocol show promising results for future application and development as an in vitro model for drug screening.
Evaluation of protocols for derivation of cardiac progenitors from human induced pluripotent stem cells
(2015) Pernevik, Elin; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Myocardial infarction and heart failure are major causes of death worldwide. The adult heart has limited potential for repair and regeneration of cardiomyocytes damaged by these pathologies and existing drugs and mechanical devices do not always provide long-term solutions. This creates a demand for advanced in vitro models of human cardiac cells that can be used in the search for long term treatments of heart damage. Existing systems commonly used for research include in vitro models using cancerous cell lines, sometimes of non-human origin, and in vivo murine models, raising questions about the translatability of these systems for human cardiac treatments. Ideally human cardiac cells would be used; however, obtaining primary human cells in sufficient numbers for research is very difficult. One solution is use of human induced pluripotent stem cells (iPSC) that theoretically have the potential to differentiate into any cell type of the body and can be expanded to sufficient numbers. Recently cardiac progenitor cells (CPC) were discovered to be present in adult mammalian cardiac tissue at low ratios (Beltrami et al., 2003). CPC have the potential, following specific stimulation, to proliferate and become new cardiomyocytes; therefore providing a novel strategy for the treatment of heart damage. The aim of this project was to evaluate protocols for derivation of CPC from iPSC as this would provide an in vitro model for drug discovery aiming towards cardiac regeneration by small molecule stimulation of CPC already present in the adult heart. Several protocols have already been published for differentiation of stem cells to cardiomyocytes, and some were evaluated for their ability to derive functional CPC from iPSC in comparison to commercial iCell® CPC. The CPC populations during differentiation were characterized with immunocytochemistry (ICC) and real-time quantitative PCR (qPCR) and expression of relevant cardiac progenitor markers PDGFRα, KDR, Isl1, Nkx2.5 among others was detected for the selected Protocol B CPC and cells derived with a commercial CVP medium. Both these CPC populations are bipotent towards the cardiomyocyte and smooth muscle lineages, but an improved endothelial differentiation is required to prove potential multipotency. The Protocol B progenitors also displayed proliferative capacity confirming their progenitor state. There is a need for optimization of robustness and preservation in the progenitor stage, and further comparison to in vivo derived cardiac cells for evaluation of relevance, but these CPC populations show promise for future use as in vitro models of CPC for drug discovery.
High Content Screening of Cellular Blebbing to Predict Sensitizing Potential of Contact Allergens
(2014) Wang, Yuanmo; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Contact allergy is one of the most prevalent forms of immunotoxicity found in the Western world. Every day, we are exposed to a plenitude of chemicals. In order to replace animal testing which today is used for screening of skin sensitization, it would be great to develop a potential alternative in vitro tool. This thesis project was trying to develop an alternative, non-animal method to detect and predict contact allergens. It combined cellular cultivation with high content screening microscopy to monitor blebbing of keratinocytes (i.e. HEKn) exposed to chemicals in vitro. HEKn cells were seeded in 96-well plates and exposed to four sensitizing chemicals (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, 1-chloro-2, 4-dinitrobenzene,1, 2-Benzisothiazol-3(2H)-one and benzyl benzoate) at five different concentrations (i.e. 0.05 mM, 0.25 mM, 0.5mM, 1 mM and 2 mM) for 24 hours. This whole process was monitored using a high concent screening microscope. It was found that HEKn cells began bleb after exposure to chemicals with moderate or higher sensitizing potency. The strongest sensitizer caused the largest bleb-cell ratio. The order of sensitizing potency matched the order of bleb-cell ratio among these four chemicals. In addition, cell viability was investigated. It can be concluded that monitoring bleb formation from HEKn cells exposed to chemicals might be a potential alternative method to evaluate the sensitizing potency of chemicals.
Human liver spheroid cultures in microfluidic chip co-cultures and comparative MetID studies
(2017) Asserlind, Johanna; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
The liver is the most important site of drug metabolism in the human body. During drug development, it is therefore of high importance to employ a robust in vitro model that resembles the in vivo microenvironment, and is able to accurately predict the metabolism and disposition of compounds. This thesis is divided into two major parts. First, the metabolism of a set of compounds was characterized qualitatively in spheroids made from primary human hepatocytes (PHH), and the resulting data was compared to previous data from hepatocyte suspension culture and human in vivo data. The metabolites formed during a 72 hour incubation period was analyzed by LC-MS. Second, a co-culture between human liver spheroids, made from HepaRG cells and primary hepatic stellate cells, and primary human pancreatic islets in microfluidic 2-organ-chips was established, and the liver spheroids were functionally characterized. A total of 5 chip experiments were performed, where each chip experiment lasted for 7 days, and different conditions such as medium composition where examined. Various staining techniques and measurements of secreted albumin and LDH were used to assess long-term sustainability, function and viability of the spheroids. Some of the metabolites seen in humans were also formed in PHH spheroids, but the spheroid model was not able to fully predict the human in vivo metabolism. Spheroids were not shown to be a significantly better model to use compared to suspension cultures during these experiments. CYP activity analysis showed that decreasing metabolic function after a change in medium composition might be a factor. HepaRG spheroids were able to display several liver-like functions when cultured in the multi-organ chips, but the large size of the spheroids led to the formation of necrotic cores. Overall, while certain parameters need to be improved, the liver spheroids are promising models for studying several different aspects of liver functions as well as for establishing organ system models.
In vitro culture of human monocytederived macrophages with regards to M1/M2 polarization
(2016) Gutman, Julia; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)
Wound healing is a complex process where macrophages are highly involved, both by clearing the tissue from bacteria and debris, but also by regulating the inflammation and healing outcome by production of various cell mediators. A characteristic feature of macrophages is their ability to display a spectrum of different phenotypes. They can roughly be divided into two extreme phenotypes; M1 macrophages which produce pro-inflammatory cytokines and metalloproteinases and is strongly antimicrobial and M2 macrophages which produce anti-inflammatory cytokines, resolving inflammation and promoting tissue repair. Although both phenotypes are key in wound healing, an imbalance of M1 macrophage domination can result in nonhealing ulcers. At Mölnlycke Health Care effort is being paid at developing wound care products that could enhance the healing. In order to develop such concepts, an in vitro inflammatory model with M1/M2 macrophages could be utilized. The aim of the thesis is to identify and study the relation of cell mediators and markers related to M1/M2 polarization in cultured human monocyte-derived macrophages and also to create a theoretical link of relevance of chosen markers to data in murine models and the human diabetic ulcer. Monocytes were isolated from buffy coats, obtained from healthy blood donors. To induce M1 and M2 macrophages, cells were stimulated with LPS and IFN- (M1) or IL-4 (M2) for 6, 24, 48 or 72 hours prior to analysis. M1 cells where further reversibly polarized with IL-4 after 48 hours in order to switch phenotype from M1 to M2 cells. Secreted markers of M1/M2 macrophages were studied with ELISA. Production of TNF- and IL-1 indicated a M1 phenotype and CCL18 production indicated a M2 phenotype. Production of IL-10 which is a M2 phenotype marker was produced by M1-stimulated macrophages. No production of IL-23 and IL-1ra was seen. Further, no significant CCL18 production was seen after reversible polarization, indicating that M1 macrophages did not switch phenotype to M2 macrophages. Different assay setups of lactate and reactive oxygen species production (M1 markers) did not display any significant results in this study.
In vitro studies of hydrogels as cell carriers for human mesenchymal stem cells intended for transplantation into degenerated intervertebral discs
(2014) Hansson, Axelia; Wenger, Anna; Chalmers tekniska högskola / Institutionen för teknisk fysik; Chalmers University of Technology / Department of Applied Physics
Low back pain is a major public health issue in the western world, which afflicts 70-85% of all people at some point in their life, and the prevalence of low back pain is reported to be as high as 84%. One main cause of low back pain is believed to be degeneration of the intervertebral discs in the spine. Disc degeneration is a combination of increased cell death, presence of matrix degrading enzymes, lack of nutrient supply, loss of gel-like structure in the nucleus pulposus (located in the center of the disc), leading to tissue dehydration, reduced disc height and fissure formation. Current treatments for low back pain include physiotherapy and surgery, but they are not very effective and the surgical process of fusing vertebrae is very invasive. New treatments for halting or diminishing disc degeneration are therefore needed and transplantation of cells, so called cell therapy, has been suggested. Hydrogels as a cell carrier and the incorporation of growth factors have been proposed to improve the efficacy of reducing the disc degeneration. The idea is that the transplanted cells will repopulate the disc, differentiate into chondrocyte-like cells, which are similar to nucleus pulposus cells naturally present in the disc, and produce extracellular matrix. This will restore the gel-like properties of the nucleus pulposus and thus reverse the degeneration of the disc. This thesis work examined two hydrogels, Puramatrix and Hydromatrix, in vitro as cell carriers for human mesenchymal stem cells intended for transplantation into degenerated intervertebral discs. Mesenchymal stem cells and degenerated disc cells from three patients were seeded in the hydrogels and in pellet cultures (control system) and analyzed at day 7, 14 and 28. The properties of mesenchymal stem cells in the hydrogels were studied regarding their ability to attach as well as proliferate in the hydrogel, produce extracellular matrix and differentiate into chondrocyte-like cells. The methods used to examine these properties were mainly immunohistochemistry, RT-PCR and TUNEL assay, but methods including FACS, SEM and rheology measurements were also employed. The results showed that more than 90% of the cells cultured in Puramatrix and Hydromatrix were viable for at least 28 days (end point of experiment). Both mesenchymal stem cells and disc cells were shown with immunohistochemistry to express integrin β1 and attach to Puramatrix and Hydromatrix respectively. Both cell types were also observed to proliferate in Hydromatrix as seen by immunohistochemistry staining of PCNA. The growth hormone Genotropin increased the expression of PCNA by mesenchymal stem cells from all patients and might therefore increase the proliferation, but further studies are needed to verify this limited setup. Differentiation was not observed in this study for mesenchymal stem cells (from all patients) cultured in Puramatrix and in pellet culture, for the two oldest patients. Mesenchymal stem cells from all three patients differentiated into chondrocyte-like cells in Hydromatrix and in pellet culture for the youngest patient and produced extracellular matrix similar to cartilage. This was verified by expression on gene level and protein level by the chondrogenic markers Sox-9, collagen type II and the proteoglycan aggrecan. Chondrogenesis in Hydromatrix was, by some mechanism, up regulated in the hydrogel compared to the pellet control system, which showed decreasing chondrogenic potential with increasing donor age. We conclude that Hydromatrix is a promising cell carrier for chondrogenesis of mesenchymal stem cells and a candidate for regenerating degenerated discs.
Investigating flow-related effects of Chronic Kidney Disease on renal drug toxicity in a human-derived proximal tubule microphysiological system
(2020) Magnusson, Otto; Chalmers tekniska högskola / Institutionen för fysik; Gold, Julie; Nieskens, Tom; Sjögren, Anna-Karin
The kidney proximal tubule is responsible for the active expulsion of drugs from the blood to the urine and is, therefore, of great importance when evaluating drug safety. Chronic kidney disease (CKD) is a major cause of reduced renal filtration function, reducing the fluid forces experienced by human renal proximal tubule eptihelial cells (HRPTEC). Developing a physiologically representative in vitro model for drug toxicity studies in both healthy and diseased HRPTEC would therefore be highly valuable. As CKD is a major cause of reduced renal function and may affect the function of proximal tubule cells, this thesis focused on the investigation of how flow-related aspects of chronic kidney disease affects drug transport and toxicity in HRPTEC cultured in 3D in the Nortis microphysiological system. qPCR was used to evaluate differential gene expression of drug transporters, proximal tubule and stress markers in response to flow exposure and nutrient deprivation in 3D and 2D cultured HRPTEC, alongside LDH in the supernatant and Live/Dead stain to evaluate cell viability. Phenotype by gene expression remained unchanged in the 3D proximal tubule model when exposed to a 5 week 2-fold increase to fluid shear stress (FSS) from 0.9 to 1.7 dyne/cm2, as well as in 2D cultures exposed to orbital flow (1.9 or 5.3 dyne/cm2) for 1 week compared to static cultures. 2D cultured cells also displayed no change to transport (P-gp) function in response to orbital flow when evaluated with Calcein-AM. Interestingly, gene expression of several drug transporters was increased in 3D HRPTEC cultures (1, 5 weeks) compared to 2D, including regained expression of OAT1 and OAT3 and upregulation of OCT2 (SLC22A2, 3.6 ± 1.1 fold [5 weeks]), MATE1 (SLC47A1, 41.3 ± 3.4 fold, 33.2 ± 13.2 fold), MATE2-K (SLC47A2, 71.9 ± 8.9 fold , 99.6 ± 61.7 fold) and the endocytosis receptor Megalin (LRP2, 46.7 ± 13.1 fold, 67.5 ± 34.1 fold). Moreover, this phenotype remained stable from 1 to 5 weeks in culture. The antibiotic polymyxin B (50 μM, 48 h) showing reduced viability to 83 ± 7.0% as evaluated by Live/Dead stain, demonstrating sensitivity to know nephrotoxicants in 3D cultured HRPTEC. Phenotype by gene expression was unaffected by nutrient and flow deprivation for 3 days. Although the aim of replicating flow related aspects of chronic kidney disease in the 3D proximal tubule model used in this study was not achieved, it was established that the 3D model displayed stable phenotype for up to five weeks with regained and significantly increased expression of drug transporter and endocytosis receptor genes when compared to 2D cultured cells. This suggests that the 3D proximal tubule model used in this study is a more physiologically relevant model for future long-term drug toxicity studies.

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