Browsing by Subject "3D cell culture"
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(2023)Three-dimensional (3D) cellular cultures have been shown to represent tissue formations and functions more accurately than two-dimensional (2D) cultures and have been successfully utilized more accurately in model organisms, e.g., to understand cellular modular functions. However, the applications in non-model organisms are limited, and to our knowledge have not been implemented in ectotherms. At an ecological scale, the technique can enhance our understanding by providing insights on cellular and tissue level molecular mechanisms. A potential implementation of this method in Atlantic salmon is to elucidate the molecular function of the vestigial-like 3 (vgll3) gene, which plays a central role in salmonid maturity, potentially by regulating energy allocation via regulating adipogenesis. In this thesis, a proof of principle study was implemented, where the feasibility of obtaining and maintaining a suspension 3D adipose tissue culture in Atlantic salmon (Salmo salar) was assessed. The harvested visceral white adipose tissue from around the intestinal tissue of mature Atlantic salmon (salmon past smolt stage) was first separated into stromal vascular fraction (SVFs) and mature adipose fraction (MAFs). SVFs contain preadipocytes (precursors of adipocyte cells) in addition to a variety of other cell types. MAFs are mature adipocytes. Both MAFs and SVFs were successfully maintained in-vitro for over four weeks. SVFs were then successfully differentiated into mature adipocytes, demonstrating the feasibility of studying adipogenesis in Atlantic salmon. Proof of this methodology and its further implications may help us to understand the cellular functions of vgll3 and may subsequently help to better understand its causal relation to the maturation process in Atlantic salmon.
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(2019)Despite the advances in the management of breast cancer, discovery of novel and targeted drugs remains a challenge. It has been suggested that drug failure rates in clinical trials might be diminished by improving the predictive potential of preclinical cancer models. Three-dimensional (3D) scaffold-based cell culture has emerged as an attractive platform for mimicking tissue-like microenvironment, since it is well-known that cells respond to the cues in the extracellular matrix (ECM). The aim of this thesis was to develop fibrin-based hydrogels and evaluate their performance in 3D cell culture of breast cancer cells. The fibrin gel formulation was first optimized by testing the effect of different buffers on gel properties. Structural properties were examined with scanning electron microscopy and mechanical properties measured with oscillatory rheometry. Three different fibrin concentrations of the optimized formulation were then used as scaffolds for DU4475 breast cancer cells. After seven days of culture, the morphology, phenotype and proliferation of the resulting cell structures were assessed by using techniques such as light microscopy, immunofluorescent confocal microscopy and Western blot analysis. The desired properties for 3D cell culture were obtained by preparing fibrin gels at high pH in the absence of calcium. The main finding of the thesis was that fibrin concentration strongly affected the phenotype of DU4475 cells, with cells cultured in the softest gel retaining their original characteristics to the greatest extent. In the future, the developed scaffold could possibly be used in drug discovery and personalized medicine by culturing tumor explants from patients. However, the methods used in the study must be further optimized and the results validated with other breast cancer cell lines and with primary tissues.
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(2023)Chronic wounds are a worldwide health problem that produce a lot of costs for society and can have a substantial impact on patients’ quality of life. Human adipose stem cells (hASCs) have been studied as a treatment option for chronic wounds as they can induce wound healing in many ways. Extracellular vesicles (EVs) produced by hASCs are a great solution to acquire the benefits of hASCs while avoiding their problems such as possible mutagenicity. HASC-EVs have been found to induce wound healing by for example enhancing angiogenesis and fibroblast proliferation. HASCs can be grown in 2D where the cells attach to the bottom of the cell culture vessel or in 3D where the cells attach to each other and create a spheroid. 2D cell culturing is easy and inexpensive but 3D cultured cells resemble in vivo –like conditions more. Because of these in vivo -like features, hASCs grown in 3D might produce EVs that resemble the properties of host cells in natural environment more than 2D. The aim of this thesis was to compare 2D culture, matrix-based nanofibrillar cellulose (NFC) hydrogel culture, and matrix-free suspension culture in ultra-low attachment (ULA) wells as growing platforms for hASCs and as continuous EV production methods. During culturing, the conditioned media was collected after which, the EVs were isolated, and the EV concentration and size range was measured with nanoparticle tracking analysis (NTA). After culturing, the metabolic activity of hASCs was measured and the cells were collected for immunocytochemistry (ICC) assay, western blot (WB) assay, and for quantitative PCR (qPCR) to examine the stemness and differentiation of hASCs grown in different cell cultures. The hypothesis of this thesis was that the NFC cell culture would produce the best EV yield and the best EVs for therapeutic use. Based on the acquired results, this hypothesis could not be supported. When visually inspecting the cells, all three cell cultures were viable but the metabolic activity of hASCs in NFC hydrogel was low compared to 2D and suspension cultures. Also, the EV, protein and RNA yield were lower in NFC. ICC, western blotting, and qPCR results were inadequate to make a straightforward implication of what cell culturing condition is the best for EV production and they would need repetition and optimization. Looking at the overall results, 2D cell culturing produced the best EV and RNA yield, had the highest metabolic activity and was least laborious cell culturing method which makes it a good option for continuous EV production. Suspension culture on the other hand resembles in vivo -like environment which could possibly produce better EVs for therapeutic use. The metabolomic assays on the EVs would be interesting to perform in the future to examine if the in vivo –like features affect the quality of EVs.
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(2024)Gastrointestinal stromal tumours (GISTs) are rare mesenchymal neoplasms that arise from the interstitial cells of Cajal, the so-called pacemaker cells of the intestine. GISTs typically contain an oncogenic driver mutation either in the proto-oncogene KIT or platelet-derived growth factor receptor A (PDGFRA), which belong to the class III receptor tyrosine kinases. Patients with a high-risk or advanced disease are standardly treated with a tyrosine kinase inhibitor imatinib. Despite this molecularly targeted treatment, many patients experience disease relapse, after which the prognosis is poor. Personalised treatment is rarely offered to patients as a first-line treatment option, even though several targeted therapies have been approved for GIST. Increasing treatment personalisation could improve treatment outcomes, yet the lack of patient-specific research models for GIST hinders the research. Three-dimensional (3D) cell culture models are widely used in cancer research to study the molecular mechanisms underlying tumorigenesis. Their ability to mimic the tumour biology and microenvironment is superior compared to the traditional two-dimensional (2D) cell culture model. For several cancers, these cell culture models have also been researched as platforms for personalised treatment selection with promising results. This thesis project aimed to study UPM Biomedicals’ GrowDex-based 3D cell culture model as a potential platform for personalised treatment selection for GIST patients. GrowDex is a plant-derived hydrogel that resembles the extracellular matrix. Another aim of this project was to set up a Sanger sequencing protocol covering frequently mutated areas in GIST to facilitate the validation of this cell culture model through drug testing on patient samples. To assess the GrowDex microenvironment, the viability and proliferation of two GIST cell lines, GIST-T1 and GIST48 were monitored. Furthermore, the imatinib response of GIST-T1 in GrowDex was assessed and compared to the response in other cell culturing conditions. The Sanger sequencing protocol was optimised using the aforementioned cell lines and then applied to GIST patient samples. The results of this project demonstrated that GrowDex provides a suitable microenvironment for culturing GIST cells and supports their 3D growth. GIST-T1 cells were less sensitive to imatinib when cultured in GrowDex in comparison to the 2D culturing condition, which is likely explained by the 3D organisation of the cells. Finally, the Sanger sequencing protocol was used to uncover the KIT/PDGFRA mutation status of several GIST patient samples. In conclusion, these results provide important information for further development of this cell culture model with patient samples.
Now showing items 1-4 of 4