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Fluorescent Methods in Research of Eukaryotic Cells
Chmelíková, Larisa ; Babula, Petr (referee) ; Pešl,, Martin (referee) ; Provazník, Ivo (advisor)
Tato práce zkoumá aplikaci fluorescenčních metod používaných v in vitro studiích v oblasti regenerace srdeční tkáně. Konfokální fluorescenční mikroskopie je vhodnou mikroskopickou technikou pro výzkum v této oblasti, protože umožňuje vizualizaci 3D struktur a distribuce buněk ve 3D modelech. Používané fluorescenční markery by měly být dlouhodobě stabilní, biokompatibilní a netoxické pro živé buňky. V současné době je použití nanočástic jako superparamagnetické nanočástice oxidu železa (SPION) velmi populární; velké množství studií ukazuje, že jsou vhodné pro dlouhodobé experimenty. Tento výzkum využívá superparamagnetické maghemitové nanočástice svázaným rhodaminem na jejich povrchu (SAMN-R) a popisuje jejích excitační a emisní spektrum, velikost a lokalizaci vbuňkách. Stanovení toxicity bylo provedeno měřením reaktivních forem kyslíku (ROS) a nekvantitativním měřením pomocí fluorescenční mikroskopie bylo zjištěno, že hodnota dávky 20 µg·cm-2 je optimální pro aplikaci na živé buňky. Dále byl zkoumán vliv aplikace SAMN-R na buněčnou proliferaci a motilitu, kdy ve studii buněčné proliferace a scratch assay byla použita buněčná linie fibroblastů 3T3. Poté byla studována migrace jednotlivých buněk s použitím mezenchymálních kmenových buněk (MSCs), izolovaných zlidské tukové tkáně. Následná statistická analýza nepotvrdila, že by aplikace SAMN-R měla významný vliv na buněčnou proliferaci, kolektivní migraci nebo na migraci jednotlivých buněk. Lze tedy předpokládat, že SAMN-R jsou vhodným fluorescenčním markerem pro výzkum živých buněk, včetně experimentů voblasti regenerace tkáně. MSC buňky izolované z tukové tkáně mají velký potenciál v regeneraci srdeční tkáně. Jejich interakce s buněčnou linií srdečních svalových buněk HL-1 byly studovány pomocí scratch assay, kdy se tento model jeví jako nadějný a vhodný pro studium buněčných kontaktů a jejich roli přiregeneraci buněk.
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Study of mesenchymal stem cell migration in the extracellular matrix based on principles of chemotaxis
Scholasterová, Viktorie ; Fohlerová, Zdenka (referee) ; Chmelíková, Larisa (advisor)
This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.
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Study of mesenchymal stem cell migration based on principles of chemotaxis
Pošustová, Veronika ; Skopalík, Josef (referee) ; Chmelíková, Larisa (advisor)
The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.
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Comparison of immunomodulatory properties of mesenchymal stem cells and Sertoli cells
Porubská, Bianka ; Krulová, Magdaléna (advisor) ; Komrsková, Kateřina (referee) ; Filipp, Dominik (referee)
Cell therapies are increasingly considered in preclinical studies and in the future of medicine. The main cell type investigated in this manner is mesenchymal stem cells (MSCs), because of their strong immunomodulatory properties. The efficacy of the therapy depends on various aspects, such as the viability and source of MSCs, the purity of the cell suspension and many more. There is a need for more tailored therapy and the use of cell type better fitting for the specific pathology. Sertoli cells (SCs) are deemed by some authors to be a kind of MSCs, namely because of their similar immunomodulatory properties. Because they reside in the seminiferous tubules in the testes, they are a promising candidate for the treatment of inflammatory pathologies of testicular tissue, such as bacterial infection-induced infertility. In vitro comparison of the ability of MSCs and SCs to differentiate into mesenchymal cell lineages such as osteocyte, chondrocyte, and adipocyte showed success in the case of SCs, providing evidence for their mesenchymal origin. The effect of MSCs or SCs on activated immune cells in vitro showed immunosuppression in both cases with distinct features. MSCs suppressed Th17 cell activation and IL-17 production by CD4+ T cells and SCs down-regulated TNFα and IL-2 production by these cells,...
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Modern therapy of chondral defects
Neckař, Pavel ; Havlas, Vojtěch (advisor) ; Zeman, Petr (referee) ; Tuček, Michal (referee)
The thesis describes the application of cultured bone marrow stem cells in the therapy of focal chondral defect of the knee joint. In the experimental part of the work, the goal was to quantitatively and qualitatively compare two sampling sites of bone marrow monocytic aspirates, the iliac crest bone and the proximal tibia, in order to determine a suitable cell source for advanced cell therapy. The sample analysis showed that the amount of monocytic cells and the yield of stem cells from the aspirate obtained were significantly higher in the bone marrow from the iliac crest. We did not find significant qualitative differences between the two sources of stem cells. In the clinical part of the work, I present a description of the surgical procedure and the results of a 1-year follow-up of patients after the implantation of cultured stem cells from the bone marrow, under the name BiCure® orthoMSCp (Bioinova, Prague, Czech Republic), fixed on a commercially available 3D scaffold Chondrotissue® (BioTissue AG, Geneva, Switzerland) using coagulated autologous platelet-rich plasma. The primary objective of the clinical study included the evaluation of the short-term and long-term safety of the applied medical product. The secondary objective of the work included the assessment of the effectiveness of the...
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Human Dental Pulp Stem Cells Cultured in Xenogeneic-Free Supplemented Media
Suchánková Kleplová, Tereza ; Jouklová, Nela (advisor) ; Merglová, Vlasta (referee) ; Bartoňová, Marie (referee)
Human dental pulp stem cells cultured in xenogeneic-free supplemented media Summary Introduction: The topic of the study is the cultivation of dental pulp stem cells (hDPSCs) in a xenogeneic-free culture medium. It is not permissible to use cells upon growing under the influence of xenogeneic (extraneous) substances in human clinical practice. The most frequently used in cultivation of hDPSCs is fetal calf serum (FCS/FBS). Unfortunately, these supplements are widespread in hMSCs cultivation, and all gold standard hMSCs properties were postulated in cells cultivated using these supplements. This raises the basic question if and how xenogeneic blood derivatives affect the properties of cells and their growth characteristics. There are two options for replacing these xenogeneic substances in the culture medium: the so-called serum-free media, or human blood supplements, ideally autologous ones. The conducted research was aimed at identifying the effects of xenogeneic and human blood supplements on basic hDPSCs characteristics that are fundamental to introduce the cell therapy into regular medical practice. Method: By culturing 12 hDPSC lines obtained from adult, deciduous, and natal teeth in 12 different culture media, we investigated the effect of FCS, human blood derivatives, i.e., blood plasma (HP), and...
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Mitochondrial transfer and its role in regenerative properties of mesenchymal stem cells
Jaborová, Natálie ; Krulová, Magdaléna (advisor) ; Nahácka, Zuzana (referee)
Mitochondrial transfer represents a form of intercellular communication. In this process, mitochondria are delivered from the donor cell to the recipient cell through several structures. The transfer of mitochondria is observed under pathological and physiological conditions and is accompanied by specific signaling. Mitochondria uptake by injured cells promotes tissue regeneration. This bachelor thesis discusses general knowledge of mitochondrial transfer focusing on mesenchymal stem cells (MSCs) as a donor cell type. Furthermore, the regenerative effect of MSC-derived mitochondria transfer to cells damaged by various pathologies is summarized. In this regard, MSCs have significant therapeutic potential that could be used in future clinical strategies for a number of diseases. Keywords: mitochondria, transfer, mesenchymal stem cells, regeneration
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Immunomodulatory and differentiation properties of MSCs in a mouse model of the injured cornea and retina
Kössl, Jan ; Holáň, Vladimír (advisor) ; Vištejnová, Lucie (referee) ; Heissigerová, Jarmila (referee)
Stem cells, in general, represent the potential for treating many diseases and disorders that are currently difficult to treat or the therapy has many side effects. One of the stem cells widely investigated these days are mesenchymal stem cells (MSCs). MSCs have the considerable immunomodulatory and regenerative potential for treating degenerative disorders and severe damage to various parts of the eye or other organs. Likewise, their application could serve as supportive therapy in corneal transplantation and other eye inflammatory conditions. In this study of immunomodulatory properties of MSCs, we have focused mainly on their ability to differentiate into cells of different tissue types (in our case, corneal epithelium and retina), their production of immunomodulatory molecules in the inflammatory environment, their ability to migrate to the site of the injury, and their local anti-inflammatory, regenerative, and anti-apoptotic effects. In addition, we tested the therapeutic effects of MSCs in a mouse model of ocular surface injury and a model of retinal degeneration. Finally, we investigated the mechanism of this effect in in vitro models with explants of these tissues. Limbal stem cells (LSCs) are already used to treat severe corneal damage as limbal stem cell deficiency. However, this...
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Effect of oxidative stress on the differentiation of mesenchymal stem cells.
Bura, Radek ; Tlapáková, Tereza (advisor) ; Pacherník, Jiří (referee)
Mesenchymal stem cells are capable of forming different types of tissue such as muscle, bone, fat or cartilage tissue, thanks to the ability to divide and transform into another cell type. Mesenchymal stem cells obtained from various tissues are used for cell therapy and regenerative medicine. Knowledge of the influence of various factors on the differentiation of these multipotent cells is important. Currently, little is known about the effect of reactive oxygen species (ROS) on the differentiation potential of mesenchymal stem cells. The aim of this bachelor's thesis is to summarize the knowledge of the effect of oxidative stress on the differentiation of mesenchymal stem cells and the affected signaling pathways.
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