Bayer, Robin
(2021).
Development of a novel in-vitro vascular model for determination of physiological and pathophysiological mechanobiology.
PhD thesis, Universität zu Köln.
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Abstract
Background/Aims The aim of the study was to develop a biological and technological method to investigate in-vitro the physiology and pathophysiology biomechanical phenomena of a vascular wall. In particular, cellular contraction and relaxation as a biomechanical response to vasoactive substances and different mechanical stimulation intervals were studied to provide data for basic research and pharmacological developments in cardiovascular diseases such as arterial hypertension. Methods: Methodologically, the study is based on CellDrum technology, which is a method to determine cellular stress changes of a few kilo Pascal(kPa). Especially for this study, a new approach was developed to characterize the cell stresses of monolayers and multilayer tissue equivalents in a standardized way. A monolayer model consisting of human aortic smooth muscle cells (haSMC) was primarily developed for CellDrum as a vascular in-vitro cell culture model. Also, a model of human aortic endothelial cells (haEC) was established, and an approach for a 3D co-culture model of both cell types was developed. Vasoactive substances with different mechanisms of action and concentrations were tested to represent the physiological properties of the model. For the first time, the biomechanical influence of blood sera was analyzed on the CellDrum models to test the potential possibility of a laboratory screening procedure. The PulSElect system was developed, which exposes the CellDrum models to a defined, cyclical, mechanical stress by stretching, to simulate the symptoms of mechanically induced hypertension. The influence of the mechanical stress was observed by cytoskeletal alignment quantification, transcriptome analysis, gene expression of mechanosensitive as well as biomechanically relevant genes and biomechanical stress evaluation to elucidate cellular stiffening and cellular stress management. Results The haSMC cell models showed significant physiological and biomechanical changes in cell tone after application of the vasoactive substances, sera and conditioned media (~-6-10% relative to initial tension). Mechanical stimulation of the cells allowed quantification of both mechanical and transcriptomic changes as well as morphological adaptation. Furthermore, it was possible to present the obtained results in a time-dependent manner. Also, mechanical stimulation has been shown to induce the development of the contractile phenotype of haSMC and improve its cellular integrity, resulting in increased basal tension and overall contractility. As an extension of a well-established haSMC CellDrum model, an approach for direct co-cultivation of human aortic smooth muscle cells and endothelial cells was elaborated. Conclusion Different CellDrum models have been established to replicate biomechanical processes of the vascular system. The study showed that the CellDrum technology is a suitable method to analyze biomechanical stress changes caused by different stimuli using haSMC. The analysis of blood sera using CellDrums allows for possible future use as a screening method for pharmacological and medical laboratory research. Since the CellDrum technology is not limited to the use of monolayers, it is possible to think about an extension of cell models with additional cell types and cell layers. Although we have already been able to show partial co-cultivation of smooth muscle cells and endothelial cells, further research is needed to establish this sufficiently. Increased expression levels of mechanosensitive genes have been shown to correlate with literature data on the pathogenesis of hypertension, using microarray analysis (Affymetrix) and qPCR. Nevertheless, it remains a speculative reflection of the cellular changes due to induced hypertension. The data and findings obtained to provide the promising potential supporting research and development of personalized medication, sports medicine, cell biology and stem cell research using CellDrum technology.
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-362212 | ||||||||
| Date: | January 2021 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | Faculty of Medicine > Physiologie und Pathophysiologie > Institut für Neurophysiologie | ||||||||
| Subjects: | Natural sciences and mathematics Life sciences Medical sciences Medicine |
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| Date of oral exam: | 15 January 2021 | ||||||||
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| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/36221 |
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