Endothelzellen bilden eine monozellulare Grenzschicht in Blutgefäßen. Ihre Migration ist ein kritischer Teilschritt bei der Gefäßbildung, zum Beispiel während der Wundheilung. Obwohl bereits eine Reihe der dafür relevanten Mediatoren und pathogenen Determinanten bekannt sind, fehlt bisher eine quantitative Analyse der molekularen Mechanismen der Gefäßbildung und Zellmigration. Voraussetzung dafür sind Verfahren zur automatisierten Bestimmung von Zelltrajektorien in Sequenzen von Mikroskopaufnahmen migrierender Zellverbände. Dazu wurde ein statistisches Modell entwickelt, welches die Segmentierung und Verfolgung von Zellen in Bildsequenzen ermöglicht. Im vorliegenden Beitrag stellen wir dieses Modell vor, diskutieren die sich daraus ergebenden Lern- und Erkennungsalgorithmen und präsentieren erste Resultate. / Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials.

Endothelzellen bilden eine monozellulare Grenzschicht in Blutgefäßen. Ihre Migration ist ein kritischer Teilschritt bei der Gefäßbildung, zum Beispiel während der Wundheilung. Obwohl bereits eine Reihe der dafür relevanten Mediatoren und pathogenen Determinanten bekannt sind, fehlt bisher eine quantitative Analyse der molekularen Mechanismen der Gefäßbildung und Zellmigration. Voraussetzung dafür sind Verfahren zur automatisierten Bestimmung von Zelltrajektorien in Sequenzen von Mikroskopaufnahmen migrierender Zellverbände. Dazu wurde ein statistisches Modell entwickelt, welches die Segmentierung und Verfolgung von Zellen in Bildsequenzen ermöglicht. Im vorliegenden Beitrag stellen wir dieses Modell vor, diskutieren die sich daraus ergebenden Lern- und Erkennungsalgorithmen und präsentieren erste Resultate. / Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials.

The present thesis aimed to fabricate a liver sinusoid model by combining different components and techniques to closely mimic the physiological microenvironment of the hepatic cells. Since liver is a complex multicellular organ, 3D extrusion bioprinting was employed as well as core-shell 3D bioprinting for creating more complex constructs with relevant physiological microarchitecture to the in vivo liver sinusoid. Figure 1 illustrates the concept of the aimed model fabrication and the combination of components required to achieve this model. To create an initial model, HepG2, a human carcinoma-derived liver cell line was used as a model cell line for hepatocytes. Biocompatible inks based on a printable alginate-methylcellulose (algMC) blend were aimed to be developed for the encapsulation of hepatocytes by functionalization with bioactive molecules to better recapitulate the hepatocytes biochemical microenvironment, supporting cellular functions. Towards tissue complexity, a further aim was to employ core-shell bioprinting to establish a coculture model of hepatocytes and fibroblasts, which acted as supportive cells; by coaxially printing HepG2 encapsulated in the shell with fibroblasts in the core of a single core-shell strand. Different bioinks were investigated as core for the fibroblasts encapsulation, whereby plasma and fibrin were utilized to functionalize the algMC blends with the aim of enhancing the fibroblasts attachment, proliferation and spreading. Moreover, the influence of functionalized core bioinks on the hepatocytes performance and function would be demonstrated, as well as the influence of the coculture with the fibroblasts. As a final step towards integrating vascularized structures in the liver sinusoid model, endothelial cells (EC) were to be cocultured with the supporting fibroblasts in the core of the core-shell constructs to create a triple-culture model with the hepatocytes in the shell. Based on collagen-fibrin matrices, suitable to support angiogenesis, a natural extracellular matrix-like Introduction 5 core bioink, which is independently printable and allows for the printing of stable core-shell vascularized constructs is aimed to be developed. The culture and coculture parameters of the ECs will be optimized and evaluated. Optimization of the shell bioink encapsulating the hepatocytes is aimed to be investigated with the goal of enhancing the HepG2 microenvironment. Printing parameters and crosslinking procedures as well as culture conditions for all the cells were to be optimized for the model. The final triple-culture core-shell printed in vitro model is aimed to characterize the ability of this triple-culture construct to support vascularization by the HUVECs printed in the core, the physiological functions of the hepatocytes printed in the shell bioink, as well as to evaluate the cellular interactions between core and shell compartments. Through engineering and modifying the bioinks which represent the extra-cellular matrix and adjusting the culture conditions for the cells, cell-cell and cell-matrix interactions can be studied in such coculture models, providing new insights towards clinical and therapeutic biomedical applications.:Abbreviations 1 1 Motivation 3 2 Introduction and state of the art 6 2.1 Liver and its microarchitecture 6 2.2 Tissue Engineering 9 2.2.1 Liver Tissue Engineering 9 2.3 From two- to three-dimensional cell cultures 10 2.3.1 2D and 3D hepatocytes coculture models 12 2.4 3D Bioprinting 14 2.4.1 Biomaterial inks and bioinks for 3D bioprinting 15 2.4.2 Core-shell 3D bioprinting 17 2.4.3 3D bioprinting of in vitro liver models 18 3 Materials and methods 20 3.1 Biomaterials for ink preparation 20 3.2 Cell lines used for bioink encapsulation 20 3.3 Cell culture media 21 3.4 3D bioprinting 22 3.5 Characterization assays 23 3.5.1 Rheological and mechanical characterization of the inks and printed scaffolds 23 3.5.2 Characterization of cell viability 23 3.5.3 Characterization of cell metabolic activity 24 3.5.4 Determination of cell number and proliferation 24 3.5.5 Quantitative analysis of hepatocytes functionality 25 3.5.6 Immunostaining 26 3.5.7 Imaging 27 3.5.8 Statistics 28 3.6 Specific experimental procedures and characterizations 28 3.6.1 Bioink development for bioprinting of hepatocytes 28 3.6.1.1 Bioink preparation 3.6.1.2 Bioprinting and crosslinking 3.6.1.3 Rheological and mechanical characterization 3.6.1.4 Biological characterization 3.6.2 Coculture of hepatocytes (HepG2) and fibroblasts (NIH 3T3) in core-shell bioprinted scaffolds 3.6.2.1 Bioink preparation 3.6.2.2 Core-shell bioprinting and crosslinking 3.6.2.3 Rheological and mechanical characterization 3.6.2.4 Biological characterization 3.6.3 Vascularization of bioprinted HepG2-laden constructs 3.6.3.2 Collagen : Fibrin (CF)-based core bioinks preparation and characterization 32 3.6.3.3 Optimization of shell bioink for HepG2 encapsulation 34 3.6.3.4 Influence of shell bioink on endothelial tube formation in the core 35 3.6.3.5 Transwell experiment for analysis of triple-cultures 35 3.6.3.6 Core-shell bioprinting of triple-culture in vitro liver sinusoid model 37 4 Results and Discussion 38 4.1 Bioink development for encapsulation of hepatocytes by 3D bioprinting 38 4.2 Spatially defined pattern of hepatocyte-fibroblast co-culture in a core-shell bioprinted system 46 4.2.1 Establishment of core-shell bioprinting 47 4.2.2 Simultaneous embedding of HepG2 and NIH 3T3 cells in core-shell strand scaffolds 49 4.2.3 Functionalization of the core bioink – enhancing fibroblast network formation 51 4.2.4 Influence of the microenvironment on expression of hepatic marker proteins in the core-shell bioprinted co-culture system 56 4.3 Vascularization strategies of an in vitro bioprinted liver sinusoid model 61 4.3.1 Preliminary investigation of prevascular-tube formation in fibrin gels 63 4.3.2 Optimization of culture conditions for HUVECs pre-vascular network formation 65 4.3.2.1 Collagen : Fibrin composite gels 65 4.3.2.2 Optimization of CF network density and influence of supportive cells on HUVECs pre-vascular network formation 70 4.3.3 Core-shell bioprinting: development of a core bioink to support formation of pre-vascular structures 75 4.3.3.1 Collagen : fibrin-based ink development for printability 75 4.3.3.2 Formation of pre-vascular structures in 3D bioprinted CFG core 83 4.3.4 Optimization of the shell bioink: HepG2 encapsulation in Plasma vs. Matrigel functionalized algMC 85 4.3.5 Vascularization in different shell biomaterial inks 94 4.3.6 Triple-culture of HepG2, HUVECs and NHDFs: analysis of shell and core bioinks in transwell-coculture 100 4.3.7 Core-shell bioprinting of a triple-culture 3D in vitro liver sinusoid model 111 5 Conclusions and Outlook 120 5.1 Bioink development for hepatocytes encapsulation 120 5.2 Establishment of core-shell bioprinting to fabricate a liver sinusoid model 120 5.3 Coculture of hepatocytes with supportive fibroblasts 121 5.4 Establishment of a vascularized liver sinusoid model 121 5.4.1 Optimization of culture conditions for endothelial cells 121 5.4.2 Development of CF-based printable bioink for endothelial cells encapsulation in the core 122 5.4.3 Optimizing hepatocytes encapsulation bioink 124 5.4.4 Establishment of the complex triple-culture liver sinusoid model 124 5.5 Outlook and future prospects 125 Summary 129 Zusammenfassung 132 References 135 List of Figures 157 List of Tables 159 List of publications