Gewebe sind nicht nur durch ihre biochemische Zusammensetzung definiert, sondern auch durch ihre individuellen mechanischen Eigenschaften. Inzwischen ist es weithin akzeptiert, dass Zellen ihre mechanische Umgebung spüren und darauf reagieren. Zum Beispiel werden Zellmigration und die Differenzierung von Stammzellen durch die Umgebungssteifigkeit beeinflusst. Um diese Effekte in vitro zu untersuchen, wurden viele Zellkulturstudien auf 2D Hydrogelsubstraten durchgeführt. Zusätzlich dazu steigt die Anzahl von Studien an, die hydrogelbasierte 3D-Scaffolds nutzen, um 2D Studien zu validieren und die experimentellen Bedingungen der Situation in vivo anzunähern. Jedoch erweist es sich weiterhin als schwierig den Effekt von Mechanik in 3D in vitro zu untersuchen, da in den gemeinhin genutzten 3D Hydrogelsystemen immer eine Kopplung zwischen Gelporosität und Steifigkeit besteht. Zusätzlich hängt die Konzentration der biologisch aktiven Bindungsstellen für Zellen oft ebenfalls von der Steifigkeit ab.
Diese Arbeit präsentiert die Entwicklung und Optimierung neuer 3D Hydrogelkugel-Scaffolds, in denen die Steifigkeit von der Porosität schließlich entkoppelt wird. Mit Hydrogelkugeln als Scaffold-Bausteine ist es nun möglich 3D Scaffolds mit definierten mechanischen Eigenschaften und konstanter Porengröße zu generieren. Während der Methodenentwicklung wurden verschiedene Prinzipien und Kultivierungskammern konstruiert und überarbeitet, gefolgt von der theoretischen Betrachtung der Sauerstoffdiffusion, um die
Eignung der gewählten Kammer hinsichtlich Zellvitalität und Zellwachstum zu überprüfen. Eine Kombination aus mehreren getesteten Filtern wurde ausgewählt um HydrogelkugelScaffolds erfolgreich in der ausgewählten Kammer zu generieren. Im Weiteren wurden verschiedene Hydrogelmaterialien untersucht hinsichtlich der erfolgreichen Produktion monodisperser Hydrogelkugeln und der Erzeugung stabiler Scaffolds. Hydrogelkugeln aus Polyacrylamid (PAAm) wurden als Scaffold-Bausteine ausgewählt um damit die Eignung des entwickelten Systems zu demonstrieren lebende Zellen zu mikroskopieren. Außerdem wurde
das Überleben von Fibroblasten über vier Tage in unterschiedlich steifen HydrogelkugelScaffolds erfolgreich gezeigt. Weiterhin war es möglich erste Zellmigrationsexperimente durchzuführen. Dafür wurden sowohl einfache PAAm-Hydrogelkugeln als auch mit Adhäsionsmolekülen funktionalisierte Hydrogelkugeln genutzt, um unterschiedlich steife Schichten in einem Scaffold zu erzeugen. Dadurch war es möglich nicht nur Zellmigration anhand von Zelladhäsion in 3D Scaffolds mit Steifigkeitsgradienten zu beobachten, sondern auch Zellmigration ohne Zelladhäsion.:1 Introduction
1.1 Mechanics play a role in biology
1.2 3D cultures and scaffolds
1.3 3D hydrogel systems to study effects of mechanics
1.4 Decoupling stiffness and porosity in 3D scaffolds
2 Materials
3 Methods
3.1 Laser scanning microscopy and microscopy data processing
3.2 Atomic force microscopy (AFM)
3.3 Refractive index matching of PMMA beads
3.4 Regular PMMA bead scaffolds for developing analysis algorithm
3.5 Cell culture standards
3.6 Fluorescent labelling of ULGP agarose
3.7 Production of polydisperse ULGP agarose beads
3.8 Hydrogel bead production via microfluidics
3.9 PAAm bead functionalization
3.10 Real-time fluorescence and deformability cytometry (RT-fDC)
3.11 3D scaffolds made from hydrogel beads
3.12 Statistics
4 Results
4.1 Design of a suitable scaffold device
4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation
4.3 Further optimization of 3D scaffold device
4.3.1 PMMA beads can be arranged in stable scaffolds
4.3.2 Regular PMMA bead scaffolds can be achieved and analysed
4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads
4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds
4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads
4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds
5 Discussion and Concluding Remarks
6 Bibliography
List of Figures
List of Tables
Eigenständigkeitserklärung
Appendix A
Appendix B
FIJI macro for FFT analysis maxima
Python script to determine regularity of PMMA bead scaffolds
Excel macro to determine number of peaks for regularity analysisTissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. For example, cell migration and stem cell differentiation is affected by stiffness. To study these effects in vitro, many cell culture studies have been performed on 2D hydrogel substrates. Additionally, the amount of 3D studies based on hydrogels as 3D scaffold is increasing to validate 2D in vitro studies and adjust experimental conditions closer to the situation in vivo. However, studying the effects of mechanics in vitro in 3D is still challenging as commonly used 3D hydrogel assays always link gel porosity with stiffness. Additionally, the concentration of biologically active adhesion sides often also
depends on the stiffness.
This work presents the development and optimization of novel 3D hydrogel bead scaffolds where the stiffness is finally decoupled from porosity. With hydrogel beads as scaffold building blocks it was possible to generate 3D scaffolds with defined mechanical properties and a constant pore size. During the method development, different culture devices were constructed and revised, followed by oxygen diffusion simulations to proof the suitability of the chosen device for cell survival and growth. A combination of different filter approaches was selected to generate hydrogel bead scaffolds in the culture device. Furthermore, different
hydrogel materials were investigated regarding successful production of monodisperse beads and stable scaffold generation. Polyacrylamide (PAAm) hydrogel beads were chosen as scaffold building blocks to demonstrate live-cell imaging and successful cell survival over four days in differently compliant hydrogel bead scaffolds. Moreover, first cell migration experiments were performed by using plain PAAm hydrogel beads as well as PAAm hydrogel beads functionalized with adhesion molecules with differently stiff layers in one scaffold. Thereby fibroblast migration was observed not only in adhesion-dependent migration manner, but also in an adhesion-independent mode .:1 Introduction
1.1 Mechanics play a role in biology
1.2 3D cultures and scaffolds
1.3 3D hydrogel systems to study effects of mechanics
1.4 Decoupling stiffness and porosity in 3D scaffolds
2 Materials
3 Methods
3.1 Laser scanning microscopy and microscopy data processing
3.2 Atomic force microscopy (AFM)
3.3 Refractive index matching of PMMA beads
3.4 Regular PMMA bead scaffolds for developing analysis algorithm
3.5 Cell culture standards
3.6 Fluorescent labelling of ULGP agarose
3.7 Production of polydisperse ULGP agarose beads
3.8 Hydrogel bead production via microfluidics
3.9 PAAm bead functionalization
3.10 Real-time fluorescence and deformability cytometry (RT-fDC)
3.11 3D scaffolds made from hydrogel beads
3.12 Statistics
4 Results
4.1 Design of a suitable scaffold device
4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation
4.3 Further optimization of 3D scaffold device
4.3.1 PMMA beads can be arranged in stable scaffolds
4.3.2 Regular PMMA bead scaffolds can be achieved and analysed
4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads
4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds
4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads
4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds
5 Discussion and Concluding Remarks
6 Bibliography
List of Figures
List of Tables
Eigenständigkeitserklärung
Appendix A
Appendix B
FIJI macro for FFT analysis maxima
Python script to determine regularity of PMMA bead scaffolds
Excel macro to determine number of peaks for regularity analysi
