Osteozyten stehen vermehrt im Fokus als wesentliche Regulatoren der Knochenmineralisierung. Das ähnlich einem neuronalen Netzwerk aufgebaute lakunokanalikuläre Netzwerk der Osteozyten breitet sich im Knochen in drei Ebenen aus. Es wurde in dieser Arbeit ein 3D-Kollagengel-Modell verwendet und dort die Osteoblasten- bzw. Osteozytenzelllinien MLO-A5 und MLO-Y4, sowie humane mesenchymale Stammzellen aus Hüftköpfen eingebettet. Es wurden die optimalen Kulturbedingungen entwickelt und die Zellen über mehrere Wochen kultiviert, beobachtet und mit dem herkömmlichen 2D-Kulturmodell verglichen. MLO-A5 und MLO-Y4 bilden die zelltypischen Zellfortsätze. Die Gele kontrahieren, wenn hMSC und MLO-A5 eingebettet sind, mit MLO-Y4 zeigt sich über den gesamten Kultivierungszeitraum keinerlei Kontraktion der Kollagengele. Die Zellen wurden zudem osteogen differenziert und mit FGF23 und Klotho stimuliert. Es ergaben sich erste Hinweise auf eine FGF23 / Klotho-abhängige Inhibierung der lokalen Mineralisierung in osteogen differenzierten MLO-A5. Es konnten einige osteogene Marker durch PCR und in den histologischen Schnitten mittels Antikörperfärbungen nachgewiesen werden, eindeutige Expressionsmuster und deren zeitliche Verläufe im Vergleich der osteogenen Differenzierungen und Zugabe von FGF23 und Klotho sind allerdings noch nicht identifizierbar und bedürfen womöglich höherer Fallzahlen und weiterer Untersuchungsmethoden. Insgesamt gesehen erweist sich das System aber als einfach und mit niederschwellig erreichbaren Methoden und Materialien durchzuführen. / Osteocytes increasingly establish as the key regulators of bone mineralisation. The network of osteocytes - similar to the neuronal network - is spreaded in three dimensions in bone. In this work the osteoblast/osteocyte cell lines MLO-A5 and MLO-Y4 were embedded in a 3D collagen gel model and compared to human MSCs. Optimal culture conditions were developed, cells cultivated and observed over several weeks and compared to 2D cell culture models. MLO-A5 and MLO-Y4 build the typical cell dendrites. Gels contract with MSCs and MLO-A5 whereas MLO-Y4 do not contract the gels. Cells were differentiated in osteogenetic pathway and stimulated with FGF23 and Klotho. First results suggest that FGF23 and Klotho can inhibit local mineralisation in MLO-A5 but more reproductions need to be made. Osteogenetic markers were identified by mRNA-PCR and antibody stainings of paraffin slices. Clear demarcation throughout osteogenetic differentiation and stimulation with FGF23 and Klotho could not be identified. Reproductions of the model prove to be easy and with low-threshold resources.

Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods. / Zahlreiche Arthropoden wie Stechmücken, Zecken, Wanzen und Fliegen sind Überträger für krankheitserregende Parasiten, Bakterien und Viren. Hierzu gehört der einzellige Parasit Trypanosoma brucei (T. brucei), welcher durch Tsetsefliegen übertragen wird und die Afrikanische Trypanosomiasis bei Menschen und Tieren verursacht. Der Entwicklungszyklus des Parasiten in der Fliege ist komplex und endet in der Speicheldrüse mit der Differenzierung in das metazyklische Lebensstadium. Diese metazyklischen Formen werden durch den Biss der blutsaugenden Tsetsefliege in die dermale Hautschicht des Säugetierwirts injiziert. Die zellzyklusarretierten metazyklischen Formen werden in der Dermis aktiviert und der Widereintritt in den Zellzyklus sowie die Differenzierung zu proliferativen Trypanosomen eingeleitet. Anschließend breitet sich der Parasit systemisch im Säugetierwirt aus. Obwohl T. brucei bereits seit Jahrzehnten erforscht wird, ist nur sehr wenig über das frühe Infektionsgeschehen in der Haut bekannt. Der genaue Zeitpunkt und die Mechanismen, die der Differenzierung des Parasiten in der Haut zugrunde liegen, sind unbekannt. Ebenso wurden die proliferativen Trypanosomen in der Haut bisher nur unzureichend charakterisiert. Das Verständnis über die ersten Schritte einer Infektion ist jedoch von entscheidender Bedeutung für die Entwicklung von neuen Strategien, die die Krankheitsentstehung und deren Fortschreiten verhindern sollen. Ein großes Hindernis bei der Erforschung der humanen Afrikanischen Trypanosomiasis ist der Mangel an geeigneten Infektionsmodellen, die den Krankheitsverlauf authentisch nachbilden. Außerdem werden für die Erzeugung der infektiösen metazyklischen Parasiten Tsetsefliegen benötigt, die aufwändig zu züchten sind. Tiermodelle haben es ermöglicht - hauptsächlich Mäuse -, viele Erkenntnisse über die Pathogenese von Trypanosomen im Säugetierwirt zu erlangen. Allerdings wurden diese überwiegend durch Nadelinjektion in den Bauchraum oder die Kaudalvene infiziert, wodurch die Haut als erste Eintrittspforte umgangen wurde. Darüber hinaus lassen Tiermodelle nicht immer Rückschlüsse auf den Infektionsverlauf beim Menschen zu. Zusätzlich erschwert die geringe Anzahl von metazyklischen Parasiten, die von Tsetsefliegen injiziert werden, die Isolation, Nachweis und Untersuchung im tierischen Wirt. Das Ziel der vorliegenden Arbeit war es, ein rekonstruiertes menschliches Hautäquivalent zu entwickeln und als Infektionsmodell zu validieren, um die Entwicklung von natürlich übertragenen metazyklischen Parasiten von T. brucei in der Säugetierhaut zu untersuchen. Der erste Teil dieser Arbeit beschreibt die Entwicklung und Charakterisierung eines primären menschlichen Hautäquivalents mit verbesserten mechanischen Eigenschaften. Zu diesem Zweck wurde ein computergesteuertes Kompressionssystem entworfen und hergestellt. Dieses System ermöglichte die gleichzeitige Verbesserung der mechanischen Stabilität von zwölf kollagenbasierten dermalen Äquivalenten durch plastische Kompression, die mittels Rheologie evaluiert wurden. Die verbesserten dermalen Äquivalente dienten als Fundament für die Erzeugung der Hautäquivalente und reduzierten deren Kontraktion und Gewichtsverlust während der Gewebebildung. Dadurch wurde ein hohes Maß an Standardisierung und Reproduzierbarkeit erreicht. Die Hautäquivalente wurden durch immunhistochemische und histologische Techniken charakterisiert und bildeten wichtige anatomische, zelluläre und funktionelle Aspekte der nativen menschlichen Haut nach. Des Weiteren wurde die zelluläre Heterogenität durch Einzelzell-RNA-Sequenzierung untersucht. Mit dieser Technik wurde ein umfangreiches Spektrum an extrazellulären Matrix-assoziierten Genen identifiziert, die von verschiedenen Zellsubpopulationen in der künstlichen Haut exprimiert werden. Zusätzlich wurden experimentelle Bedingungen etabliert, damit Tsetsefliegen eingesetzt werden konnten, um die Hautäquivalente auf natürlichem Weg mit Trypanosomen zu infizieren. Im zweiten Teil dieser Arbeit wurde die Entwicklung der Trypanosomen in der künstlichen Haut im Detail untersucht. Dies umfasste die Etablierung von Methoden zur erfolgreichen Isolierung der Trypanosomen aus der Haut, um deren Proteinsyntheserate, Zellzyklus- und Stoffwechselstatus, sowie Morphologie und Transkriptom zu bestimmen. Zusätzlich wurden Mikroskopietechniken zur Untersuchung der Trypanosomenmotilität und migration in der Haut optimiert. Nach der Injektion in die künstliche Haut durch Tsetsefliegen wurden die metazyklischen Parasiten schnell aktiviert und etablierten innerhalb eines Tages eine proliferative Population. Dieser Entwicklungsprozess wurde begleitet von (I) einer Reaktivierung der Proteinsynthese, (II) einem Wiedereintritt in den Zellzyklus, (III) einer Veränderung der Morphologie und (IV) einer erhöhten Motilität. Des Weiteren wurden diese Beobachtungen mit potentiell zugrundeliegenden entwicklungsbiologischen Mechanismen in Verbindung gebracht, indem eine Einzelzell RNA-Sequenzierung der Trypanosomen zu fünf verschiedenen Zeitpunkten nach der Infektion durchgeführt wurde. Nach der ersten proliferativen Phase traten die Tsetse-übertragenen Trypanosomen in der Haut in ein reversibles Ruhestadium ein. Diese ruhenden Trypanosomen waren durch eine sehr langsame Zellteilung, einen stark reduzierten Stoffwechsel und ein Transkriptom gekennzeichnet, dass sich deutlich von dem der injizierten metazyklischen Formen und der ersten proliferativen Trypanosomen unterschied. Durch Nachahmung der Migration von der Haut in den Blutkreislauf konnte dieser Phänotyp reaktiviert werden und die Parasiten kehrten in einen aktiven, proliferierenden Zustand zurück. Unter Berücksichtigung, dass vorangegangene Forschungsarbeiten die Haut als anatomisches Reservoir für T. brucei während des Krankheitsverlaufs identifiziert haben, ist anzunehmen, dass das Ruheprogramm eine authentische Facette im Verhalten des Parasiten in einem infizierten Wirt darstellt. Zusammenfassend zeigt diese Arbeit, das primäre menschliche Hautäquivalente eine neue und vielversprechende Möglichkeit bieten, vektorübertragene Parasiten unter naturnahen Bedingungen als Alternative zu Tierversuchen zu untersuchen. Durch die Verwendung des natürlichen Infektionsweges - dem Biss einer infizierten Tsetsefliege -, konnten die frühen Prozesse einer Trypanosomen-Infektion mit noch nie dagewesener Detailtiefe nachvollzogen werden. Des Weiteren könnte der hier erbrachte Nachweis einer ruhenden, hautresidenten Trypanosomen-Population die Persistenz von T. brucei in der Haut von aparasitämischen und asymptomatischen Personen erklären. Dies könnte eine wichtige Rolle bei der Aufrechterhaltung einer Infektion über lange Zeiträume spielen.

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.