@phdthesis{Gensler2023,
  author    = {Gensler, Marius E.},
  title     = {Simultaneous printing of tissue and customized bioreactor},
  doi       = {10.25972/OPUS-28019},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally. As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination.},
  subject      = {3 D bioprinting},
  language  = {en}
}
@phdthesis{Malkmus2023,
  author    = {Malkmus, Christoph},
  title     = {Establishment of a 3D \(in\) \(vitro\) skin culture system for the obligatory human parasite \(Onchocerca\) \(volvulus\)},
  doi       = {10.25972/OPUS-31717},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317171},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Onchocerciasis, the world's second-leading infectious cause of blindness in humans -prevalent in Sub-Saharan Africa - is caused by Onchocerca volvulus (O. volvulus), an obligatory human parasitic filarial worm. Commonly known as river blindness, onchocerciasis is being targeted for elimination through ivermectin-based mass drug administration programs. However, ivermectin does not kill adult parasites, which can live and reproduce for more than 15 years within the human host. These impediments heighten the need for a deeper understanding of parasite biology and parasite-human host interactions, coupled with research into the development of new tools - macrofilaricidal drugs, diagnostics, and vaccines. Humans are the only definitive host for O. volvulus. Hence, no small-animal models exist for propagating the full life cycle of O. volvulus, so the adult parasites must be obtained surgically from subcutaneous nodules. A two-dimensional (2D) culture system allows that O. volvulus larvae develop from the vector-derived infective stage larvae (L3) in vitro to the early pre-adult L5 stages. As problematic, the in vitro development of O. volvulus to adult worms has so far proved infeasible. We hypothesized that an increased biological complexity of a three-dimensional (3D) culture system will support the development of O. volvulus larvae in vitro. Thus, we aimed to translate crucial factors of the in vivo environment of the developing worms into a culture system based on human skin. The proposed tissue model should contain 1. skinspecific extracellular matrix, 2. skin-specific cells, and 3. enable a direct contact of larvae and tissue components. For the achievement, a novel adipose tissue model was developed and integrated to a multilayered skin tissue comprised of epidermis, dermis and subcutis. Challenges of the direct culture within a 3D tissue model hindered the application of the three-layered skin tissue. However, the indirect coculture of larvae and skin models supported the growth of fourth stage (L4) larvae in vitro. The direct culture of L4 and adipose tissue strongly improved the larvae survival. Furthermore, the results revealed important cues that might represent the initial encapsulation of the developing worm within nodular tissue. These results demonstrate that tissue engineered 3D tissues represent an appropriate in vitro environment for the maintenance and examination of O. volvulus larvae.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Leikeim2022,
  author    = {Leikeim, Anna},
  title     = {Vascularization Strategies for Full-Thickness Skin Equivalents to Model Melanoma Progression},
  doi       = {10.25972/OPUS-27295},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272956},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Malignant melanoma (MM) is the most dangerous type of skin cancer with rising incidences worldwide. Melanoma skin models can help to elucidate its causes and formation or to develop new treatment strategies. However, most of the current skin models lack a vasculature, limiting their functionality and applicability. MM relies on the vascular system for its own supply and for its dissemination to distant body sites via lymphatic and blood vessels. Thus, to accurately study MM progression, a functional vasculature is indispensable. To date, there are no vascularized skin models to study melanoma metastasis in vitro, which is why such studies still rely on animal experimentation. In the present thesis, two different approaches for the vascularization of skin models are employed with the aim to establish a vascularized 3D in vitro full-thickness skin equivalent (FTSE) that can serve as a test system for the investigation of the progression of MM. Initially, endothelial cells were incorporated in the dermal part of FTSEs. The optimal seeding density, a spheroid conformation of the cells and the cell culture medium were tested. A high cell density resulted in the formation of lumen-forming shapes distributed in the dermal part of the model. These capillary-like structures were proven to be of endothelial origin by staining for the endothelial cell marker CD31. The established vascularized FTSE (vFTSE) was characterized histologically after 4 weeks of culture, revealing an architecture similar to human skin in vivo with a stratified epidermis, separated from the dermal equivalent by a basement membrane indicated by collagen type IV. However, this random capillary-like network is not functional as it cannot be perfused. Therefore, the second vascularization approach focused on the generation of a perfusable tissue construct. A channel was molded within a collagen hydrogel and seeded with endothelial cells to mimic a central, perfusable vessel. The generation and the perfusion culture of the collagen hydrogel was enabled by the use of two custom-made, 3D printed bioreactors. Histological assessment of the hydrogels revealed the lining of the channel with a monolayer of endothelial cells, expressing the cell specific marker CD31. For the investigation of MM progression in vitro, a 3D melanoma skin equivalent was established. Melanoma cells were incorporated in the epidermal part of FTSEs, representing the native microenvironment of the tumor. Melanoma nests grew at the dermo-epidermal junction within the well stratified epidermis and were characterized by the expression of common melanoma markers. First experiments were conducted showing the feasibility of combining the melanoma model with the vFTSE, resulting in skin models with tumors at the dermo-epidermal junction and lumen-like structures in the dermis. Taken together, the models presented in this thesis provide further steps towards the establishment of a vascularized, perfusable melanoma model to study melanoma progression and metastasis.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Fey2022,
  author    = {Fey, Christina},
  title     = {Establishment of an intestinal tissue model for pre-clinical screenings},
  doi       = {10.25972/OPUS-24410},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244107},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed. In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the "gold standard" in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs. In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes. Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture. Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive. With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10\% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies. Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.},
  subject      = {D{\"u}nndarm},
  language  = {en}
}
@phdthesis{WeigelgebSchneider2022,
  author    = {Weigel [geb. Schneider], Verena},
  title     = {Entwicklung eines \(in\) \(vitro\) Modells f{\"u}r Verbrennungen ersten Grades zur Testung einer zellbasierten Wundauflage},
  doi       = {10.25972/OPUS-26151},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261514},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Mit j{\"a}hrlich circa 11 Millionen F{\"a}llen weltweit, stellen schwere Brandwunden bis heute einen großen Anteil an Verletzungen dar, die in Kliniken behandelt werden m{\"u}ssen. W{\"a}hrend leichte Verbrennungen meist problemlos heilen, bedarf die Behandlung tieferer Verbrennungen medizinischer Intervention. Zellbasierte Therapeutika zeigen hier bereits große Erfolge, aufgrund der eingeschr{\"a}nkten {\"U}bertragbarkeit von Ergebnissen aus Tiermodellen ist jedoch sowohl die Testung neuer Produkte, als auch die Erforschung der Wundheilung bei Brandwunden noch immer schwierig. Aufgrund dessen wurden in dieser Arbeit zwei Ziele verfolgt: Die Etablierung von Methoden, um ein zellbasiertes Therapeutikum produzieren zu k{\"o}nnen und die Entwicklung eines Modells zur Untersuchung von Verbrennungswunden. Zun{\"a}chst wurden hierf{\"u}r die Kulturbedingungen und -protokolle zur Isolation und Expansion von Keratinozyten so angepasst, dass sie g{\"a}ngigen Regularien zur Produktion medizinischer Produkte entsprechen. Hier zeigten die Zellen auch in anschließenden Analysen, dass charakteristische Merkmale nicht verloren hatten. Dar{\"u}ber hinaus gelang es, die Zellen mithilfe verschiedener protektiver Substanzen erfolgreich einzufrieren und zu konservieren. Des Weiteren konnte ein Modell etabliert werden, das eine Verbrennung ersten Grades widerspiegelt. {\"U}ber einen Zeitraum von zwei Wochen wurde seine Regeneration hinsichtlich verschiedener Aspekte, wie der Histomorphologie, dem Metabolismus und der Reepithelialisierungsrate, untersucht. Die Modelle zeigten hier viele Parallelen zur Wundheilung in vivo auf. Um die Eignung der Modelle zur Testung von Wirkstoffen zu ermitteln wurde außerdem eine Behandlung mit 5\% Dexpanthenol getestet. Sie resultierte in einer verbesserten Histomorphologie und einer erh{\"o}hten Anzahl an proliferativen Zellen in den Modellen, beschleunigte jedoch die Reepithelialisierung nicht. Zusammengefasst konnten in dieser Arbeit zun{\"a}chst Methoden etabliert werden, um ein medizinisches Produkt aus Keratinozyten herzustellen und zu charakterisieren. Außerdem wurde ein Modell entwickelt, anhand dessen die Wundheilung und Behandlung von Verbrennungen ersten Grades untersucht werden kann und welches als Basis zur Entwicklung von Modellen von tieferen Verbrennungen dienen kann.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@phdthesis{Schmidt2021,
  author    = {Schmidt, Stefanie},
  title     = {Cartilage Tissue Engineering - Comparison of Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells in Agarose and Hyaluronic Acid-Based Hydrogels},
  doi       = {10.25972/OPUS-25171},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251719},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Articular cartilage damage caused by sports accidents, trauma or gradual wear and tear can lead to degeneration and the development of osteoarthritis because cartilage tissue has only limited capacity for intrinsic healing. Osteoarthritis causes reduction of mobility and chronic pain and is one of the leading causes of disability in the elderly population. Current clinical treatment options can reduce pain and restore mobility for some time, but the formed repair tissue has mostly inferior functionality compared to healthy articular cartilage and does not last long-term. Articular cartilage tissue engineering is a promising approach for the improvement of the quality of cartilage repair tissue and regeneration. In this thesis, a promising new cell type for articular cartilage tissue engineering, the so-called articular cartilage progenitor cell (ACPC), was investigated for the first time in the two different hydrogels agarose and HA-SH/P(AGE-co-G) in comparison to mesenchymal stromal cells (MSCs). In agarose, ACPCs´ and MSCs´ chondrogenic capacity was investigated under normoxic (21 \% oxygen) and hypoxic (2 \% oxygen) conditions in monoculture constructs and in zonally layered co-culture constructs with ACPCs in the upper layer and MSCs in the lower layer. In the newly developed hyaluronic acid (HA)-based hydrogel HA-SH/P(AGE-co-G), chondrogenesis of ACPCs and MSCs was also evaluated in monoculture constructs and in zonally layered co-culture constructs like in agarose hydrogel. Additionally, the contribution of the bioactive molecule hyaluronic acid to chondrogenic gene expression of MSCs was investigated in 2D monolayer, 3D pellet and HA-SH hydrogel culture. It was shown that both ACPCs and MSCs could chondrogenically differentiate in agarose and HA-SH/P(AGE-co-G) hydrogels. In agarose hydrogel, ACPCs produced a more articular cartilage-like tissue than MSCs that contained more glycosaminoglycan (GAG), less type I collagen and only little alkaline phosphatase (ALP) activity. Hypoxic conditions did not increase extracellular matrix (ECM) production of ACPCs and MSCs significantly but improved the quality of the neo-cartilage tissue produced by MSCs. The creation of zonal agarose constructs with ACPCs in the upper layer and MSCs in the lower layer led to an ECM production in zonal hydrogels that lay in general in between the ECM production of non-zonal ACPC and MSC hydrogels. Even though zonal co-culture of ACPCs and MSCs did not increase ECM production, the two cell types influenced each other and, for example, modulated the staining intensities of type II and type I collagen in comparison to non-zonal constructs under normoxic and hypoxic conditions. In HA-SH/P(AGE-co-G) hydrogel, MSCs produced more ECM than ACPCs, but the ECM was limited to the pericellular region for both cell types. Zonal HASH/P(AGE-co-G) hydrogels resulted in a native-like zonal distribution of ECM as MSCs in the lower zone produced more ECM than ACPCs in the upper zone. It appeared that chondrogenesis of ACPCs was supported by hydrogels without biological attachment sites such as agarose, and that chondrogenesis of MSCs benefited from hydrogels with biological cues like HA. As HA is an attractive material for cartilage tissue engineering, and the HA-based hydrogel HA-SH/P(AGE-co-G) appeared to be beneficial for MSC chondrogenic differentiation, the contribution of HA to chondrogenic gene expression of MSCs was investigated. An upregulation of chondrogenic gene expression was found in 2D monolayer and 3D pellet culture of MSCs in response to HA supplementation, while gene expression of osteogenic and adipogenic transcription factors was not upregulated. MSCs, encapsulated in a HA-based hydrogel, showed upregulation of gene expression for chondrogenic, osteogenic and adipogenic differentiation markers as well as for stemness markers. In a 3D bioprinting process, using the HA-based hydrogel, gene expression levels of MSCs mostly did not change. Nevertheless, expression of three tested genes (COL2A1, SOX2, CD168) was downregulated in printed in comparison to cast constructs, underscoring the importance of closely monitoring cellular behaviour during and after the printing process. In summary, it was confirmed that ACPCs are a promising cell source for articular cartilage engineering with advantages over MSCs when they were cultured in a suitable hydrogel like agarose. The performance of the cells was strongly dependent on the hydrogel environment they were cultured in. The different chondrogenic performance of ACPCs and MSCs in agarose and HA-SH/P(AGE-co-G) hydrogels highlighted the importance of choosing suitable hydrogels for the different cell types used in articular cartilage tissue engineering. Hydrogels with high polymer content, such as the investigated HA-SH/P(AGE-co-G) hydrogels, can limit ECM distribution to the pericellular area and should be developed further towards less polymer content, leading to more homogenous ECM distribution of the cultured cells. The influence of HA on chondrogenic gene expression and on the balance between differentiation and maintenance of stemness in MSCs was demonstrated. More studies should be performed in the future to further elucidate the signalling functions of HA and the effects of 3D bioprinting in HA-based hydrogels. Taken together, the results of this thesis expand the knowledge in the area of articular cartilage engineering with regard to the rational combination of cell types and hydrogel materials and open up new possible approaches to the regeneration of articular cartilage tissue.},
  subject      = {Hyaliner Knorpel},
  language  = {en}
}
@phdthesis{Lodes2021,
  author    = {Lodes, Nina Theresa},
  title     = {Tissue Engineering f{\"u}r seltene Erkrankungen mit St{\"o}rungen des mukozili{\"a}ren Transports},
  doi       = {10.25972/OPUS-20017},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200178},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Bei der zystischen Fibrose (CF) sowie der prim{\"a}ren Ziliendyskinesie (PCD) handelt es sich um zwei seltene Erkrankungen, die unter anderem den mukozili{\"a}ren Transport beeintr{\"a}chtigen. CF geh{\"o}rt hierbei zu den am h{\"a}ufigsten vorkommenden angeborenen Stoffwechselerkrankungen, wobei Betroffene unter einem Defekt des Cystic Fibrosis Transmembrane Conductor Regulator (CFTR)-Gens leiden, der durch die Produktion von hochviskosem Sekret in muzinproduzierenden Organen, wie dem gastrointestinalen Trakt und der Lunge, gekennzeichnet ist. Patienten, die an PCD leiden, weisen Defekte in, zum jetzigen Zeitpunkt, ca. 38 bekannten und PCD-assoziierten Genen auf, die in strukturellen Defekten des zili{\"a}ren Apparats und somit in dysfunktionalen Kinozilien resultieren. Da aktuell weder f{\"u}r die CF noch f{\"u}r die PCD eine Heilung m{\"o}glich ist, steht bei der Therapie vor allem die Linderung der Symptome im Fokus. Grundlegendes Ziel ist der langfristige Erhalt der Lungenfunktion sowie die Pr{\"a}vention bakterieller Infekte. Als bisherige Modellsysteme zur Erforschung m{\"o}glicher Therapeutika gelten Tiermodelle, die den humanen Ph{\"a}notyp aufgrund von Speziesdiversit{\"a}t nicht vollst{\"a}ndig abbilden k{\"o}nnen. Als vielversprechende Testsysteme f{\"u}r die zystische Fibrose gelten humane intestinale Organoidkulturen. Nachdem allerdings vorwiegend respiratorische Symptome f{\"u}r die Mortalit{\"a}t der Patienten verantwortlich sind, stellen CF-Atemwegsmodelle bessere Testsysteme f{\"u}r zuk{\"u}nftige Therapeutika dar. Atmungsorganoidkulturen wurden verwendet, um die CFTR-Funktionalit{\"a}t zu untersuchen, repr{\"a}sentieren aber nicht vollst{\"a}ndig die in vivo Situation. Deshalb werden zur Entwicklung neuer Therapiestrategien patientenspezifische 3D in vitro Testsysteme der humanen Atemwege ben{\"o}tigt, die insbesondere im Hinblick auf personalisierte Medizin ihren Einsatz finden. In der vorliegenden Arbeit wurde eine f{\"u}r den Lehrstuhl neue Methode zur Zellgewinnung aus nasalen Schleimhautabstrichen etabliert, die eine standardisierte Versorgung mit humanem Prim{\"a}rmaterial garantiert. Zur Generierung einer krankheitsspezifischen Zelllinie, wie beispielsweise einer PCD-Zelllinie mit Hilfe des CRISPR/Cas9-Systems, ist eine Atemwegszelllinie erforderlich, die die in vivo Situation vollst{\"a}ndig repr{\"a}sentiert. So wurden vier verschiedene respiratorische Epithelzelllinien (HBEC3-KT, Calu-3, VA10 und Cl-huAEC) auf ihren mukozili{\"a}ren Ph{\"a}notyp hin untersucht, wobei lediglich die Zelllinie HBEC3-KT in zilientragende Zellen differenzierte. Diese zeigten jedoch nur auf ca. 5 \% der Modelloberfl{\"a}che Kinozilien, wodurch die humane respiratorische Mukosa nicht komplett abgebildet werden konnte und die HBEC3-KT-Zelllinie keine geeignete Zelllinie zur Generierung einer PCD-Zelllinie darstellte. Mit Hilfe des Tissue Engineering war es m{\"o}glich, 3D in vitro Testsysteme basierend auf zwei unterschiedlichen Matrices, der biologischen SIS (small intestinal submucosa) und der synthetischen Polyethylenterephthalat (PET)-Membran, aufzubauen. Es wurden 3D Atemwegstestsysteme mit humanen prim{\"a}ren nasalen und tracheobronchialen Epithelzellen generiert. Erg{\"a}nzend zu histologischen Untersuchungen und zur Charakterisierung spezifischer Marker des respiratorischen Systems mittels Immunfluoreszenz, wurde die Ultrastruktur der Modelle, mit speziellem Fokus auf zili{\"a}re Strukturen, analysiert. Um R{\"u}ckschl{\"u}sse auf die zili{\"a}re Funktionalit{\"a}t ziehen zu k{\"o}nnen und somit eine hohe in vivo Korrelation zu best{\"a}tigen, wurde im Rahmen dieser Arbeit am Lehrstuhl f{\"u}r Tissue Engineering und Regenerative Medizin die Methode der Hochgeschwindigkeitsvideomikroskopie etabliert, welche die Analyse der Zilienschlagfrequenz sowie des mukozili{\"a}ren Transports erm{\"o}glicht. Ebenfalls wurde der Einfluss von isotoner Kochsalzl{\"o}sung und des � 2-adrenergen Agonisten Salbutamol, das vor allem als Bronchodilatator bei Asthmapatienten eingesetzt wird, auf die Zilienschlagfrequenz analysiert. Es konnte gezeigt werden, dass beide Substanzen den Zilienschlag im Atemwegsmodell erh{\"o}hen. Zur Generierung der Testsysteme der beiden seltenen Erkrankungen CF und PCD wurden Epithelzellen der betroffenen Patienten zun{\"a}chst mittels nicht-invasiver Raman-Spektroskopie auf einen potentiellen Biomarker untersucht, welcher Einsatz in der Diagnostik der beiden Krankheiten finden k{\"o}nnte. Es konnte jedoch weder f{\"u}r die CF noch f{\"u}r die PCD ein Biomarker aufgedeckt werden. Jedoch zeigten PCD-Zellen eine geringe Auftrennung gegen{\"u}ber nicht-PCD Zellen. Anschließend wurden 3D-Atemwegstestsysteme basierend auf Patientenzellen aufgebaut. Der Ph{\"a}notyp der CF-Modelle wurde mittels immunhistologischer F{\"a}rbung und der Analyse des gest{\"o}rten mukozili{\"a}ren Transports verifiziert. Strukturelle zili{\"a}re Defekte konnten durch die ultrastrukturelle Analyse von Zilienquerschnitten in drei donorspezifischen PCD-Modellen identifiziert werden. Dar{\"u}ber hinaus konnte die zili{\"a}re Funktionalit{\"a}t mit Hilfe der Hochgeschwindigkeitsvideomikroskopie nicht nachgewiesen werden. Zusammenfassend ist es in dieser Arbeit gelungen, eine neue Methode zur vollst{\"a}ndigen Charakterisierung von 3D-Atemwegstestsystemen zu etablieren, die die Analyse der Zilienschlagfrequenz sowie des mukozili{\"a}ren Transports erm{\"o}glicht. Es konnte erstmalig gezeigt werden, dass mit Hilfe des Tissue Engineering ein personalisiertes Krankheitsmodell f{\"u}r die PCD auf Segmenten eines dezellularisierten porzinen Jejunums generiert werden kann, das zuk{\"u}nftig ein Testsystem f{\"u}r potentielle Therapeutika darstellen kann.},
  subject      = {In-vitro-Kultur},
  language  = {de}
}
@phdthesis{Wallstabe2020,
  author    = {Wallstabe, Julia},
  title     = {Induktion von GvHD-artigen Gewebesch{\"a}den an humanen artifiziellen Hautmodellen},
  doi       = {10.25972/OPUS-15396},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153966},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Graft-versus-Host Disease (GvHD) stellt einen h{\"a}ufigen, den Gesamterfolg einer allogenen h{\"a}matopoetischen Stammzelltransplantation limitierenden Faktor dar. Bei dieser Komplikation attackieren vor allem alloreaktive T-Lymphozyten des Stammzellspenders gesunde K{\"o}rperzellen des Patienten. Infolgedessen kommt es zu Gewebesch{\"a}den in den Zielorganen Haut, Leber und Darm. Die Behandlung der GvHD erfordert eine effektive Immunsuppression, was wiederum Graft-versusTumor-Effekte kompromittiert und den R{\"u}ckfall der malignen Grunderkrankung bedingen kann. Viele Patienten sprechen aus bisher ungekl{\"a}rten Gr{\"u}nden nicht auf die klassische immunsuppressive Therapie mit Steroiden oder second-line Therapien an. Neue zellul{\"a}re Therapien zur Behandlung der refrakt{\"a}ren GvHD sind auf dem Vormarsch, bed{\"u}rfen aber einer weiterf{\"u}hrenden klinischen Testung, auch um die exakten Wirkungsmechanismen zu verstehen. Idealerweise k{\"o}nnten neue Testsysteme das GvHD-Potential von allogenen Stammzellpr{\"a}paraten oder aber das immunsuppressive Potential von neuen GvHD-Therapien vorhersagen, bevor diese in klinischen Studien eingesetzt werden. Ziel der vorliegenden Arbeit war es, ein erstes, in multiplen Replikaten einsetzbares, humanes organotypisches Gewebemodell zur Simulation einer GvHD-Reaktion am Beispiel der Haut zu etablieren. Zu diesem Zweck wurden artifizielle humane Hautmodelle unter statischen (KollagenHautmodelle) und dynamischen Kulturbedingungen (vaskularisierte Hautmodelle) generiert. Die Injektion unstimulierter PBMCs (engl. peripheral blood mononuclear cells) f{\"u}hrte zu keinen histomorphologischen Ver{\"a}nderungen in den KollagenHautmodellen. Im Gegensatz dazu hatte die Injektion vorstimulierter allogener PBMCs eine Zerst{\"o}rung der epidermalen Strukturen der Kollagen-Hautmodelle zur Folge, welche vergleichbar waren mit Gewebesch{\"a}den bei einer akuten GvHD der Haut. Dieselben Sch{\"a}digungen der Epidermis wurden durch die Injektion von Medium{\"u}berst{\"a}nden vorstimulierter PBMCs in die Kollagen-Hautmodelle erreicht. Im Kulturmedium der Kollagen-Hautmodelle wurden hohe Konzentrationen von Interleukin 2 und 17, Interferon gamma sowie Tumornekrosefaktor alpha gemessen, wodurch auf die Beteiligung von Zytokinen an der inflammatorischen Reaktion geschlossen werden konnte. Auch im komplexeren vaskularisierten Hautmodell verursachte die Injektion vorstimulierter PBMCs histomorphologische Ver{\"a}nderungen entsprechend einer akuten Haut-GvHD sowie einen zeitabh{\"a}ngigen Anstieg proinflammatorischer Zytokine. Zusammenfassend zeigen die Resultate dieser Arbeit, dass die Induktion einer starken Inflammations- und Immunreaktion in artifiziellen humanen Hautmodellen, welche histomorphologisch eine GvHD imitiert, m{\"o}glich ist. Dieses Modell k{\"o}nnte als Grundlage f{\"u}r die Entwicklung eines klinisch relevanten Testsystems zur Bestimmung des GvHD-Restpotentials oder zur Festlegung der immunsuppressiven Kapazit{\"a}t innovativer Zellpr{\"a}parate dienen. Somit k{\"o}nnten humane artifizielle GvHDModelle in klinischen Studien eingesetzt werden und die Erfahrungen aus Tiermodellen erg{\"a}nzen sowie erste in vitro Ergebnisse im humanen System liefern, welche dann mit dem tats{\"a}chlichen klinischen Resultat verglichen werden k{\"o}nnten.},
  subject      = {Graft-versus-host-disease},
  language  = {de}
}
@phdthesis{Wiesner2020,
  author    = {Wiesner, Miriam},
  title     = {Stem Cell-based Adipose Tissue Engineering - Engineering of Prevascularized Adipose Tissue Constructs In Vitro \& Investigation on Gap Junctional Intercellular Communication in Adipose-derived Stem Cells},
  doi       = {10.25972/OPUS-18500},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185005},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In reconstructive and plastic surgery, there exists a growing demand of adequate tissue implants, since currently available strategies for autologous transplantation are limited by complications including transplant failure and donor site morbidity. By developing in vitro and in vivo autologous substitutes for defective tissue sites, adipose tissue engineering can address these challenges, although there are several obstacles to overcome. One of the major limitations is the sufficient vascularization of in vitro engineered large constructs that remains crucial and demanding for functional tissues. Decellularized jejunal segments may represent a suitable scaffolding system with preexisting capillary structures that can be repopulated with human microvascular endothelial cells (hMVECs), and a luminal matrix applicable for the adipogenic differentiation of human adipose-derived stem cells (hASCs). Hence, co-culture of these cells in jejunal segments, utilizing a custom-made bioreactor system, was characterized in terms of vascularization and adipose tissue development. Substantial adipogenesis of hASCs was demonstrated within the jejunal lumen in contrast to non-induced controls, and the increase of key adipogenic markers was verified over time upon induction. The development of major extracellular matrix components of mature adipose tissue, such as laminin and collagen IV, was shown within the scaffold in induced samples. Successful reseeding of the vascular network with hMVECs was demonstrated in long-term culture and co-localization of vascular structures and adipogenically differentiated hASCs was observed. Therefore, these results represent a novel approach for in vitro engineering of vascularized adipose tissue constructs that warrants further investigations in preclinical studies. Another still existing obstacle in adipose tissue engineering is the insufficient knowledge about the applied cells, for instance the understanding of how cells can be optimally expanded and differentiated for successful engineering of tissue transplants. Even though hASCs can be easily isolated from liposuction of abdominal fat depots, yielding low donor site morbidity, huge numbers of cells are required to entirely seed complex and large 3D matrices or scaffolds. Thus, cells need to be large-scale expanded in vitro on the premise of not losing their differentiation capacity caused by replicative aging. Accordingly, an improved differentiation of hASCs in adipose tissue engineering approaches remains still desirable since most engineered constructs exhibit an inhomogeneous differentiation pattern. For mesenchymal stem cells (MSCs), it has been shown that growth factor application can lead to a significant improvement of both proliferation and differentiation capacity. Especially basic fibroblast growth factor (bFGF) represents a potent mitogen for MSCs, while maintaining or even promoting their osteogenic, chondrogenic and adipogenic differentiation potential. As there are currently different contradictory information present in literature about the applied bFGF concentration and the explicit effect of bFGF on ASC differentiation, here, the effect of bFGF on hASC proliferation and differentiation capacity was investigated at different concentrations and time points in 2D culture. Preculture of hASCs with bFGF prior to adipogenic induction showed a remarkable effect, whereas administration of bFGF during culture did not improve adipogenic differentiation capacity. Furthermore, the observations indicated as mode of action an impact of this preculture on cell proliferation capacity, resulting in increased cellular density at the time of adipogenic induction. The difference in cell density at this time point appeared to be pivotal for increased adipogenic capacity of the cells, which was confirmed in a further experiment employing different seeding densities. Interestingly, furthermore, the obtained results suggested a cell-cell contact-mediated mechanism positively influencing adipogenic differentiation. As a consequence, subsequently, studies were conducted focusing on intercellular communication of these cells, which has hardly been investigated to date. Despite the multitude of literature on the differentiation capacity of ASCs, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in hASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in hASCs was demonstrated histologically and on the gene and protein expression level and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of hASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, these results demonstrate the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of hASCs and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Nelke2019,
  author    = {Nelke, Lena},
  title     = {Establishment and optimization of 3-dimensional mamma carcinoma models for therapy simulation and drug testing},
  doi       = {10.25972/OPUS-17228},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172280},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Breast cancer is the most common cancer among women worldwide and the second most common cause of cancer death in the developed countries. As the current state of the art in first-line drug screenings is highly ineffective, there is an urgent need for novel test systems that allow for reliable predictions of drug sensitivity. In this study, a tissue engineering approach was used to successfully establish and standardize a 3-dimensional (3D) mamma carcinoma test system that was optimized for the testing of anti-tumour therapies as well as for the investigation of tumour biological issues. This 3D test system is based on the decellularised scaffold of a porcine small intestinal segment and represents the three molecular subsets of oestrogen receptor-positive, HER2/Neu-overexpressing and triple negative breast cancer (TNBC). The characterization of the test system with respect to morphology as well as the expression of markers for epithelial-mesenchymal transition (EMT) and differentiation indicate that the 3D tumour models cultured under static and dynamic conditions reflect tumour relevant features and have a good correlation with in vivo tumour tissue from the corresponding xenograft models. In this respect, the dynamic culture in a flow bioreactor resulted in the generation of tumour models that exhibited best reflection of the morphology of the xenograft material. Furthermore, the proliferation indices of 3D models were significantly reduced compared to 2-dimensional (2D) cell culture and therefore better reflect the in vivo situation. As this more physiological proliferation index prevents an overestimation of the therapeutic effect of cytostatic compounds, this is a crucial advantage of the test system compared to 2D culture. Moreover, it could be shown that the 3D models can recapitulate different tumour stages with respect to tumour cell invasion. The scaffold SISmuc with the preserved basement membrane structure allowed the investigation of invasion over this barrier which tumour cells of epithelial origin have to cross in in vivo conditions during the process of metastasis formation. Additionally, the data obtained from ultrastructural analysis and in situ zymography indicate that the invasion observed is connected to a tumour cell-associated change in the basement membrane in which matrix metalloproteinases (MMPs) are also involved. This features of the model in combination with the mentioned methods of analysis could be used in the future to mechanistically investigate invasive processes and to test anti-metastatic therapy strategies. The validation of the 3D models as a test system with respect to the predictability of therapeutic effects was achieved by the clinically relevant targeted therapy with the monoclonal antibody trastuzumab which induces therapeutic response only in patients with HER2/Neu-overexpressing mamma carcinomas due to its specificity for HER2. While neither in 2D nor in 3D models of all molecular subsets a clear reduction of cell viability or an increase in apoptosis could be observed, a distinct increase in antibody-dependent cell-mediated cytotoxicity (ADCC) was detected only in the HER2/NEU-overexpressing 3D model with the help of an ADCC reporter gene assay that had been adapted for the application in the 3D model in the here presented work. This correlates with the clinical observations and underlines the relevance of ADCC as a mechanism of action (MOA) of trastuzumab. In order to measure the effects of ADCC on the tumour cells in a direct way without the indirect measurement via a reporter gene, the introduction of an immunological component into the models was required. This was achieved by the integration of peripheral blood mononuclear cells (PBMCs), thereby allowing the measurement of the induction of tumour cell apoptosis in the HER2/Neu-overexpressing model. Hence, in this study an immunocompetent model could be established that holds the potential for further testing of therapies from the emergent field of cancer immunotherapies. Subsequently, the established test system was used for the investigation of scientific issues from different areas of application. By the comparison of the sensitivity of the 2D and 3D model of TNBC towards the water-insoluble compound curcumin that was applied in a novel nanoformulation or in a DMSO-based formulation, the 3D test system was successfully applied for the evaluation of an innovative formulation strategy for poorly soluble drugs in order to achieve cancer therapy-relevant concentrations. Moreover, due to the lack of targeted therapies for TNBC, the TNBC model was applied for testing novel treatment strategies. On the one hand, therapy with the WEE1 kinase inhibitor MK 1775 was evaluated as a single agent as well as in combination with the chemotherapeutic agent doxorubicin. This therapy approach did not reveal any distinct benefits in the 3D test system in contrast to testing in 2D culture. On the other hand, a novel therapy approach from the field of cellular immunotherapies was successfully applied in the TNBC 3D model. The treatment with T cells that express a chimeric antigen receptor (CAR) against ROR1 revealed in the static as well as in the dynamic model a migration of T cells into the tumour tissue, an enhanced proliferation of T cells as well as an efficient lysis of the tumour cells via apoptosis and therefore a specific anti-cancer effect of CAR-transduced T cells compared to control T cells. These results illustrate that the therapeutic application of CAR T cells is a promising strategy for the treatment of solid tumours like TNBC and that the here presented 3D models are suitable for the evaluation and optimization of cellular immunotherapies. In the last part of this work, the 3D models were expanded by components of the tumour stroma for future applications. By coculture with fibroblasts, the natural structures of the intestinal scaffold comprising crypts and villi were remodelled and the tumour cells formed tumour-like structures together with the fibroblasts. This tissue model displayed a strong correlation with xenograft models with respect to morphology, marker expression as well as the activation of dermal fibroblasts towards a cancer-associated fibroblast (CAF) phenotype. For the integration of adipocytes which are an essential component of the breast stroma, a coculture with human adipose-derived stromal/stem cells (hASCs) which could be successfully differentiated along the adipose lineage in 3D static as well as dynamic models was established. These models are suitable especially for the mechanistic analysis of the reciprocal interaction between tumour cells and adipocytes due to the complex differentiation process. Taken together, in this study a human 3D mamma carcinoma test system for application in the preclinical development and testing of anti-tumour therapies as well as in basic research in the field of tumour biology was successfully established. With the help of this modular test system, relevant data can be obtained concerning the efficacy of therapies in tumours of different molecular subsets and different tumour stages as well as for the optimization of novel therapy strategies like immunotherapies. In the future this can contribute to improve the preclinical screening and thereby to reduce the high attrition rates in pharmaceutical industry as well as the amount of animal experiments.},
  subject      = {Brustkrebs},
  language  = {en}
}