@phdthesis{Schwedhelm2019,
  author    = {Schwedhelm, Ivo Peter},
  title     = {A non-invasive microscopy platform for the online monitoring of hiPSC aggregation in suspension cultures in small-scale stirred tank bioreactors},
  doi       = {10.25972/OPUS-19298},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192989},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The culture of human induced pluripotent stem cells (hiPSCs) at large-scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Suspension cul- tures of hiPSCs are characterized by the self-aggregation of single cells into macroscopic cell aggre- gates that increase in size over time. The development of these free-floating aggregates is dependent on the culture vessel and thus represents a novel process parameter that is of particular interest for hiPSC suspension culture scaling. Further, aggregates surpassing a critical size are prone to spon- taneous differentiation or cell viability loss. In this regard, and, for the first time, a hiPSC-specific suspension culture unit was developed that utilizes in situ microscope imaging to monitor and to characterize hiPSC aggregation in one specific CSTR setup to a statistically significant degree while omitting the need for error-prone and time-intensive sampling. For this purpose, a small-scale CSTR system was designed and fabricated by fused deposition modeling (FDM) using an in-house 3D- printer. To provide a suitable cell culture environment for the CSTR system and in situ microscope, a custom-built incubator was constructed to accommodate all culture vessels and process control devices. Prior to manufacture, the CSTR design was characterized in silico for standard engineering parameters such as the specific power input, mixing time, and shear stress using computational fluid dynamics (CFD) simulations. The established computational model was successfully validated by comparing CFD-derived mixing time data to manual measurements. Proof for system functionality was provided in the context of long-term expansion (4 passages) of hiPSCs. Thereby, hiPSC aggregate size development was successfully tracked by in situ imaging of CSTR suspensions and subsequent automated image processing. Further, the suitability of the developed hiPSC culture unit was proven by demonstrating the preservation of CSTR-cultured hiPSC pluripotency on RNA level by qRT-PCR and PluriTest, and on protein level by flow cytometry.},
  subject      = {Induzierte pluripotente Stammzelle},
  language  = {en}
}
@phdthesis{RamaniMohan2021,
  author    = {Ramani Mohan, Ramkumar},
  title     = {Effect of Mechanical Stress On Stem Cells to Improve Better Bone Regeneration},
  doi       = {10.25972/OPUS-24013},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240134},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Critical size bone defects and nonunion fractures remain difficult to treat. Although cell-loaded bone substitutes have improved bone ingrowth and formation, the lack of methods for achieving viability and the uniform distribution of cells in the scaffold limits their use as bone grafts. In addition, the predominant mechanical stimulus that drives early osteogenic cell maturation has not been clearly identified. Further, it is challenging to evaluate mechanical stimuli (i.e., deformation and fluid-flow-induced shear stress) because they are interdependent. This thesis compares different mechanical stimuli applied to cell-seeded scaffolds to develop bone grafts efficiently for the treatment of critical size bone defects. It also seeks to understand how deformation strain and interstitial fluid-flow-induced shear stress promote osteogenic lineage commitment. In this thesis, different scaffolds were seeded with primary human bone marrow mesenchymal stem cells (BM-MSCs) from different donors and subjected to static and dynamic culture conditions. In contrast with the static culture conditions, homogenous cell distributions were accomplished under dynamic culture conditions. Additionally, the induction of osteogenic lineage commitment without the addition of soluble factors was observed in the bioreactor system after one week of cell culture. To determine the role of mechanical stimuli, a bioreactor was developed to apply mechanical deformation force to a mesenchymal stem sell (MSC) line (telomerase reverse transcriptase (TERT)) expressing a strain-responsive AP-1 luciferase reporter construct on porous scaffolds. Increased luciferase expression was observed in the deformation strain compared with the shear stress strain. Furthermore, the expression of osteogenic lineage commitment markers such as osteonectin, osteocalcin (OC), osteopontin, runt-related transcription factor 2 (RUNX2), alkaline phosphate (AP), and collagen type 1 was significantly downregulated in the shear stress strain compared with the deformation strain. These findings establish that the deformation strain was the predominant stimulus causing skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation. Finally, these findings were used to develop a bioreactor in vitro test system in which the effect of medication on osteoporosis could be tested. Primary human BM-MSCs from osteoporotic donors were subjected to strontium ranelate (an osteoporotic drug marketed as Protelos®). Increased expression of collagen type 1 and calcification was seen in the drugtreated osteoporotic stem cells compared with the nondrug-treated osteoporotic stem cells. Thus, this bioreactor technology can easily be adapted into an in vitro osteoporotic drug testing system.},
  language  = {en}
}
@phdthesis{Jannasch2019,
  author    = {Jannasch, Maren Annika},
  title     = {In vitro Fremdk{\"o}rpermodellsysteme zur Vorhersage von biomaterialinduzierten Immunreaktionen},
  doi       = {10.25972/OPUS-16289},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162893},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die Implantation eines Medizinprodukts in den menschlichen K{\"o}rper ruft eine Immunreaktion hervor, die zur fibr{\"o}sen Einkapselung f{\"u}hren kann. Makrophagen in direktem Kontakt mit der Oberfl{\"a}che des Implantats erfassen sensorisch den Fremdk{\"o}rper und {\"u}bersetzten das Signal in die Freisetzung zahlreicher l{\"o}slicher Mediatoren. Das generierte Entz{\"u}ndungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erh{\"o}hung der Matrixsyntheserate in der Wundumgebung f{\"u}hren. Eine dichte fibr{\"o}se Kapsel um ein Medizinprodukt beeintr{\"a}chtigt den Ersatz von K{\"o}rperstrukturen, das Unterst{\"u}tzen physiologischer K{\"o}rperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterst{\"u}tzt werden. Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begr{\"u}ndet die Komplexi-t{\"a}t der ablaufenden Fremdk{\"o}rperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verf{\"u}gbarkeit validierter, verl{\"a}sslicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen w{\"a}hrend einer Fremdk{\"o}rperreaktion abbildet und sich zur Testung {\"u}ber l{\"a}ngere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch k{\"o}nnen in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine h{\"o}here {\"U}bertragbarkeit auf den Menschen aufweisen. Zus{\"a}tzlich adressiert die Pr{\"a}ferenz zu in vitro Testmethoden den Aspekt „Reduzierung" der 3R-Prinzipien „Replacement, Reduction, Refinement" (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begr{\"u}ndeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium f{\"u}r die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellul{\"a}rer Matrix in der Wundumgebung stellen wichtige Teilprozesse w{\"a}hrend einer Fremdk{\"o}rperreaktion dar. F{\"u}r beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von l{\"o}slichen Mediatoren durch materialadh{\"a}rente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden. Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialst{\"a}ndige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberfl{\"a}chenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix erm{\"o}glichte die {\"U}bersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Dar{\"u}ber hinaus konnte in einem dreidimensionalen Fremdk{\"o}rpermodell die komplexe dreidimensionale Struktur der extrazellul{\"a}ren Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdk{\"o}rpermodelle erm{\"o}glichten die Testung von Biomaterialien {\"u}ber l{\"a}ngere Testphasen und k{\"o}nnen in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen erm{\"o}glichen, (II) die Identifizierung verl{\"a}sslicher Testbedingungen unterst{\"u}tzen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verl{\"a}ssliche Unterscheidungskapazit{\"a}t zwischen Biomaterialien aufweist.},
  subject      = {Biomaterial},
  language  = {de}
}
@phdthesis{Weigel2019,
  author    = {Weigel, Tobias Maximilian},
  title     = {Entwicklung von 3D-Herzschrittmacher-Elektroden auf Basis von Kohlenstoffnanofasern},
  doi       = {10.25972/OPUS-17636},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176362},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Herzschrittmachersysteme sind eine weitverbreitete M{\"o}glichkeit Herz-Kreislauf-Erkrankungen zu behandeln. Wegen der nat{\"u}rlichen Reaktion des Immunsystems auf Fremdk{\"o}rper, erfolgt aber eine fortschreitende Verkapselung der Herzschrittmacherelektrode. Die Folge ist eine ansteigende Verminderung der Stimulationseffizienz durch Erh{\"o}hung der Anregungsschwelle. Die Integration der Elektrode in das Gewebe ist dabei mangelhaft und wird bestimmt durch Implantateigenschaften wie Gr{\"o}ße, Flexibilit{\"a}t und Dimensionalit{\"a}t. Um die Integration zu verbessern, stellen dreidimensionale (3D) bzw. gewebeartige Elektroden eine Alternative zu den derzeit verwendeten planaren Metallelektroden dar. Zur Entwicklung einer leitf{\"a}higen, 3D und faserf{\"o}rmigen Elektrode wurden in dieser Arbeit Kohlenstoff-Nanofaser-Scaffolds {\"u}ber Elektrospinnen hergestellt. Durch die Modifikation des Faserger{\"u}stes mit Natriumchlorid (NaCl) w{\"a}hrend der Scaffoldherstellung, konnte das Fasernetzwerk aufgelockert und Poren generiert werden. Die Kohlenstofffaser-Elektroden zeigten einen effizienten Energie{\"u}bertrag, welcher vergleichbar mit heutigen Titannitrid (TiN) -Elektroden ist. Die Auflockerung des Fasergewebes hatte eine verbesserte Flexibilit{\"a}t des Faserscaffolds zu Folge. Neben der Flexibilit{\"a}t, konnte auch die Infiltration von Zellen in das por{\"o}se Faserscaffold erheblich verbessert werden. Dabei konnten Fibroblasten durch das gesamte Scaffold migrieren. Die Kompatibilit{\"a}t mit kardialen Zellen, die Grundvoraussetzung von Herzschrittmacherelektroden, wurde in vitro nachgewiesen. Durch die Kombination aus dem 3D-Elektrodenger{\"u}st mit einer Co-Kultur aus humanen Kardiomyozyten, mesenchymalen Stammzellen und Fibroblasten, erfolgte eine Einbettung der Elektrode in funktionelles kardiales Gewebe. Dadurch konnte ein lebender Gewebe-Elektroden-Hybrid generiert werden, welcher m{\"o}glicherweise die Elektrode vor Immunzellen in vivo abschirmen kann. Eine Zusammenf{\"u}hrung der hybriden Elektrode mit einen Tissue-Engineerten humanen kardialen Patch in vitro, f{\"u}hrte zu Bildung einer nahtlosen Elektronik-Gewebe-Schnittstelle. Die fusionierte Einheit wurde abschließend auf ihre mechanische Belastbarkeit getestet und konnte {\"u}ber einen Elektroden-Anschluss elektrisch stimuliert werden.},
  subject      = {Herzschrittmacher},
  language  = {de}
}
@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{SchmidtgebSchmid2023,
  author    = {Schmidt [geb. Schmid], Freia Florina},
  title     = {Ein dreidimensionales kutanes Melanommodell f{\"u}r den Einsatz in der pr{\"a}klinischen Testung},
  doi       = {10.25972/OPUS-32925},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-329255},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Das maligne Melanom nimmt als Tumorerkrankung mit hoher Metastasierungsrate und steigenden Inzidenzraten bei h{\"o}chster Mortalit{\"a}t aller Hauttumoren eine zunehmende Bedeutung in der modernen Onkologie ein. Fr{\"u}hzeitige Diagnosem{\"o}glichkeiten und moderne Behandlungen konnten das {\"U}berleben der Patienten bereits erheblich verbessern. Jedoch besteht nach wie vor Bedarf an geeigneten Modellen, um die Melanomprogression vollst{\"a}ndig zu verstehen und neue wirksame Therapien zu entwickeln. Hierf{\"u}r werden h{\"a}ufig Tiermodelle verwendet, diese spiegeln jedoch nicht die menschliche Mikroumgebung wider. Zweidimensionalen Zellkulturen fehlen dagegen entscheidende Elemente der Tumormikroumgebung. Daher wurde in dieser Arbeit ein dreidimensionales epidermales Tumormodell des malignen Melanoms, welches aus prim{\"a}ren humanen Keratinozyten und verschiedenen Melanomzelllinien besteht, entwickelt. Die eingesetzten Melanomzelllinien variieren in ihren Treibermutationen, wodurch das Modell in der Lage ist, Wirkstoffe zu untersuchen, die spezifisch auf diese Mutationen wirken. Mit Techniken des Tissue Engineerings konnte ein dreidimensionales Hautmodell aufgebaut werden, das alle charakteristischen Schichten der Epidermis aufweist und im Bereich des stratum basale Melanomcluster ausbildet. Diese reichen je nach Gr{\"o}ße und Ausdehnung bis in suprabasale Epidermisschichten hinein. Die Tumor-Histopathologie, der Tumorstoffwechsel sowie tumorassoziierte Proteinsekretionen ließen sich im in vitro Modell nachweisen. Dar{\"u}ber hinaus konnte ein Protokoll entwickelt werden, mit dem einzelne Zellen aus den Modellen reisoliert werden k{\"o}nnen. Dies erm{\"o}glichte es, den Proliferationszustand innerhalb des jeweiligen Modells zu charakterisieren und die Wirkung von Antitumortherapien gezielt zu bewerten. Die Anwendbarkeit als Testsystem im Bereich der Tumortherapeutika wurde mit dem in der Klinik h{\"a}ufig verwendeten v-raf-Maus-Sarkom-Virus-Onkogen-Homolog B (BRAF)-Inhibitor Vemurafenib demonstriert. Der selektive BRAF-Inhibitor reduzierte erfolgreich das Tumorwachstum in den Modellen mit BRAF-mutierten Melanomzellen, was durch eine Verringerung der metabolischen Aktivit{\"a}t, der proliferierenden Zellen und des Glukoseverbrauchs gezeigt wurde. F{\"u}r die Implementierung des Modells in die pr{\"a}klinische Therapieentwicklung wurde B-B-Dimethylacrylshikonin, ein vielversprechender Wirkstoffkandidat, welcher einen Zellzyklusarrest mit anschließender Apoptose bewirkt, im Modell getestet. Bei einer Anwendung der Modelle im Bereich der Testung topischer Behandlungen ist eine Barrierefunktion der Modelle notwendig, die der in vivo Situation nahe kommt. Die Barriereeigenschaften der Haut{\"a}quivalente wurden durch die Melanomzellen nachweislich nicht beeinflusst, sind aber im Vergleich zur in vivo Situation noch unzureichend. Eine signifikante Steigerung der Hautbarriere konnte durch die Bereitstellung von Lipiden und die Anregung hauteigener Regenerationsprozesse erreicht werden. {\"U}ber den Nachweis des transepidermalen Wasserverlusts konnte eine Messmethode zur nicht-invasiven Bestimmung der Hautbarriere etabliert und {\"u}ber den Vergleich zur Impedanzspektroskopie validiert werden. Hierbei gelang es, erstmals die Korrelation der Hautmodelle zur in vivo Situation {\"u}ber ein solches Verfahren zu zeigen. Das entwickelte epidermale Modell konnte durch die Integration eines dermalen Anteils und einer Endothelzellschicht noch weiter an die komplexe Struktur und Physiologie der Haut angepasst werden um Untersuchungen, die mit der Metastierung und Invasion zusammenh{\"a}ngen, zu erm{\"o}glichen. Die artifizielle Dermis basiert auf einem Kollagen-Hydrogel mit prim{\"a}ren Fibroblasten. Eine dezellularisierte Schweinedarmmatrix ließ sich zur Erweiterung des Modells um eine Endothelzellschicht nutzen. Dabei wanderten die prim{\"a}ren Fibroblasten apikal in die nat{\"u}rliche Schweindarmmatrix ein, w{\"a}hrend die Endothelzellen basolateral eine geschlossene Schicht bildeten. Die in dieser Arbeit entwickelten Gewebemodelle sind in der Lage, die Vorhersagekraft der in vitro Modelle und die in vitro - in vivo Korrelation zu verbessern. Durch die Kombination des Melanommodells mit einer darauf abgestimmten Analytik wurde ein neuartiges Werkzeug f{\"u}r die pr{\"a}klinische Forschung zur Testung von pharmazeutischen Wirkstoffen geschaffen.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@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{Fey2023,
  author    = {Fey, Philipp},
  title     = {KI-gest{\"u}tzte MR-Klassifizierung von Zellen und zellul{\"a}rer Differenzierung},
  doi       = {10.25972/OPUS-34516},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-345164},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {F{\"u}r die Verwendung von zellbasierten Therapeutika ist vor allem die korrekt Identifikation sowohl vom Ausgangsmaterial wie auch dem produziertem Material von zentraler Wichtigkeit. In dieser Arbeit wurde eine Methodik entwickelt, welche eine nicht-invasive Klassifizierung von Zellen und zellul{\"a}rer Entwicklung aufgrund ihrer zweidimensionalen Magnetresonanz-Korrelationsspektren erm{\"o}glichte. Hierzu wurde ein mobiler MR-Scanner mit einer Feldst{\"a}rke von 0.5T und einem Isozentrum von 1 cm3 verwendet. Aufgrund der kompakten und leichten Bauweise war es m{\"o}glich, das System in normalen Zellkulturlaboren zu verwenden. Von den Proben wurde ein zweidimensionales T1/T2 -Korrelationsspektrum aufgenommen, anhand dessen die Zellen klassifiziert werden sollten. Mithilfe von Agarose-Dotagraf® -Zell- Phantomen konnte die Stabilit{\"a}t und Reproduzierbarkeit des Messsystems und der verwendeten Sequenz validiert werden. Aufgrund der unter Umst{\"a}nden recht langen Messzeiten der MR-Technologie war auch die Handhabung und Kultur der Zellproben w{\"a}hrend des Messprozesses von großer Bedeutung. Um hierf{\"u}r den Durchsatz an Proben zu erh{\"o}hen, wurde eine kosteng{\"u}nstige und ebenfalls mobile Robotikanlage entwickelt. Diese basierte auf dem kommerziell erh{\"a}ltlichen Roboterarm Braccio, welcher durch einen Arduino Mega Mikrocontroller gesteuert wurde. Mit bis zu 24 Proben pro Tag konnte durch die Automatisierung der Durchsatz an Proben um den Faktor 3 - 4 gesteigert werden. Durch den entwickelten Prozess war es m{\"o}glich, eine umfangreiche Datenbank - bestehend aus 362 unabh{\"a}ngigen Messungen (biologische Replikate) - aufzubauen. Die Datenbank enthielt Messungen von zehn unterschiedlichen Zelllinien. Zus{\"a}tzlich wurden T1/T2 -Korrelationsspektren von mesenchymalen Stromazellen (MSCs) vor und nach deren Differenzierung zu Adipocyten aufgenommen, um ihre zellul{\"a}re Entwicklung nicht-invasiv charakterisieren zu k{\"o}nnen. Die aufgenommenen Daten wurden mithilfe einer geeigneten Support Vector Machine wie auch angepassten k{\"u}nstlichen neuronalen Netzwerken klassifiziert. Mithilfe dieser Methoden konnten die Zelllinien und MSCs anhand ihrer aufgenommenen Korrelationsspektren mit einer Genauigkeit von bis zu 98\% klassifiziert werden. Diese hohe Treffsicherheit legte den Schluss nahe, dass die Kombination aus nichtinvasiver, zweidimensionaler T1/T2 -MR-Relaxometrie und der Verwendung von geeigneten Methoden des machine learning und der k{\"u}nstlichen Intelligenz eine effiziente Methodik f{\"u}r die nicht-invasive Klassifizierung von Zellen sowie zellul{\"a}rer Entwicklung darstellt.},
  subject      = {Kernspintomografie},
  language  = {de}
}
@phdthesis{Andelovic2024,
  author    = {Andelovic, Kristina},
  title     = {Characterization of arterial hemodynamics using mouse models of atherosclerosis and tissue-engineered artery models},
  doi       = {10.25972/OPUS-30360},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303601},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted: 1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI 2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis 3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice. The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition. In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly - at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe-/- mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD. Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis. Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models - two parameters highly influencing plaque development and progression - there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis. Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development. Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis. Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models. In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.},
  subject      = {H{\"a}modynamik},
  language  = {en}
}
@phdthesis{Choi2024,
  author    = {Choi, Jihyoung},
  title     = {Development of an Add-On Electrode for Non-Invasive Monitoring in Bioreactor Cultures and Medical Devices},
  doi       = {10.25972/OPUS-35823},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358232},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies. In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.},
  subject      = {Monitoring},
  language  = {en}
}