Engineering Blood and Lymphatic Microvascular Networks in Fibrin Matrices

Vascular network engineering is essential for nutrient delivery to tissue-engineered constructs and, consequently, their survival. In addition, the functionality of tissues also depends on tissue drainage and immune cell accessibility, which are the main functions of the lymphatic system. Engineering both the blood and lymphatic microvasculature would advance the survival and functionality of tissue-engineered constructs. The aim of this study was to isolate pure populations of lymphatic endothelial cells (LEC) and blood vascular endothelial cells (BEC) from human dermal microvascular endothelial cells and to study their network formation in our previously described coculture model with adipose-derived stromal cells (ASC) in fibrin scaffolds. We could follow the network development over a period of 4 weeks by fluorescently labeling the cells. We show that LEC and BEC form separate networks, which are morphologically distinguishable and sustainable over several weeks. In addition, lymphatic network development was dependent on vascular endothelial growth factor (VEGF)-C, resulting in denser networks with increasing VEGF-C concentration. Finally, we confirm the necessity of cell–cell contact between endothelial cells and ASC for the formation of both blood and lymphatic microvascular networks. This model represents a valuable platform for in vitro drug testing and for the future in vivo studies on lymphatic and blood microvascularization

Chip-based human liver-intestine and liver-skin co-culture : A first step toward systemic repeated dose substance testing in vitro

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called “human-on-a-chip” concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air–liquid interface for the skin model during their co-culture with the liver equivalents respectively at 1/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver–skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance – troglitazone – to the chip-based co-cultures.BMBF/0315569/GO-Bio 3: Multi-Organ-Bioreaktoren für die prädiktive Substanztestung im Chipforma

A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.BMBF, 0315569, GO-Bio 3: Multi-Organ-Bioreaktoren für die prädiktive Substanztestung im Chipforma

Die Nachbildung der humanen Vaskulatur in einer Multi-Organ-Chip Plattform : Rheologie und Vaskulogenese

The Multi-Organ-Chip platform is a microphysiological system developed to evaluate toxicity and efficacy of drugs, and adverse effects of cosmetics, chemicals and alike in a sub-systemic mode. At the scale of a microscope glass slide, it comprises several compartments for the co-cultivation of human 3D tissue constructs. The organoids are physically separated, yet, interconnected through perfused microfluidics. The incorporated on chip micropump provides pulsatile circulation at a microliter scale – enough to provide oxygen, nutrition and deplete excreted products from the cells. The system contains a minute volume of medium enabling crosstalk and interaction of the organoids. The resulting tissue-to-fluid ratio is more physiological-like than in comparable systems. The organoid cultures are, however, not sufficiently vascularised to overcome limitations in size and complexity. Hence, this dissertation’s objective is to contribute to the recreation of a continuous endothelial barrier throughout the system. For this, three major aspects were addressed: (1) Implementing a near-physiological, pulsatile flow. It should provide an in vivo-like shear stress regime, which is required for a phenotypical behaviour of some of the incorporated cell types, specifically the endothelial cells. The complex fluid dynamics created by the micropump were characterised and – where possible – optimised. (2) Creating an endothelial lining within the chip’s microfluidic system. A prerequisite already set up in previous works. (3) Establishing capillary-like vessels in the cultivation compartments, preferably interconnected with the endothelialised microfluidics, as a direct route to the organoids. Fibrin hydrogels containing an endothelial / stromal cell co culture enabled the self-organised formation of microcapillaries. This work will address issues of dynamic versus static cultivation environments, the stability of the hydrogel, along with the influence of the medium constituents on the cell behaviour. The work will show that basic features of blood vessels could be emulated inside the Multi-Organ-Chip platform. A continuous endothelium is crucial for physiological-like interactions, regulation and homeostasis within organoid (co-)cultures as well as for long-term tissue cultivation. Moreover, it is a requirement for replacing medium with a full blood surrogate and to enable immunological queries.Die Multi-Organ-Chip Plattform ist ein mikrophysiologisches System, welches für die Beurteilung von Toxizität und Wirksamkeit von Medikamenten, sowie von negativen Auswirkungen von Kosmetika, Chemikalien und ähnlichem entwickelt wurde. Auf Größe eines Objektträgers enthält sie mehrere Kompartimente für jedwede subsystemische Co-Kultur dreidimensionaler Gewebekonstrukte. Diese Organoide sind zwar physisch voneinander getrennt, jedoch durch ein mikrofluidisches System strömungstechnisch miteinander verbunden. Die eingebaute on-chip Mikropumpe erzeugt eine pulsatile Strömung im Mikroliter-Maßstab – genug, um den Zellen Sauerstoff und Nährstoffe bereitzustellen und ausgeschiedene Produkte abzuführen. Das System verfügt über ein sehr geringes Volumen an Nährmedium und ermöglicht so den Austausch sowie Wechselwirkungen zwischen den Organoiden. Daraus resultiert auch ein Volumenverhältnis von Gewebe zu Medium, das näher an physiologischen Maßstäben liegt als in vergleichbaren Systemen. Die Gewebekonstrukte sind jedoch unzureichend vaskularisiert, um Beschränkungen in Größe und Komplexität zu überwinden. Gegenstand dieser Dissertation soll es daher sein, einen Beitrag zur Nachbildung der endothealen Barriere im gesamten System zu leisten. Drei Bedingungen müssen dafür erfüllt werden: (1) Ein nahezu physiologischer, pulsatiler Volumenstrom muss zur Verfügung gestellt werden. Dieser soll eine geeignete Schubspannung aufbauen, welche für ein phänotypisches Verhalten der verwendeten Zelltypen – insbesondere der Endothelzellen – erforderlich ist. Die komplexen Strömungsverhältnisse, die die Mikropumpe hervorruft, wurden charakterisiert. Wo es möglich war, fand eine Optimierung statt. (2) Das mikrofluidische System der Plattform muss komplett endothealisiert sein. Eine Voraussetzung die bereits in vorhergehenden Arbeiten erfüllt wurde. (3) Als direkte Route in die Organoide hinein müssen kapillarartige Gefäße in den Kultivierungskompartimenten erzeugt werden, die möglichst mit der Mikrofluidik verbunden sind. Fibrinhydrogele, die eine Co-Kultur aus Endothel- und Stromazellen enthalten, ermöglichten die sich selbst organisierende Bildung von Mikrokapillaren. Diese Arbeit wird sich mit Fragestellungen bezüglich dynamischer versus statischer Kulturbedingungen befassen, sowie mit der Stabilität der Hydrogele und dem Einfluss der Medienzusammensetzung auf das Verhalten der Zellen. Die Dissertation zeigt, dass grundlegende Charakteristika von Blutgefäßen innerhalb der Multi-Organ-Chip Plattform nachgebildet werden konnten. Ein durchgehendes Endothelium ist entscheidend für physiologische Interaktionen, Regulation und Homöostase innerhalb der Organoid(co)kulturen. Zudem ist es essentiell für Langzeitkultivierung der Gewebe. Darüber hinaus stellt es eine Voraussetzung dar, das Nährmedium mit einem Vollblutäquivalent zu ersetzen und immunologische Fragestellungen zu ermöglichen.BMBF, 031A597A, ERA Net EuroTransBio-9: VASC-MOC - Biologische Vaskualisierung eines Knochenmark-auf-dem-Chip-Modell

Simultaneous evaluation of anti-EGFR-induced tumour and adverse skin effects in a microfluidic human 3D co-culture model

Abstract Antibody therapies targeting the epithelial growth factor receptor (EGFR) are being increasingly applied in cancer therapy. However, increased tumour containment correlates proportionally with the severity of well-known adverse events in skin. The prediction of the latter is not currently possible in conventional in vitro systems and limited in existing laboratory animal models. Here we established a repeated dose “safficacy” test assay for the simultaneous generation of safety and efficacy data. Therefore, a commercially available multi-organ chip platform connecting two organ culture compartments was adapted for the microfluidic co-culture of human H292 lung cancer microtissues and human full-thickness skin equivalents. Repeated dose treatment of the anti-EGFR-antibody cetuximab showed an increased pro-apoptotic related gene expression in the tumour microtissues. Simultaneously, proliferative keratinocytes in the basal layer of the skin microtissues were eliminated, demonstrating crucial inhibitory effects on the physiological skin cell turnover. Furthermore, antibody exposure modulated the release of CXCL8 and CXCL10, reflecting the pattern changes seen in antibody-treated patients. The combination of a metastatic tumour environment with a miniaturized healthy organotypic human skin equivalent make this “safficacy” assay an ideal tool for evaluation of the therapeutic index of EGFR inhibitors and other promising oncology candidates

Potenziale digitaler Lehre und digitaler Kooperations- und Unterstützungsangebote zur Förderung der Theorie-Praxis-Verzahnung in der Lehrer*innenbildung

Bulizek B, Freudenau T, Habicher A, et al. Potenziale digitaler Lehre und digitaler Kooperations- und Unterstützungsangebote zur Förderung der Theorie-Praxis-Verzahnung in der Lehrer*innenbildung. In: Beißwenger M, Bulizek B, Gryl I, Schacht F, eds. Digitale Innovationen und Kompetenzen in der Lehramtsausbildung. Duisburg: Universitätsverlag Rhein-Ruhr; 2020: 209-233

Chip-based human liver-intestine and liver-skin co-cultures: A first step toward systemic repeated dose substance testing in vitro

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called “human-on-a-chip” concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air–liquid interface for the skin model during their co-culture with the liver equivalents respectively at 1/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver–skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance – troglitazone – to the chip-based co-cultures

Autologous induced pluripotent stem cell-derived four-organ-chip

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing