Ilka Wagner

A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a multi-organ-chip capable of maintaining 3D tissues derived from cell lines, primary cells and biopsies of various human organs. We designed a multi-organ-chip with co-cultures of human artificial liver microtissues and skin biopsies, each a (1)/100,000 of the biomass of their original human organ counterparts, and have successfully proven its long-term performance. The system supports two different culture modes: i) tissue exposed to the fluid flow, or ii) tissue shielded from the underlying fluid flow by standard Transwell® cultures. Crosstalk between the two tissues was observed in 14-day co-cultures exposed to fluid flow. Applying the same culture mode, liver microtissues showed sensitivity at different molecular levels to the toxic substance troglitazone during a 6-day exposure. Finally, an astonishingly stable long-term performance of the Transwell®-based co-cultures could be observed over a 28-day period. This mode facilitates exposure of skin at the air-liquid interface. Thus, we provide here a potential new tool for systemic substance testing.

De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro

Across many tissues and organs, the ability to create an organoid, the smallest functional unit of an organ, in vitro is the key both to tissue engineering and preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. But hair follicle formation in vitro has been elusive. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need. By carefully analyzing and recapitulating the growth conditions of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing hair. Our microfollicles contained all relevant cell types and their structure and orientation resembled in some ways excised hair follicle specimens from human skin. This finding offers a new window onto hair follicle development. Having a robust culture system for hair follicles is an important step towards improved hair regeneration as well as to an understanding of how marketed drugs or drug candidates, including cancer chemotherapy, will affect this important organ.

Skin and hair-on-a-chip: Hair and skin assembly versus native skin maintenance in a chip-based perfusion system

Background and novelty In recent decades, substantial progress to mimic structures and complex functions of human skin in the form of skin equivalents has been achieved. Different approaches to generate functional skin models were made possible by the use of improved bioreactor technologies and advanced tissue engineering. Although various forms of skin models are successfully being used in clinical applications, in basic research, current systems still lack essential physiological properties for toxicity testing and compound screening (such as for the REACH program) and are not suitable for high-throughput processes.

Dynamic culture of human liver equivalents inside a micro-bioreactor for long-term substance testing

Published by BioMed Central: Materne, Eva-Maria et al.: Dynamic culture of human liver equivalents inside a micro-bioreactor for longterm substance testing. - In: BMC Proceedings. - ISSN 1753-6561 (online). - 7 (2012), suppl. 6, art. P72. - doi:10.1186/1753-6561-7-S6-P72.

Miniaturisierte humane organtypische Zell- und Gewebekulturen

ZusammenfassungFür die Substanztestung wurde eine Methode zur Integration menschlicher Mikrofollikel in ein menschliches Epidermis-Dermis-Modell sowie eine vollständige Systemtechnik für die automatisierte Langzeitkultivierung in miniaturisierten, dynamischen Mikrobioreaktoren etabliert.AbstractA method to integrate human micro follicles into a human epidermis-dermis-model as well as a complete system technology for automated long-time culture in a miniaturised, dynamic micro bioreactor was established for substance testing.

Assessment of troglitazone induced liver toxicity in a dynamically perfused two-organ Micro-Bioreactor system

Background The ever-growing amount of new substances released to the market and the limited predictability of current in vitro test systems has led to an ample need for new substance testing solutions. Many drugs like troglitazone, that had to be removed from the market due to drug induced liver injury, show their toxic potential only after chronic long term exposure. But for long-term multiple dosing experiments, a controlled microenvironment is pivotal, as even minor alterations in extracellular conditions may greatly influence the cell physiology. Within our research program, we focused on the generation of a micro-engineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory systems better mimics the in vivo conditions of primary cell cultures and assures steadier, more quantifiable extracellular signaling to the cells.

Aktuelle Trends bei der in vitro-Substanztestung in Deutschland und der Schweiz

o Der Bedarf an Testsystemen fur chemische und pharmakologisch aktive Substanzen ist aufgrund regulatorischer Vorgaben (EU-Chemikalienverordnung REACH, Kosmetikverordnung) immens gestiegen. Anstelle der heute noch ublichen Tierversuche sollen kunftig organotypische Gewebekulturen treten, deren Aussagekraft eine hohere Relevanz verspricht. Tierversuche werfen zur ethischen Problematik weitere Limitierungen wie die unzureichende Verfugbarkeit oder die haufig nicht gewahrleistete Ubertragbarkeit von Daten aus dem Tiermodell auf den Menschen auf. Es wird erwartet, dass durch organotypische Gewebekulturen die Medikamentenentwicklung und Wirkstofftestung sicherer und vorhersehbarer werden. Die neuen Testverfahren sind des Weiteren durch die Verwendung von patientenspezifischem Material als Erganzung zu klinischen Studien zu sehen, da dadurch die genetische Vielfalt der Patienten besser berucksichtigt werden kann. Nach einer DECHEMA-Umfrage im Jahr 2009 zu akademischen und industriellen Forschungsaktivitaten auf dem Gebiet der Zellund Gewebekulturtechnik fur die regenerative Medizin und Substanzprufungen in Deutschland [1] wurde im Jahr 2013 eine Neubewertung der Aktivitaten in diesem Bereich durchgefuhrt und um die Aktivitaten in der Schweiz erweitert. Die Umfrage wurde zudem starker auf die in vitro-Substanztestung einschlieslich der Nutzung systembiologischer Methoden ausgerichtet. Auf deutscher Seite wurde die Studie durch die Beirate der Fachgruppen „Zellkulturtechnologie“ und „Medizinische Biotechnologie“ der DECHEMA e. V., auf schweizerischer Seite vom nationalen Kompetenzzentrum TEDD (Tissue Engineering for Drug Development and Substance Testing) getragen.

Aspects of vascularization in Multi-Organ-Chips

Background Enormous efforts have been made to develop circulation systems for physiological nutrient supply and waste removal of in vitro cultured tissues. These developments are aiming for in vitro generation of organ equivalents such as liver, lymph nodes and lung or even multi-organ systems for substance testing, research on organ regeneration or transplant manufacturing. Initially technical perfusion systems based on membranes, hollow fibers or networks of micro-channels were used for these purposes. However, none of the currently available systems ensures long-term homeostasis of the respective tissue over months. This is caused by a lack of in vivo-like vasculature which leads to continuous accumulation of protein sediments and cell debris in the systems. Here, we demonstrate a closed and self-contained circulation system emulating the natural blood perfusion environment of vertebrates at tissue level.

Human hair follicle equivalents in vitro for transplantation and chip-based substance testing

The ability to create an organoid, the smallest functional unit of an organ, in vitro across many human tissues and organs is the key to both efficient transplant generation and predictive preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need. By carefully analyzing and recapitulating the growth and differentiation mechanisms of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing a hair shaft and revealed a striking similarity to their in vivo counterparts. Extensive molecular and electron microscopy analysis were used to track the assembly of follicular keratinocytes, melanocytes and fibroblasts into the final hair shaft producing microfollicle architecture. The hair follicle generation process was optimized in terms of efficiency, reproducibility and compliance with regulatory requirements for later transplantation. In addition, we developed a procedure to integrate the de novo created human microfollicles into our existing chip-based human skin equivalents for substance testing. This would allow the evaluation of the role of hair follicles in dermal substance transport mechanisms for cosmetic products. Finally, we describe the challenges and opportunities we are facing for first-in-man transplantation trials.