Katharina Schimek

The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

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.

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described. Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

Integrating skin and vasculature in a Multi-Organ-Chip Platform

Background Tests for drug development require an almost perfect fit with the human (patho-) physiological microenvironment. The majority of skin equivalents currently commercially available are based on static culture systems emulating only human epidermis, or combining epidermis and dermis in so-called full thickness skin equivalents. None of the existing systems contain important elements, such as vasculature, skin appendices or an immune system. Therefore, current in vitro and animal tests are failing to accurately predict drug toxicity. Our Multi-Organ-Chip (MOC) platform is a micro scale bioreactor providing pulsatile dynamic perfusion for microscale organoids. Here, we combine skin equivalents with vasculature in our two-organ variant (2OC). This would be needed for physiological-like interactions, regulation and, eventually, homeostasis within the chip.

Bioengineering of a Full-Thickness Skin Equivalent in a 96-Well Insert Format for Substance Permeation Studies and Organ-On-A-Chip Applications

The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays.

Automated substance testing for lab-on-chip devices

First published by BioMed Central: Kloke, Lutz ; Schimek, Katharina ; Brincker, Sven ; Lorenz, Alexandra ; Janicke, Annika ; Drewell, Christopher ; Hoffmann, Silke ; Busek, Mathias ; Sonntag, Frank ; Danz, Norbert ; Polk, Christoph ; Schmieder, Florian ; Borchanikov, Alexey ; Artyushenko, Viacheslav ; Baudisch, Frank ; Burger, Mario ; Horland, Reyk ; Lauster, Roland ; Marx, Uwe : Automated substance testing for lab-on-chip devices : From 23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health Lille, France. 23-26 June 2013. - In: BMC Proceedings. - ISSN 1753-6561 (online). - 7 (2013), suppl. 6, P28. - doi:10.1186/1753-6561-7-S6-P28.

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.