Chip-based liver equivalents for toxicity testing - organotypicalness versus cost-efficient high throughput

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Drug-induced liver toxicity dominates the reasons for pharmaceutical product ban, withdrawal or non-approval since the thalidomide disaster in the late-1950s. Hopes to finally solve the liver toxicity test dilemma have recently risen to a historic level based on the latest progress in human microfluidic tissue culture devices. Chip-based human liver equivalents are envisaged to identify liver toxic agents regularly undiscovered by current test procedures at industrial throughput. In this review, we focus on advanced microfluidic microscale liver equivalents, appraising them against the level of architectural and, consequently, functional identity with their human counterpart in vivo. We emphasise the inherent relationship between human liver architecture and its drug-induced injury. Furthermore, we plot the current socio-economic drug development environment against the possible value such systems may add. Finally, we try to sketch a forecast for translational innovations in the field

High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor

<p>Abstract</p> <p>Background</p> <p>Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT^®CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase^®Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of <it>E. coli </it>and recombinant protein production.</p> <p>Results</p> <p>Cultivation of <it>E. coli </it>in the BIOSTAT^®CultiBag RM system in a conventional batch mode without control yielded an optical density (OD₆₀₀) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD₆₀₀of 60 (20 g L^-1cell dry weight). By the use of an internal controlled glucose delivery system, EnBase^®Flo, OD₆₀₀of 30 (10 g L^-1cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase^®Flo also worked well in the CultiBag RM system with a recombinant <it>E. coli </it>RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.</p> <p>Conclusions</p> <p>Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT^®Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase^®Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.</p

Dynamic culture of human liver equivalents inside a micro-bioreactor for long-term substance testing : From 23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health Lille, France. 23-26 June 2013

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

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

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.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.BMBF, 0315569, GO-Bio 3: Multi-Organ-Bioreaktoren für die prädiktive Substanztestung im Chipforma

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

Generierung von Multi-Organ-Chip basierenden Leberäquivalenten für die Toxizitätstestung

Die Leber hat als zentrales Organ des gesamten Stoffwechsels eine einzigartige Bedeutung in der Aufrechterhaltung der metabolischen Homöostase, der Gallenproduktion und damit einhergehend dem Abbau und der Ausscheidung von Medikamenten und Giftstoffen. Diese Fülle an Stoffwechselfunktionen und die daraus resultierende, evolutionär hoch spezialisierte strukturelle Segregation der metabolischen Aktivitäten der Leber stellt eine große Herausforderung bei der frühzeitigen Erkennung von medikamenteninduzierten Leberschädigungen dar. Die Generierung miniaturisierter Leberäquivalente zur Substanztestung bedarf daher besonderer Aufmerksamkeit in Hinblick auf die Komplexität des Organs. Im Verlauf dieser Arbeit wurden Leber-Aggregate aus Hepatozyten und nicht-parenchymalen Ito-Zellen generiert und charakterisiert. Diese Leber-Äquivalente wurden in dem von uns entwickelten Multi-Organ-Chip (MOC) sowohl als Einzelorgankultur als auch als Co-Kulturen mit Hautbiopsien, Endothelzellen und Neurosphären kultiviert. Es konnte gezeigt werden, dass die Zellen der Aggregate lebertypische extrazelluläre Matrixbestandteile wie Fibronektin und Kollagen Typ I produzieren. Zudem polarisierten Hepatozyten de-novo und bildeten Gallenkapillaren. Die Verlängerung der Kulturzeit in MOCs über 14 Tage führte zu einer weiteren Verbesserung der Polarisation sowie der Expression funktioneller Marker wie der Cytochrom-P450-Enzyme. Die Analyse der Albuminkonzentration im Medium ergab eine deutlich erhöhte Produktionsrate in Leber-Äquivalenten die unter dynamischen Bedingungen im MOC kultiviert wurden im Vergleich zu Kontrollen unter statischen Bedingungen. Co-Kulturen von Leber Aggregaten mit Hautbiopsien erreichten ein metabolisch stabiles Niveau nach 5 bis 8 Tagen ein. Es konnte gezeigt werden, dass diese Co-Kulturen glukoselimitiert waren, was zu einer Verringerung der Albuminproduktionsrate führte. Ebenso führte die Co-Kultivierung mit Endothelzellen und Neurosphären zu einer Glukoselimitierung. Dennoch konnten die Zellen in einem vitalen, metabolisch kompetenten Zustand über eine Kulturzeit von 28 Tagen erhalten werden. Die Applikation von hepatotoxischen Substanzen wie Troglitazon zu Leber-Haut-Kulturen führte zu einer in vivo-ähnlichen, dosisabhängigen Hochregulation des metabolischen Enzyms Cytochrom-P450 3A4 und zur Induktion von Toxizität. Ebenso konnte die in vivo Halbwertszeit von Troglitazon erfolgreich in Leber-Haut-Endtohelzell MOC Co-Kulturen reproduziert werden. Auch die Toxizität von 2,5-Hexandion in Leber-Neurospären Co-Kultur war dosisabhängig und darüber hinaus wurde eine etwas höhere Sensitivität in Co-Kulturen im Vergleich zu Einzelorgan-kulturen im MOC beobachtet. Erste Hinweise auf eine Toxizität von n-Hexan in diesen Co-Kultursystemen wurden deutlich. Zusammengenommen stellt das präsentierte MOC Co-Kultursystem nicht nur ein geeignetes Mittel für die Aufrechterhaltung der Vitalität und metabolischen Aktivität von Hepatozyten über längere Zeiträume dar, sondern auch ein Model um Kommunikationen zwischen Organen zu untersuchen. Es konnten sowohl homöostatische als auch toxische Umgebungen für die Zellen in den MOC simuliert und analysiert werden, was für die Nutzung des Systems für Wirkstofftests von besonderer Bedeutung ist.The unique importance of the liver for organismal homeostasis and blood detoxification has led to an evolutionary optimisation of the human liver architecture at the scale of its smallest functional unit – the liver lobule. Unsurprisingly, such a super-optimised tissue is especially vulnerable to inconsistencies, such as toxic effects. The ever-growing amount of new substances released to the market and the limited predictability of current in vitro test systems for drug-induced liver injury (DILI) has led to an ample need for new substance testing solutions. Especially, the application of miniaturised, dynamically perfused chip-based systems is gaining much attention recently due to their ability of imitating in vivo-like nutrient supply and defined microenvironments of the cells. In this thesis, a novel 3D co-culture system comprising the hepatic HepaRG cell line and non-parenchymal hepatic stellate cells (hSteC) is presented and its suitability as in vitro liver test system is evaluated. These 3D tissue equivalents were, furthermore, cultivated inside a multi-organ-chip (MOC) to assess the effects of fluid flow on the cells. Single tissue cultures, as well as two to three organ co-cultures including skin biopsies, endothelial cells and neurospheres were performed, observing organ-organ crosstalk. It could be shown, that liver aggregates produced liver-typical extracellular matrix components, indicating the formation of an in vivo-like environment. Cells polarised de-novo, forming bile-canalicular like structures as shown by ZO-1 and MRP-2 expression. Prolonged culture in the MOC led to a more pronounced expression of these markers, as well as of functional markers like cytochrome P450 enzymes indicating the generation of a functional and metabolically competent organ equivalent. Furthermore, first indicators for functional liver zonation were obtained in Transwell® assisted cultures. Analysing the release of albumin to the culture medium over a culture period of up to 14 days revealed a significantly increased production rate in liver equivalents cultured under dynamic conditions in the MOC compared to standard static controls. Co-cultivating liver aggregates with skin biopsies revealed that a metabolic steady state in terms of glucose consumption and lactate production could be achieved after 5 to 8 days of co-culture in a combined medium circuit. The long-term stability of these cultures was proven by 28-day cultivation. These co-cultures were shown to be glucose limited, which led to a reduction in albumin production by the liver equivalents. Still, viability of the cells was maintained and strongly increased compared to static cultures. The stable consumption of glucose and production of lactate indicated the establishment of an artificial but balanced co-existence between the tissues, proving the feasibility of two (or even more) tissue co-cultures in a combined medium over prolonged periods of time. Similarly, the co-cultivation with endothelial cells and neurospheres led to a glucose-limited, but still viable and metabolically competent system. Exposing MOC co-cultures to pharmaceutical substances at regimens relevant to respective guidelines successfully revealed a dose-dependent response of cultures. The chronic application of troglitazone to the co-cultures over 7 to 14 days proved a sensitivity of liver equivalents to this hepatotoxic anti-diabeticum. Dose dependent in vivo-like up-regulation of metabolic enzyme cytochrome P450 3A4 and induction of toxicity could be shown. Furthermore, the in vivo elimination half-life of troglitazone could successfully be reproduced in a liver-skin-endothelial cell co-culture MOC system. Taken together, this MOC co-culture system not only presents a useful mean for maintaining hepatocyte viability and metabolic activity over prolonged periods of time, but also a tool to study organ-organ interactions under control and toxic environments useful for drug testing