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Generation of three dimensional vascular tissues and establishment of long-term cultures for establishing a multi-organ chip as an alternative to repeated dose (systemic) toxicity testing in vivo
Project
Project code: BfR-ZEBET-08-1322-601
Contract period: 01.03.2014
- 31.12.2014
Purpose of research: Applied research
In recent years a series of new alternative methods have been developed and validated that have gained regulatory acceptance. These methods are predominately in the area of skin / eye irritation and corrosion. These methods addressed topic toxicity, i.p. assessment of the irritant or corrosive activity of chemicals upon exposure of eye or skin and methods that address general mechanism of action, i.p. in respect to phototoxicty and genotoxicity that help to exclude toxic activities. More challenging is however the development of non-animal methods for the analysis of systemic toxicity after repeated exposure. Subacute toxicity studies according to OECD TG 407 (Repeated Dose 28-day Oral Toxicity Study) are usually conducted in rodents and depending on the regulatory context, in a second species, including dogs or non-human primates. After daily oral administration of the test substance, for 28 days clinical observations and histopathology of various organs and tissues including liver, kidney, lung, central nervous system, reproductive organs, the haematopoietic system, the immune system as well as the endocrine system, are used to identify dose-dependent adverse effects in order to determine a “no observed adverse effect level” (NOAEL). REACH legislation requires repeated dose toxicity studies for all substances in quantities above 10 tonnes per annum and these tests are also an integral part of preclinical testing for the evaluation of pharmaceuticals. In addition, repeated dose toxicity testing in animals is no longer possible for cosmetic ingredients marketed in Europe as a consequence of the implementation of the 7th amendment of the Cosmetics Directive (76/768/EEC). In order to keep the number of animals used for repeated dose toxicity testing to an absolute necessary minimum and to assure continued safety of cosmetic products, non-animal test systems are highly needed, but until today no validated methods are available. A promising non-animal alternative offers the multi-organ chip (MOC) technology where miniaturized organoids are connected on a chip to mimic the conditions in a whole organism. The overall aim of this project is the development of a MOC to complement and finally replace repeated dose toxicity testing in animals. The project described here focuses on the development of three-dimensional vascular tissue that is needed for the functional connection of the distinct organs on the MOC. In addition, it is proposed to subsequently optimize the system in order to allow the generation of blood-brain barrier model.
The aim of this project was mainly to establish differentiating embryonic stem cells as a suit-able tool for the analysis of early vasculogenesis and as a potential source of early endothe-lial cells for the characterization of toxic substances in microfluidic systems. During this first phase, we were able to establish (1) quantitative RT-PCR for the analysis of the endothelial differentiation process, (2) immunohistochemical methods for the characterisation of the morphological organisation of the endotheIial cell into three-dimensional structures and (3) the cultivation of the stem cell sin a three-dimensional collagen matrix. We could show that endothelial cells as well as smooth muscular cells and pericytes that are the key compon-ments of mature blood vessels are formed within a week of differentiation and are organized in complex 3D-structures that are reminiscent of blood vessels.
Section overview
Subjects
- Animal health
- Computer science
Framework programme
Funding programme
Excutive institution
BfR - Centre for Documentation and Evaluation of Alternatives to Animal Experiments (BfR - ZEBET)
