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General considerations implemented in the TAO test concept
The TAO concept for pharmacological and toxicological tests in tissue cultures was designed to meet the following targets which have been identified as mandatory prerequisites:- Reproducible tissue sample preparation
- Control of culture conditions by medium perfusion
- Capability for real-time measurements
- Polar drug application in gradient culture devices
- Acute and chronic toxicity test capacity
TAO's novel tissue culture test concept employs either precision-cut tissue slices, or epithelial explants for the investigation of drug effects in vitro.The use of mechanical tissue slicing, used for samples from massive organs, avoids the application of proteases. Protease treatment causes the disintegration of the organotypic cellular composition. By retaining the typical architecture of the tissue, the TAO test concept allows to investigate drug effects in the tissue-specific context.
For the same reason, epithelial explants of skin and hollow organs are also collected by mechanical sectioning. Epithelial explants provide attractive in vitro models for polar substance uptake, which is the common administration pathway for many medications.
In the living organism, tissues are supplied by the endogenous network of blood vessels, which deliver nutrients and oxygen and remove toxic metabolites. Any tissue culture approach has to replace these complex logistics to maintain the functionality of the tissue explant.
Continuous medium perfusion has, in many studies, proven a feasible solution for nutrient supply and metabolite removal in culture. The same way, perfusion culture offers the unprecedented opportunity to maintain tissue in vitro in a differentiated state - for extended periods.
- Encased in a culture chamber, the tissue sample is constantly perfused with liquid culture medium, which supplies nutrients and oxygen.
- Through the exchange of medium, metabolites are continuously drained from the culture chamber.
- The culture technique allows for the complete omission of serum supplements to the culture fluid. The culture medium solely consists of defined components. Interferences caused by unknown serum factors or by variability between serum lots thus can be ruled out.
Once the perfusion culture has been established, pharmacological or toxicological testing employs the continuous medium flow for the delivery of test substances, as well as for monitoring of physiological responses in the tissue.
- The test substance is delivered to the tissue with the medium flow.
- Changes in metabolite release due to specific tissue responses can be measured by real-time analysis of test parameters in the medium runoff.
The polarity of tissues, in particular of epithelia, is an important issue for pharmacology. Many medications are administered via epithelial barriers (skin, gastrointestinal tract, vessel endothel), which can influence the bioavailability of a drug and can become a serious matter of concern regarding its tolerance. Up to now, the respective properties of drug candidates could only be tested in animal experiments, or were discovered during clinical trials.The TAO test concept is capable to address this question under in vitro conditions. Under perfusion culture, epithelial sheets can be maintained with their three-dimensional, differentiated structure remaining intact.
Employing a gradient culture chamber, it becomes possible to simulate the polar uptake of test substances across epithelia.First, the tissue explant is mounted on a tissue carrier to assure easy handling and tight fit. The mounted explant is then inserted in the culture chamber, which houses an upper and lower medium compartment. By chamber design, the explant assembly divides the chamber volume and exposes the different tissue surfaces to the two compartments. Through separate sets of medium supply lines, both medium compartments can be perfused with different media compositions, and unilateral application of test substances can be accomplished.
The sensitivity of our in vitro test concept allows for fast detection of toxic effects, eventually outperforming animal trials. This was demonstrated by assessing the effects of synthetic vitreous body substitutes in an angiogenesis model (Kloth et al. 2000). The tested compound was found to cause severe deterioration of capillary networks. First damage was detected in vitro after 24 hours, while the test substance had to remain applied in the rabbit eye for several weeks to detect a comparable degree of deterioration.
The TAO test concept can equally handle tests for acute and for chronic toxicity. Short culture intervals (8 to 24 hours) may be sufficient to detect acute toxic effects. The same experimental system is capable of analysing of chronic drug effects, since prolonged culture intervals of up to several weeks (Kloth et al. 1995) allow for continuous or repeated application of test substances.
Contact and Inquiries
Inquiries regarding the TAO in vitro tissue culture test concept or licensing opportunities are kindly requested.
Find out more about the author's scientific expertise.
Sabine Kloth, PhD
Goldshausener Str. 1a
D-85417 Marzling
GERMANY
Phone: +49-8161-65968
© 2003 Sabine Kloth