In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints.

The goal of replacement, refinement and reduction of animal testing is critically dependent on the development and assessment of novel in vitro methodologies and the further development of existing methodologies. Here, we evaluated the use of a modified perfusion cell culture apparatus for application to chronic in vitro nephrotoxicity testing using DMSO, SDS, paracetamol and cyclosporine A as test compounds.
Renal epithelial monolayers were cultured on microporous growth supports and exposed to test compounds under static or perfusion conditions. Alamar Blue reduction, gamma-glutamyl transpeptidase activity (GGT), lactate dehydrogenase activity (LDH) and remnant protein were used to assay cell toxicity. There was no significant difference in IC50 values between static and perfusion cultures up to 72 hours exposure. However, the perfusion system allowed continuous real-time monitoring of plasma membrane damage, which gives important information of time, duration and scale of toxicity.
The complexity of the system restrains its use to low-throughput analysis. However, the real and theoretical advantages of this and similar systems merit further investigations.

To successfully grow cells in serum-free medium is an interesting challenge to cell biology. The use of such media for in vitro cell culture work would be a key contribution to the 3Rs concept, enabling the avoidance of the use of animals and animal products at all stages of the experiment. In addition, numerous problems related to virus infections transmitted by animal serum would be avoided, thus increasing reproducibility. Prolifix is a new reagent of plant origin. It contains a molecule (GCR 1003) that has an activity similar to that of the mitogenic molecules in serum and could be suitable to substitute serum in culture medium. Two epithelial cell lines, LLC-PK1 and Caco-2, were progressively adapted to a special culture medium containing 10% Prolifix in the absence of serum. After adaptation, cell cultures were characterised. We found that these reagents of plant origin could be promising alternatives to serum. However, more work and effort is needed to improve cell adaptation, cell attachment, growth rates as well as freezing/thawing protocols.

In the 4th edition of the European Pharmacopoeia, animal experiments are described, which are required for the quality control of pharmaceuticals. This refers to investigations regarding both biological activity and safety aspects of the medication. The latter include investigations to find contaminations with histamin and pyrogenic substances and tests for abnormal toxicity. Even though the safety of medical drugs is of great importance, it should be examined whether these tests are still indispensable (in terms of § 7 of the German Animal Protection Law of 2002). The determination of histamine content may also be possible by chemical-analytical methods, which could represent an alternative to the animal experiment. The testing of abnormal toxicity should be scientifically questioned due to its lack of specificity. Although the in vitro test detecting bacterial endotoxins is mentioned in many monographs, the in vivo test on rabbits to test for pyrogenicity is still mentioned in some other monographs. The in vivo test should be rechallenged as to whether it can be replaced. In light of the global trade in pharmaceuticals, which must also fulfill national quality standards. A reduction in animal experiments can only be reached by fruitful cooperation between pharmaceutical companies, health authorities and the commissions of the pharmacopoeias, leading to the formation of a consensus. The International Conference on Harmonization could prove an auspicious panel to take up this subject and to contribute to an internationally accepted and scientifically based reduction of animal experiments.

Vaccines must provide effective protection against disease without any harmful side effects. Antigen purity is a crucial factor which until now has had to be tested on animals. However, recent scientific developments allow the use of alternatives to animal experiments in many cases, e.g. poultry vaccine purity testing. The in vitro Polymerase Chain Reaction method (PCR) is one advance which could reduce the number of animal experiments.