The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

Long-term culture of functional liver tissue: three-dimensional coculture of primary hepatocytes and stellate cells.

One of the greatest challenges in the attempt to create functional liver tissue in vitro is the maintenance of hepatocyte-specific functions. The pharmaceutical industry has long awaited the development of engineered liver tissue, which could represent a long-term, inducible, high-fidelity model for high-throughput screening of new drug compounds. It is also anticipated that such engineered models could one day be used in liver transplants, where replacement is limited by chronic donor shortages. As isolated hepatocytes dedifferentiate rapidly in culture the use of hepatocytes in long-term studies has proved to be a difficult challenge. Here we report a system of rat hepatocytes cocultured with primary rat hepatic stellate cells on a biodegradable poly(DL-lactic acid) substratum. These coculture conditions were found to encourage the rapid self-organization of three-dimensional spheroids. The spheroids formed exhibit hepatocyte-specific functionality (CYP-450 activity and albumin secretion) after almost 2 months in static culture.

A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue.

Hepatic stellate cells (HSCs), also referred to as Ito cells, perisinusiodal cells and fat-storing cells, have numerous vital functions. They are the main extracellular matrix-producing cells within the liver and are involved in the storage of retinol. HSCs are also known to secrete a number of liver mitogens. Current isolation techniques are cumbersome and most require a pronase digestion step, which destroys any hepatocytes present. We present a simple method for isolation and culture of hepatic stellate cells from the normally discarded washings from a two-step collagenase hepatocyte isolation, which has shown a yield of more than 1.5 x 10(6) viable HSCs after 5 days in culture. The cells were positively identified as HSCs by staining for two intermediate filaments (desmin and GFAP) and observing their distinct morphology from other liver cell types. This efficient method allows rapid and consistent isolation of stellate cells to give a culture that may be passaged several times.