Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay

Xavier Ponsoda, Maria José Gómez-Lechón and José V. Castell

The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.
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Cytostatic Properties of a Novel Compound Derived from Penicillium pinophilum: An In Vitro Study

Annalaura Stammati, Rosario Nicoletti, Salvatore De Stefano, Franco Zampaglioni & Flavia Zucco

3-O-Methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, inhibits the in vitro growth of plant pathogenic fungi. This specific property suggested that the compound could be used against other fungal pathogenic activities, including dermatological ones. However, for such applications, toxicological side-effects should be taken into account, in order to prevent other types of risk to mammalian cells. Therefore, investigations were made of the basic toxicity of OMF toward a human tumour cell line. The compound was found to have a cytostatic effect, which represents a counter-indication to its use as a therapeutic agent in dermatology, but suggests that it may have potential as an antitumour agent. This study confirmed the validity of in vitro systems for preliminary assays on new compounds, in order to avoid the use of animals in toxicological studies.
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Cryopreservation of Organotypic Brain Spheroid Cultures

Wendy M. Purcell, Christopher K. Atterwill and Jinsheng Xu

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5–15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12–15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5–7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.
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