Cytotoxicity testing in vitro: investigation of 5 miniaturized, colorimetric assays

Recent years have seen the development of miniaturized, colorimetric assays for quantification of cell growth, and their use in toxixity testing has grown. These methods offer significant advantages (speed, ease of performance, sensitivity and better use of resources) over previously used methods such as the Stem Cell Assay or determination of DNA, RNA or protein synthesis. At present many such assays are used in many different areas of toxixity testing. This thesis undertook to compare 5 such assays in detail, and assess their performance in terms of linearity, sensitivity, precision, reproducibility and ease of performance. An Acid Phosphatase (AP) assay, not previously applied to toxicity testing, was developed and validated against some commonly used assays (Neutral Red (NR), MTT, and staining with Crystal Violet and Sulforhodamine B), where it was found to offer the best combinataion of sensitivity, ease of performance, reproducibility and versatility. It was later applied to quantification of growth in serum batch testing, and in determination of cytotoxicity in screening for new drugs, assessment of Multiple Drug Resistance, and screening for toxicity in samples of environmental relevance, including industrial sludges and wastewaters. While results from the ecotoxicity tests were found to correlate with invertebrate tests, difficultues with sample preparation and absence of biotransformation ability were identified. The MTT assay, exhaustatively used in the literature, was found to be least favourable of all the methods compared in this work, due to its low sensitivity and technical difficulties. It was established that problems with reproducibility of these assays do exist, identifying the need to consider 1C$q values as guide values only, and not as absolute values. Activity in each of these assays was found to vary when cells were grown under different conditions, (AP levels per cell increased in starved cultures). The NR assay however, was less prone to variation in activity than any of the other methods. Investigation into differences in assay response to chemicals with mechanisms related to their own mechanism of action showed that after 6 days' exposure to a range of relevant chemicals, no bias in assay ability to detect toxicity was evident. Conditions where each of these assays underestimated cell death were identified; after 1 or 2 days exposure to low concentrations of Vinblastine, the methods (and also trypan blue exclusion) did not register cell death in cells which were shown to be nonviable by cloning studies. A number of factors which significantly influence the i c ^q were identified. Increasing cell seeding density had a masking effect on drug toxicity, while increased drug exposure time and assay duration resulted in greater cell kill (as expected). Small variations in pretreatment regime and trypsinization time prior to drug exposure did not significantly affect cellular response to drug. Attempts made to improve culture conditions for lung tumour biopsies resulted in identification of a number of factors which must be optimized before individualized chemosensitivity testing can have an impact on selection of treatment regimes, and ultimately prognosis