Cytotoxicity tests help determine either quantitatively or qualitatively the toxicity of cells. Cytotoxicity testing forms a crucial part of the drug discovery and development process. This testing helps a pharmaceutical company get vital information about the biological attributes of new molecules while focusing on its tolerance levels.
Cytotoxicity assay is a quick way to assess a particular chemical compound’s effects on a human cell line. These are used in the drug development process as a screening tool before performing the extensive toxicological testing. Cytotoxicity tests can also be utilized for quality control purposes in the case of raw material lot testing or even testing of manufactured products.
Cytotoxicity testing makes use of the following basic qualitative and quantitative methods.
Quantitative Cytotoxicity Assays
Luciferase is an enzyme which in the presence of ATP, converts luciferin to oxyluciferin. ATP is found in direct proportion to viable cells, and this viability is an alternate readout for cytotoxicity.
The MTT cytotoxicity assay makes use of a dye known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye. This dye is commonly known as MTT. It is a water-soluble compound that is yellow in color. It is divided by mitochondrial succinate dehydrogenase to form formazan, which is violet in color.
MTT assay is done on an extract of the sample article. It is added to L-929 mouse fibroblasts. This is done with four different concentrations i.e., undiluted, in the ratio of 1:2, 1:4, and 1:10. MTT is added after 24 hours to the cells, and then the existence of formazan is analyzed with the help of a plate reader. The samples are then compared to ascertain the percentage of healthy cells.
Direct Contact Test
This method makes use of the L-929 mouse fibroblasts. The cells are usually grown to approximately 80% confluency. The test article is placed directly on these cells. A sterile paper is saturated with the sample and placed on the cell in case it is liquid or extract. These are incubated for 24 hours and then examined. The cells are graded depending on their reactivity.
Agar diffusion test
Agarose overlay test is used for very high and low-density materials and is similar in nature to the direct contact method. The only difference is that the cells are solidified by pouring agar over them, and the test sample is placed in. These are again incubated for over 24 hours and then graded.
Unlike the methods mentioned above, this method involves the application of leachable and extractable to the cells from the test sample. The sample is incubated with a liquid extraction vehicle. The test sample extract is then added to mouse fibroblasts (L-929). The incubation period is 48 hours, and scoring depends on the conditions of the cells.
These studies carried out under Cytotoxicity tests help save time and resources that go on a test molecule. These tests can efficiently assess probable toxic effects without posing any risk to humans or animals.