Measurement of the toxic effects of chemical compounds on different types of cells is essential to the development of cancer drugs that are intended to kill their target cells. Cell survival analysis is also an important task in areas such as environmental regulation to test industrial and agricultural chemicals for potentially harmful effects on healthy cells.
MIT biological engineers have now devised a new toxicity test that can measure chemical effects on cell survival with far greater sensitivity than some of the most popular tests currently used. It is also much faster than the gold standard test, which is not widely used because it takes two to three weeks to deliver results. The new test could thus help pharmaceutical companies and academic researchers to identify and evaluate new drugs more quickly.
"Cytotoxicity assays are one of the most widely used assays in life sciences," says Bevin Engelward, a biotechnology professor at MIT and the lead author of the study.
Le Ngo, a former MIT graduate student and postdoc, is the lead author of the paper, which appears from the February 5 issue of Cell Reports . Other authors include Tze Khee Chan, a former graduate student in the Singapore-MIT Alliance for Research and Technology (SMART); Jing Ge, a former MIT graduate student; and Leona Samson, Ngo's co-adviser and a MIT professor emerita of biotechnology.
The traditional test for cell survival measurement, known as the colony formation assay, involves growing cell colonies in tissue culture dishes for two to three weeks after subjecting the cells to a chemical compound or other harmful agent, such as radiation. A researcher then counts the number of colonies to determine how the treatment affects cell survival.
Part of Engelward's motivation for this study was reminiscent of the long hours she spent counting such colonies as a graduate student.
The census is really cumbersome and painfully difficult because you have to constantly judge what is a colony versus waste, "she says." Few people use the colony formation anymore because it is difficult, too slow, and requires huge amounts of cell growth media, so you need a lot of the compound being tested. "In recent years, researchers have begun to use other methods that are faster but not as precise and sensitive as the colony formation assay. These tests do not directly measure cell growth but instead analyzes mitochondrial function.
Engelward and colleagues set out to develop a test that could generate results within a few days while still matching the accuracy and sensitivity of the colony formation assay. MicroColonyChip consists of small wells on a plate, treated and untreated cells are placed in these wells and begin to form very small colonies in a given area. Within a few days before the colonies become visible to the naked eye, scientists can use a microscope to image the cells DNA that is fluorescently labeled.
By changing the code originally developed by former MIT postdoc David Wood and MIT Professor Sangeeta Bhatia, the researchers created a software program that measures the amount of fluorescent DNA in each well and then calculates how much cell growth occurred. By comparing the growth of treated and untreated cells, the researchers can determine the toxicity of whatever substance they study.
"We have an automatic scanning system to perform fluorescent imaging, and subsequently the image analysis is completely automated" Ngo says.
The researchers compared their new test to the gold standard colony formation analysis and found that the results could not be distinguished. They could also accurately reproduce data on the effects of gamma radiation on human lymphoblastoid cells collected 20 years ago using the colony formation assay. Using MicroColonyChip, the researchers obtained their data in three days instead of three weeks.
"We could reproduce radiation studies from 20 years ago using a process much easier than they did," says Engelward.
The researchers also compared their new test to the two toxicity tests most commonly used by researchers and pharmaceutical companies, known as XTT and CellTiter-Glo (CTG). Both of these tests are indirect targets for cell viability: XTT measures the ability of cells to degrade tetrazolium, a key step in cellular metabolism, and CTG measures intracellular levels of ATP, molecules that cells use to store energy.
"MicroColonyChip is much more sensitive than the XTT assay, so it really allows you to see subtle changes in cell survival, and it's as sensitive as the CTG analysis while you're more robust to artifacts," says Engelward. Using the new test, the researchers examined the effects of two DNA-damaging substances used for chemotherapy and found that they could accurately reproduce the results obtained using the conventional colony-forming assay. "We are now planning to expand these studies in the hope of demonstrating that the test works for many more types of drugs and cells," says NGO.
In addition to being useful for drug development, this test can also benefit environmental regulators responsible for testing chemical compounds for potential adverse effects, Engelward says. Another possible use is in personal medicine where it could be used to test a variety of drugs on the patient's cells before a treatment is selected.
The researchers have filed a patent for their technology. The research was funded by the National Institute of Environmental Health Sciences, including the NIEHS Superfund Basic Research Program and the National Institutes of Health.