This method can help answer key questions in the drug discovery field, such as, what is the most relevant tissue culture setting for hit to lead identification. The main advantage of this technique is that it allows the culturing of cells in three dimensions, in a high throughput manner, using 1536 well-plates. Demonstrating this protocol will be Kalyani Gampa, a senior scientist from my lab.
To set up a 3D tumor spheroid culture, detach the cancer cell lines of interest with three washes of BBS. Followed by the addition of one milliliter of trypsin per five centimeters squared area of culture flask. After five minutes, neutralize the trypsin with five times the volume of serum supplemented medium in each flask, and count the cells.
Use a sterilized, small stainless steel tipped cassette with a peristaltic pump-based system to seed a total of 500 cells in eight microliters of spheroid medium into each well of a 1, 536 well tissue culture treated plate. Then use a breathable adhesive plate seal to cover the plate. And place the plate in a spinning incubator set at 37 degrees Celsius and 10rpm with 5%carbon dioxide and 95%relative humidity for three days.
The single most critical step is ensuring that the plates are well sealed before they are placed into the incubator. The spheroids will need three days to form and this step prevents medium evaporation. At the end of the incubation, quick spin the cancer lined culture plates at 100 times gravity to collect any condensation, and remove the adhesive seals.
Put each plate to be treated onto an acoustic dispenser set up to deliver a total of eight nanoliters of each compound. Dilution to a final concentration of 0.1%DMSO per well, and a top compound dose of 10 micromolar. When the compound has been added to the appropriate plates, place a custom made, stainless steel cell culture lid on to each plate to prevent evaporation, and return the plates to the spinning incubator for another five days.
At the end of the incubation, add three microliters of pre-warmed cell lysis reagent, supplemented with luciferin to each well of each cell culture plate. And incubate the plates at room temperature for at least 15 minutes. Then capture the ATP luminesce on a plate luminometer.
When all of the plates have been analyzed, extract the raw data from the instrument and normalize all of the fields containing the test compounds to the average of all the wells containing DMSO alone as the neutral control. The percent growth inhibition of each compound can then be calculated according to the formula, and dose response curves, and inhibitory IC50s can be generated. A variety of established cancer cells known to grow well under 2D culture conditions also formed 3D structures when cultured as just demonstrated, despite their morphological differences.
When used for large scale screening, differential compound sensitivity between cultures grown in 2D versus those grown in 3D can be observed. With each dot representing the inhibitory concentration of a single compound for which a 50%cell killing response is observed in both culture settings. In this representative experiment, both tested RAF kinase inhibitory compounds were more potent in 3D conditions in a non-small cell lung cancer line culture harboring an activating mutation in the KRAS protein.
However, the compounds demonstrate a similar potency in both 3D spheroid and 2D colon cancer line cultures that contain a G13D activating mutation in KRAS. The 3D assay also permits observation of the paradoxical growth activation phenomenon, which occurs when there is an incomplete inhibition of RAF dimers in highly activated RAFs mutant lines, as evidenced by cell growth at low concentrations of the drug, that is repressed at high concentrations. After its development, this technique paved the way for researchers in the field of cancer drug discovery to explore alternative culture conditions for identifying new, efficacious chemical matter against known targets.
