This protocol describes a low cost method for performing drug viability assays using patient-derived ovarian cancer organoids. The main advantages of this technique are the use of low cost and readily available materials and lab equipment. This method can be used before undertaking expensive high-throughput drug screens.
Although the method deals with performing drug viability assays in patient-derived ovarian cancer organoids, it can be applied to any organoid system, including those that are murine derived. To begin, observe the basement membrane extract or BME containing organoids under a brightfield microscope to ensure 70 to 90%confluency. Using a pipette, add one to two milliliters of prewarmed base media to the well containing the organoids and mechanically dissociate the organoids by pipetting them up and down.
Transfer the entire organoid suspension to a 15-milliliter conical tube and vortex the tube for five to 10 seconds to further disaggregate the cells. Then, centrifuge the cells at 1, 107 G for five minutes at room temperature. Using a single-channel pipette, aspirate and discard the supernatant.
Resuspend the pellet in one milliliter of organoid dissociation reagent prewarmed to 37 degrees Celsius, and transfer the suspension to a 1.5-milliliter micro centrifuge tube. Incubate the sample for seven minutes at 37 degrees Celsius. Then centrifuge the result in cell suspension at 1, 107 G for five minutes at room temperature.
After discarding the supernatant, resuspend the cell pellet in one milliliter of prewarmed base media. Reconstitute the cells in a mix of base media and BME, maintaining a final concentration of 20, 000 cells per 10 microliters. Plate one three-microliter droplet of the resuspended cells per well of a black opaque 96 well plate.
Incubate the plate in a cell culture incubator at 37 degrees Celsius for 15 minutes. Finally, add 100 microliters of prewarmed advanced organoid media to each well before incubating the plate at 37 degrees Celsius for 24 to 72 hours. Perform drug treatment of the organoids two days after plating them.
Perform viability assay on the organoids seven days after drug treatment. Add 100 microliters of viability assay reagent to each well, bringing the total volume to 200 microliters, and shake the plate at 80 RPM for five minutes. Then allow the plate to incubate at room temperature for an additional 25 minutes.
Meanwhile, switch on the bioluminescence plate reader and open the eye control software. Navigate to connect to instrument"and select Infinite 200Pro"Select luminescence"and default script"Next from the dropdown menu, choose BD 96 FB Falcon BD Falcon 96 Flat Black"as the plate type to set the luminescence parameters. Assign attenuation as none.
Integration time as 1000 milliseconds and settle time as zero milliseconds. Finally, uncover the plate, load it onto the plate reader, and press start"to begin. Brightfield images were acquired for the two different patient-derived organoids, or PDOs, used in the protocol.
PDO one, derived from a tumor biopsy and PDO two derived from ascites. Results of the viability assay revealed the percentage of live cells in PDO one and PDO two after both were treated with varying concentrations of carboplatin. The calculated growth rate, or GR values, were plotted against the corresponding carboplatin concentrations, and the GR metrics revealed that the GR50 value for PDO one was much higher than that for PDO two.
This indicated that PDO one was more resistant to platinum chemotherapy While plating, it is crucial that the three-microliter droplet of the cell suspension is placed directly in the center of the well. If the droplet is placed to close to the edge of the well, it may cause the droplet to collapse, affecting the assay results.
