High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids recapitulate the architecture and function of tumor tissue accurately with better reproducibility of disease. Therefore, a patient's response to anti-cancer drugs can be evaluated accurately using this model. Tumor organoids are unsuitable for an in-vitro high throughput assay system or cell analysis because of large clusters formation in culture.

Using this technique, simple and more accurate HTS for evaluation of molecular targeted drugs was developed. Since the organoid culture is very different from 2D culture, one may feel confused about the culture at first, but it is important to carefully observe the morphological changes of the organoids. After removing the frozen vile from liquid nitrogen storage, gently agitate the patient-derived tumor organoids in a water bath at 37 degrees Celsius for one minute.

Remove the vile from the water bath and wipe with 70%ethanol. Then place the vial in the biosafety cabinet. Transfer the tumor organoids in a 15 milliliter tube containing the medium for organoids.

Use a three milliliter transfer pipette to gently mix the tumor organoids and medium by pipetting up and down five times. Pellet down the tumor organoids by centrifugation. Discard the supernatant and resuspend the tumor organoid pellet in five milliliters of fresh medium.

Transfer the tumor organoid suspension into a T25 flask and incubate at 37 degrees Celsius and 5%carbon dioxide. After one week of the first passage, transfer the tumor organoid suspension from two T25 flasks into two centrifuge tubes and pellet down the tumor organoids by centrifugation. Estimate the tumor organoid pellet volume and resuspend the pellet in 2.5 milliliters of fresh medium per tube.

Pool the tumor organoid suspension in one tube. Transfer the pooled suspension into a T75 flask containing 10 milliliters of fresh medium and incubate at 37 degrees Celsius and 5%carbon dioxide. After 24 hours of medium change, mince the tumor organoids using a cell fragmentation and dispersion instrument equipped with a filter holder containing a 70 micrometer mesh filter.

Dilute 15 milliliters of tumor organoid suspension 10 times. Use the cell suspension dispenser to seed 40 microliters of diluted tumor organoid suspension in a 384-well ultra low attachment spheroid microplate. After 24 hours, use a liquid handler to add 0.04 microliters of test agent solutions from 10 serial dilutions at final concentration ranges of 20 micromolar to 1.0 nanomolar in the wells, and incubate the plate for six days.

At the end of the incubation, add intracellular ATP measuring reagent in the test wells. Use a mixer to mix the contents of the plate and incubate the plate for 10 minutes at approximately 25 degrees Celsius. Use a plate reader to measure the intracellular ATP content as luminescence.

After 24 hours of the third passage, place a 384-well ultra low attachment spheroid microplate with 40 microliters of medium per well as a destination plate on the cell picking and imaging system. Build the picking chamber with six milliliters of culture medium and centrifuge at 1, 500 times G for two minutes to remove the air bubbles. Add four microliters of tumor organoid suspension in the picking chamber, and set the picking chamber on the system.

Allow the cell clusters to settle at the bottom of the chamber for one minute after dispersion and then initiate the chamber scanning. Set the picking size to 140 to 160 micrometers on the system for automatic selection of cell clusters. Next, check the quality of the selected cell clusters on the scanned images and transfer 10 cell clusters per well using picking tips into the destination plate.

In the current study, the effect of anti-cancer agents on the patient-derived tumor organoid was assessed. The high sensitivity for all the anticancer agents was indicated by the half maximal inhibitory concentration values less than two micromolar in the area under the curve values less than 282. For an antibody dependent cellular cytotoxicity assessment, percent cytolysis of tumor organoids in the presence of two different antibodies was measured.

The percent cytolysis increased with time. Without the antibodies at six hours with the effector to target cell ratio of one-to-one, the cytolysis reached 45%While at the ratio of two to one, the cytolysis reached 70%The natural killer cell mediated cytolysis using trastuzumab was approximately 60%at the effector to target cell ratio of one-to-one, and 80%at the ratio of two to one at six hours. In contrast, cetuximab had a dose dependent impact on the natural killer cell mediated cytolysis.

At the highest concentration of cetuximab, 90%cytolysis was observed at the effector to target cell ratio of one-to-one, and 100%cytolysis was observed at the ratio of two to one, indicating high effectivity of cetuximab. A high throughput assay system using patient-derived tumor organoids is superior to evaluating potential anti-cancer agents and presents opportunities for drug assessment and advances in personalized medicine.