Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Our lab aims to investigate the molecular pathogenesis of liver cancer and identify novel therapeutic targets for drug development. We would like to answer how cancer initiates metastasis and progress to drug tolerance utilizing novel disease models such as patient-derived organoids. The current experimental challenges are in two aspects.

First, there is a lack of complete set of protocols for constructing hepatocellular carcinoma organoids for the whole process. Second, the size of cultured HCC organoids is too small for subsequent experimental processing. In this protocol, we provide the details of the optimized organoid cultural and subsequent transfer and freezing procedures as well as the precautions to be taken during all the procedures.

We optimized the composition of the organoid medium for harvesting larger organoids with close to the original tissue characteristics. We will monitor the tumor evolution using the newly stabilized patient organoids and try to identify novel molecular mechanism and therapeutic targets for HCC treatment. To begin the tissue dissociation, collect one to two cubic centimeters of hepatocellular carcinoma or HCC tissue from untreated patients.

Then place the tissue in the tissue preservation solution and maintain it at four degrees Celsius until processing. Now preheat the ultra low attachment surface cell culture plates for one hour in an incubator set at 37 degrees Celsius. File the basement membrane extract overnight at four degrees Celsius.

Using surgical scissors, cut the tumor tissue into small pieces on a Petri dish inside a laminar flow hood. Transfer these pieces into a 15 milliliter conical tube and add five to 10 milliliters of basal medium. With a barotropic pipette, wash away as much blood as possible.

Allow the mixture to settle for one to two minutes before removing some of the supernatant, including any blood cells and floating fat clots. Next, centrifuge the mixture at 300 G for five minutes at room temperature. Then carefully extract the supernatant and add five milliliters of prewarmed digestion solution to the trimmed tissue.

Place the tube at 37 degrees Celsius in a rotor for optimum digestion. After 30 minutes of initial digestion, use a microscope to look for small cell clusters. To stop the digestion, add five milliliters of cold basal medium to the tube.

Then use a 100 micrometer cell filter to sieve into a new 50 milliliter tube. Fill up the tube with cold basal medium to reach a total volume of 50 milliliters. Next, centrifuge the cells at two G for 10 minutes at eight degrees Celsius.

Once complete, carefully remove the supernatant liquid. Resuspend the pellet in 50 milliliters of cold basal medium to obtain the cell pellet for organoid plating. To perform organoid plating, first remove the supernatant from the centrifuge and washed hepatocellular carcinoma cells.

Resuspend the cells in the 50 microliters of basement membrane extract, or BME, per well for plating. Next, suspend the cells in advanced DMEM/F12. Then gently pipette the cell aggregates until an even suspension is seen.

Add BME to the suspension, ensuring that the BME concentration is between 30 and 50%Now, seed 50 microliters of BME droplets with cell clusters in the center of a 24 well plate. Allow the droplets to solidify at 37 degrees Celsius for 20 minutes. Add 500 microliters of prewarmed medium to each well and incubate in a cell incubator at 37 degrees Celsius.

Refresh the culture medium every two to three days. After two weeks, replace the isolation medium with the organoid expansion medium. After seven to 10 days of culturing, once the organoids have reached the appropriate density, process the cultures as needed.

To passage the organoids, first preheat ultra low attachment surface cell culture plates in an incubator. Next, thaw the frozen BME overnight at four degrees Celsius until right before usage. Prewarm the organoid harvest solution and trypsin substitute at 37 degrees Celsius for 30 minutes.

Following the preparation stage, remove the culture medium from the organoid culture plate. Then transfer the organoid suspension into a 15 milliliter tube. Now, add organoid harvesting solution in proportion to the amount of BME.

To mix the suspension, use a 1000 microliter pipette gun to scrape and pipette up and down. After incubating the tube at room temperature for 30 minutes, use a one milliliter pipette to carefully aspirate the BME. Ensure that the BME is fully dissolved.

Monitor the extract every 10 minutes until a clear organoid cell cluster is visible. Then centrifuge at room temperature at 400 G for five minutes. Remove as much of the supernatant as feasible.

To start enzymatic digestion of the hepatocellular carcinoma organoids, add one to five milliliters of the prewarmed trypsin substitute to the cultured organoid pellet. Incubate the suspension at 37 degrees Celsius for two minutes. Use a microscope to verify if organoids dissociate into small clusters of two to 10 cells.

To stop the digestion, add an appropriate volume of cold basal medium. Then centrifuge the organoids at 400 G for five minutes at eight degrees Celsius. Carefully remove as much supernatant as possible.

Resuspend the desired number of organoids in the proper matrix for plating. Every 10 days, passage the organoids depending on the density of organoid growth. HCC organoid spheroids were observed within three days of culture.

Compact spheroids with rounded edges and permeable cytosol were seen on the initial day of establishment. Organoids were similarly sized and had the largest diameter when cultured in 30 to 50%BME. The BME was most fragmented at 10%resulting in the smallest organoids.

The BME was most intact at 100%but resulted in organoids with intermediate diameter. The proliferating HCC organoid attained a size exceeding 500 micrometers in each culture after three generations. Substantially sized HCC organoids of higher than 1000 micrometers were obtained within a 20 day culture period.