Generation and Single-Cell Transcriptomic Analysis of Hepatocellular Carcinoma Organoids following Drug Treatment

We focus on how HCC organoids respond to drug treatment and how their translational studies change at work. This protocol is useful for HCC organoids, and can also be adapted to other tumor organoid systems. To begin, establish hepatocellular carcinoma or HCC-patient-derived organoids, and prepare them for therapeutic perturbation.

Prepare the lenvatinib stock solution in dimethyl sulphoxide, or DMSO, according to the manufacturer's instructions. Dilute the stock solution in prewarm culture medium immediately before use to the predefined working concentration. Prepare enough solution to achieve equal final volumes in all wells, ensuring the same DMSO concentration across each well.

Next, add lenvatinib-containing medium at 20 micromolar along the wall of each well to avoid disturbing the matrix domes. Include a vehicle control with the same final DMSO concentration. Incubate the organoids under standard culture conditions for the predefined treatment period.

For treatments exceeding 72 hours, replace the drug-containing medium every 48 to 72 hours, ensuring a consistent replacement schedule across all wells. Record baseline bright-field images before treatment. Acquire images at fixed intervals during treatment using identical microscope settings, magnification and field positions.

For morphology-based evaluation of the treatment response, use identical exposure settings, magnification and analysis thresholds for all images. Measure the organoid area growth curve by calculating the total organoid area, per well or field, at each time point, and normalizing the values to baseline. To measure the mean organoid diameter, determine the diameter of individual intact organoids and calculate the mean value per well.

Next, determine the surviving organoid count by counting morphologically identifiable intact organoids, while excluding any debris or collapsed fragments. Perform a three-dimensional luminescence viability assay at the treatment endpoint if additional bulk viability assessment is required following the manufacturer's instructions. Then, plot the organoid area growth curves over time.

Compare the mean organoid diameter between vehicle-treated and lenvatinib-treated groups. Also, compare surviving organoid counts across treatment groups. Ensure using consistent imaging schedules, inclusion criteria and analysis parameters to ensure reproducibility.

Select organoid wells with intact 3D morphology, sufficient material for single cell capture, and no visible contamination. Record bright-field images of each selected well before dissociation using identical microscope settings, magnification and field selection criteria. Collect organoids at matched time points across control and treated groups, while maintaining identical plating density, treatment schedule, and medium replacement conditions across all samples.

Aspirate the culture medium completely from each well. Wash each well twice with ice-cold PBS to remove residual medium and debris. Add one milliliter of ice-cold cell recovery solution to each well, and incubate on ice for 20 to 30 minutes.

Pipette gently every five to seven minutes during incubation to facilitate matrix dissolution. Transfer the dissolved material into pre-chilled tubes using a wide bore or cut pipette tip to minimize sheer stress. Proceed to enzymatic dissociation only after the matrix is largely dissolved and minimal visible gel residue remains.

Centrifuge the recovered organoid suspension at 300 G for five minutes at four degrees Celsius. Remove the supernatant carefully without disturbing the pellet. Re-suspend the pellet in one milliliter of recombinant cell dissociation enzyme, and incubate at 37 degrees Celsius for five to 10 minutes.

Pipette gently eight to 10 times every two to three minutes, using a wide bore P1000 tip, to promote dissociation. Stop the digestion when most organoid fragments have dispersed into single cells, and only small residual clusters remain. Next, add four milliliters of ice-cold PBS containing 2%fetal bovine serum to stop the digestion.

Then, filter the suspension through a 40-micrometer cell strainer. Wash once with PBS to remove residual enzyme, aggregates and debris. Count the cells using trypan blue exclusion.

After ensuring the cell viability of 85%or above, adjust the final cell concentration to 700 to 1, 200 cells per microliter. Exclude samples with excessive debris, abundant dead cells, large visible aggregates or incomplete dissociation. Repeat filtration before loading if aggregates are present.

Process control and treated samples under identical conditions, including the dissociation enzyme, digestion time, pipetting frequency, filtration method, cell concentration, and loading strategy. Finally, after single-cell library amplification, perform single-cell sequencing. Organoid survived and expanded under standard three-dimensional culture conditions from day one to day six post-recovery.

At day one, organoids appeared as small compact structures, whereas by day six, they exhibited increased size and well-defined morphology. Lenvatinib-treated organoids were smaller, and displayed altered morphology compared to DMSO-treated controls. Quantitative analysis showed a reduction in mean organoid diameter, following lenvatinib treatment, compared to DMSO controls.

This protocol allows us to study changes in cell state, cellular composition and gene expression after treatment. The biggest challenge is preserving cell viability, keeping simple processing consistent across all groups. Cellular downstream analysis can be conducted following this procedure, including clustering, differential gene experiments analysis, pathway enrichment analysis, and treasury inference.