What is the main advantage of using organoids in this protocol?

Organoids closely mimic the three-dimensional structure and cellular heterogeneity of tumors, enabling more physiologically relevant drug response studies compared to traditional cell lines.

How is drug treatment standardized across samples?

Drug solutions are prepared to ensure equal final volumes and DMSO concentrations in all wells, with consistent medium replacement schedules and imaging parameters for reproducibility.

What are the key steps for preparing organoids for single-cell RNA sequencing?

Key steps include gentle matrix dissolution, enzymatic dissociation to single cells, viability assessment, filtration to remove aggregates, and precise cell concentration adjustment before library preparation.

How is treatment response quantitatively assessed in organoids?

Response is measured by analyzing organoid area growth curves, mean diameter, and surviving organoid counts, using consistent imaging and analysis parameters across groups.

What challenges are addressed by this protocol?

The protocol addresses challenges in preserving cell viability, ensuring consistent processing, and enabling reproducible single-cell transcriptomic analysis after drug treatment.

Can this workflow be adapted to other cancer types?

Yes, the protocol is adaptable to other tumor organoid systems beyond hepatocellular carcinoma, supporting broader applications in cancer research.

What downstream analyses are enabled by this workflow?

The workflow supports clustering, differential gene expression analysis, pathway enrichment, and inference of cellular trajectories from single-cell RNA sequencing data.

Entstehung und einzellige transkriptomische Analyse von hepatozellulären Karzinom-Organoiden nach medikamentöser Behandlung

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08:18 min

May 26th, 2026

10.3791/70511-v

May 26th, 2026

256 views

Dan He*¹ , Zhong Liang*¹^,²^,³ , Cheng-Yang Zhang*¹^,²^,³ , Jie-Shen Xian*⁴ , Yuan-Xiang Yang⁵ , Yuan-Feng Gong³ , Hui Tang³ , Guang-Ding Zhou¹^,²^,³ , Chen-Yu Jiang¹^,²^,³ , Le Xing¹^,²^,³ , Dong-Mei Li¹^,²^,³ , Ying-Chen Hou¹^,²^,³ , Chen Yue¹^,²^,³ , Wei Cheng¹^,² , Yun-Qiang Tang³ , Ming Liu¹^,²^,³ , Xiao-Feng Zhang¹^,²^,³^,⁶

¹School of Basic Medical Science, Guangzhou Medical University, ²Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, Guangzhou Medical University, ³Affiliated Cancer Hospital and Institute, Guangzhou Medical University, ⁴The First Clinical School of Guangzhou Medical University, Guangzhou Medical University, ⁵Department of Obstetrics, Guangzhou Eighth People's Hospital, Guangzhou Medical University, ⁶Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University

Dieses Protokoll beschreibt eine optimierte Methode zur Erzeugung von hepatozellulären Karzinom-Organoiden, zur Anwendung einer medikamentösen Behandlung und zur Durchführung einer Einzelzell-RNA-Sequenzierung vor und nach der Behandlung, um therapieassoziierte transkriptionale Veränderungen zu charakterisieren.