This research was supported by the National Natural Science Foundation of China (82122048; 82003773; 82203380) and Guangdong Basic and Applied Basic Research Foundation (2023A1515011416).

Human-biopsied tissues were obtained from the respective patient at the Affiliated Cancer Hospital and Institute of Guangzhou Medical University, and informed consent was obtained from the patients. See the Table of Materials for details about all materials, reagents, and instruments used in this protocol.
1. Establishing patient-derived HCC organoids from surgical samples
NOTE: The establishment of HCC organoids encompasses various stages, namely tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation. The process of tissue dissociation requires a duration of 2 h, while the seeding of organoids onto a plate takes approximately 40 min. Following this, the initial generation of HCC organoids undergoes a culture period of 10-14 days using HCC isolation medium. Once a satisfactory density is achieved, organoid passages are conducted, which requires 1 h. Subsequent cultures of the organoids are then maintained using HCC expansion medium for 7-10 days, which may vary depending on the growth rate and condition of the organoids.
<ol>
	<li>Tissue dissociation
	<ol>
		<li>Preparation of supplies and materials
		<ol>
			<li>Collect 1-2 cm3 of HCC tissue from patients who have not received any previous local or systemic treatment before the operation. After surgical resection, keep the tissue at 4 &#176;C in tissue preservation solution (Table 1) until processing. 
			NOTE: High-quality fresh tissue samples are essential for the successful establishment of organoids. It is important to process samples promptly, ideally within 1-4 h after surgical resection to maintain tissue viability. Perform all subsequent procedures in an aseptic environment using sterile materials and reagents.</li>
			<li>Preheat ultra-low attachment surface cell culture plates (24 wells) for 1 h in an incubator at 37 &#176;C. Thaw the frozen basement membrane extract (BME) overnight at 4 &#176;C until just before use. 
			NOTE: To ensure complete thawing of the BME, it must be placed on ice for at least 3 h. Complete melting of the BME is essential for successful organoid culture in the subsequent steps.</li>
			<li>Before use, ensure that the digestion solution (Table 1) is warmed to a temperature of 37 &#176;C. 
			NOTE: Prepare the digestion solution freshly in an aseptic environment and utilize it promptly.</li>
		</ol>
		</li>
		<li>Organizational processing flow
		<ol>
			<li>In a laminar flow cabinet, cut the tumor tissue into small pieces (0.5-1 mm3) using surgical scissors on a Petri dish. Transfer the clipped tissue to a 15 mL conical tube and add 5-10 mL of basal medium (Table 1). 
			NOTE: The basal medium (Table 1) can be stored at 4 &#176;C for up to 1 month.</li>
			<li>Use a barotropic pipette to wash away as much blood as possible and let it stand for 1-2 min to remove some of the supernatant, including any blood cells and floating fat clots. Repeat this procedure twice.</li>
			<li>Centrifuge the mixture at 300 &#215; g for 5 min at room temperature, and carefully remove the supernatant. Add 5 mL of the prewarmed digestion solution (Table 1) to the trimmed tissue.</li>
			<li>Rotate the tube at 37 &#176;C for digestion.</li>
			<li>After 30 min of initial digestion, look for small clusters of cells under the microscope. Check every 5-10 min to avoid overdigestion. 
			NOTE: The time required for tissue digestion will depend on the size and type of tissue. Tissue digestion should not exceed 90 min. Overdigested tissue will appear under the microscope as a large sheet of individual cells. The appropriate level of digestion is indicated by the fact that most of these are cell clusters and have a grape-like appearance.</li>
			<li>Stop digestion by adding cold basal medium (Table 1) and filter into a new 50 mL tube with a 100 &#181;m cell filter. Add cold basal medium to a volume of 50 mL.</li>
			<li>Centrifuge the cells at 2 &#215; g for 10 min at 8 &#176;C. Carefully remove the supernatant and resuspend the pellet by adding a further 50 mL of cold basal medium (Table 1). Repeat this step twice. 
			NOTE: Low-speed centrifugation is used to allow small cell clusters to settle down, while blood cells and cell debris are still suspended in the supernatant; the process is repeated several times to obtain a relatively pure tissue cell mass.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Organoid plating
	<ol>
		<li>After the second wash, remove as much of the supernatant as possible and resuspend the desired number of cells (1,000-5,000 cells per well of a 24-well plate) in the appropriate BME for plating. 
		NOTE: Always keep the BME at 4 &#176;C before use.</li>
		<li>Add BME and suspend small cell clusters in Advanced DMEM/F-12 by adding BME and gently pipetting up and down until the cell aggregates are completely suspended. Control the concentration of the BME between 30% and 50%.</li>
		<li>Seed 50 &#181;L BME droplets mixed with cell clusters in the center of 24-well culture plates. 
		NOTE: As far as possible, do not let the droplets spread to the sidewall of the well plate. The sidewall of the low-adsorption plate is not in a low-adsorption state, and contact between the droplets and the sidewall will lead to adhesion, which is not conducive to the subsequent incubation.</li>
		<li>Allow the plates with the added droplets to solidify at 37 &#176;C for 20 min. At the end of the incubation, add 500 &#181;L of prewarmed medium (Table 1) to each well and incubate in a cell incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Organoid culture
	<ol>
		<li>Refresh the culture medium (Table 1) once every 2-3 days. At the beginning of the culture, culture the HCC organoids in HCC isolation medium (Table 1) for 2 weeks.</li>
		<li>After 2 weeks of incubation with HCC isolation medium, replace the medium with HCC organoid expansion medium (Table 1) (stored at 4 &#176;C for up to 2 weeks).</li>
		<li>Refresh the culture medium once every 3 days.</li>
		<li>After 7-10 days of culturing with HCC expansion medium when the organoid has reached the appropriate density or size, initiate experimental interventions or passage or lyophilize the organoids as needed.</li>
	</ol>
	</li>
	<li>Organoid passaging
	<ol>
		<li>Preparation of supplies and materials
		<ol>
			<li>Preheat ultra-low attachment surface cell culture plates (24 wells) for 1 h in an incubator at 37 &#176;C. Thaw frozen BME overnight at 4 &#176;C until just before use.</li>
			<li>Prewarm organoid harvest solution and trypsin substitute at 37 &#176;C for 30 min.</li>
		</ol>
		</li>
		<li>passaging process
		<ol>
			<li>After removing the culture medium from the well plate, transfer the organoid suspension to a 15 mL tube.</li>
			<li>Add organoid harvesting solution according to the amount of BME (500 &#181;L of organoid harvesting solution per 50 &#181;L of BME) by scraping and pipetting up and down using a 1,000 &#181;L pipette gun. 
			NOTE: Avoid air bubbles throughout the process to prevent loss of organoids as the presence of air bubbles indicates higher pipetting pressure.</li>
			<li>Incubate for 30 min at room temperature.</li>
			<li>Gently aspirate the BME with a 1,000 &#181;L pipette and observe that the BME is completely dissolved. Observe every 10 min until a clear organoid cell cluster is observed, then centrifuge at 400 &#215; g for 5 min at room temperature and remove as much of the supernatant as possible. 
			NOTE: At this stage, the organoid can be frozen and preserved.</li>
			<li>For enzymatic digestion of organoids, add 1-5 mL of the trypsin substitute (according to the number of organoids) and incubate at 37 &#176;C for 2 min. Observe under the microscope the degree of enzymatic digestion to determine whether the organoids dissociate into small clusters of 2-10 cells; if not, continue the enzymatic digestion. 
			NOTE: Overdigestion will result in the lysis of organoid cell clusters into single cells, decreasing their viability and prolonging subsequent culture time.</li>
			<li>Add the appropriate volume of cold basal medium (Table 1) to stop the digestion.</li>
			<li>Centrifuge the organoids at 400 &#215; g for 5 min at 8 &#176;C; remove the supernatant as much as possible.</li>
			<li>Resuspend the desired number of organoids (1,000-5,000 organoids per well of a 24-well plate) in the appropriate matrix for plating. Follow the same steps as in section 1.2. 
			NOTE: The plating density should be optimized according to the growth rate and size of the organoids.</li>
			<li>Passage the organoids every 10 days at a ratio of 1:3 or 1:4 depending on the density of organoid growth.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Organoid cryopreservation and resuscitation
	<ol>
		<li>Cryopreservation of organoids
		<ol>
			<li>Prepare lyophilization tubes, each tube confluent with two wells of a 24-well plate containing organoids.</li>
			<li>Follow steps 1.4.2.1 to 1.4.2.4 for organoid passaging to obtain organoids without BME, and gently resuspend the organoids by adding 500 &#181;L/well of organoid lyophilization solution to a 24-well plate. 
			&#8203;NOTE: Depending on the growth status and size of the organoids, it is recommended to cryopreserve the organoids in culture up to the third generation.</li>
			<li>Transfer the suspension into lyophilization tubes and place it in a gradient-cooled box at -80 &#176;C. After gradient cooling at -80 &#176;C for at least 24 h, transfer the tubes to liquid nitrogen for long-term storage.</li>
		</ol>
		</li>
		<li>Resuscitation of organoids
		<ol>
			<li>Incubate lyophilized tubes in a 37 &#176;C water bath and stop incubation when only a small block of ice remains.</li>
			<li>Centrifuge at 400 &#215; g for 5 min at 8 &#176;C to remove the supernatant completely.</li>
			<li>Follow sections 1.2 and 1.3 for subsequent operations.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Optimizing HCC Organoid Formation for Target Identification and Drug Development

<ol><li>Vogel, A., Meyer, T., Sapisochin, G., Salem, R., Saborowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatocellular+carcinoma%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Hepatocellular carcinoma.</a> Lancet. 400 (10360), 1345-1362 (2022).</li>
<li>Craig, A. J., von Felden, J., Garcia-Lezana, T., Sarcognato, S., Villanueva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour+evolution+in+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Nature+Reviews.+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">Tumour evolution in hepatocellular carcinoma.</a> Nature Reviews. Gastroenterology & Hepatology. 17 (3), 139-152 (2020).</li>
<li>Yang, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+global+view+of+hepatocellular+carcinoma%3A+trends%2C+risk%2C+prevention+and+management%5BTitle%5D)+AND+%22Nature+Reviews.+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">A global view of hepatocellular carcinoma: trends, risk, prevention and management.</a> Nature Reviews. Gastroenterology & Hepatology. 16 (10), 589-604 (2019).</li>
<li>Huang, A., Yang, X. R., Chung, W. Y., Dennison, A. R., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+therapy+for+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Signal+Transduction+and+Targeted+Therapy%22%5BJournal%5D)">Targeted therapy for hepatocellular carcinoma.</a> Signal Transduction and Targeted Therapy. 5 (1), 146(2020).</li>
<li>Cancer Genome Atlas Research Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+and+Integrative+Genomic+Characterization+of+Hepatocellular+Carcinoma%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma.</a> Cell. 169 (7), 1327.e23-1341.e23 (2017).</li>
<li>Broutier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+primary+liver+cancer-derived+organoid+cultures+for+disease+modeling+and+drug+screening%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.</a> Nature Medicine. 23 (12), 1424-1435 (2017).</li>
<li>Driehuis, E., Kretzschmar, K., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+patient-derived+cancer+organoids+for+drug-screening+applications%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Establishment of patient-derived cancer organoids for drug-screening applications.</a> Nature Protocols. 15 (10), 3380-3409 (2020).</li>
<li>Peng, W. C., Kraaier, L. J., Kluiver, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatocyte+organoids+and+cell+transplantation%3A+What+the+future+holds%5BTitle%5D)+AND+%22Experimental+%26+Molecular+Medicine%22%5BJournal%5D)">Hepatocyte organoids and cell transplantation: What the future holds.</a> Experimental & Molecular Medicine. 53 (10), 1512-1528 (2021).</li>
<li>Nuciforo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+models+of+human+liver+cancers+derived+from+tumor+needle+biopsies%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Organoid models of human liver cancers derived from tumor needle biopsies.</a> Cell Reports. 24 (5), 1363-1376 (2018).</li>
<li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hepatocyte+differentiation+model+reveals+two+subtypes+of+liver+cancer+with+different+oncofetal+properties+and+therapeutic+targets%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (11), 6103-6113 (2020).</li>
<li>Kong, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+tumor+lineage+plasticity+in+hepatocellular+carcinoma+using+an+anti-CLDN6+antibody-drug+conjugate%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Targeting tumor lineage plasticity in hepatocellular carcinoma using an anti-CLDN6 antibody-drug conjugate.</a> Science Translational Medicine. 13 (579), eabb6282(2021).</li>
<li>Li, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+functional+characterization+of+potential+oncofetal+targets+in+human+hepatocellular+carcinoma%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Identification and functional characterization of potential oncofetal targets in human hepatocellular carcinoma.</a> STAR Protocols. 3 (4), 101921(2022).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+stem+cell-mediated+therapeutic+resistance+in+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Hepatoma+Research%22%5BJournal%5D)">Cancer stem cell-mediated therapeutic resistance in hepatocellular carcinoma.</a> Hepatoma Research. 8, 36(2022).</li>
</ol>

The authors have no conflicts of interest to disclose.

One notable benefit of patient-derived organoid models lies in their capacity to faithfully replicate the biological characteristics of tumors, encompassing tissue structure and genomic landscape. These models demonstrate a remarkable level of accuracy and effectively mirror the heterogeneity and progression of tumors, even over extended periods of cultivation6,8,9. Through the utilization of this refined organoid culture protoc...

Hepatocellular carcinoma (HCC), a prevalent and extensively diverse tumor1, has garnered considerable attention within the medical community. The presence of lineage plasticity and substantial heterogeneity in HCC suggests that tumor cells originating from various patients and even distinct lesions within the same patient may manifest dissimilar molecular and phenotypic traits, thereby presenting formidable obstacles in the advancement of innovative therapeutic approaches2,3,4,5. Consequently, there is an imperative need for enhanced comprehension of the biological attributes and mechanisms of drug resistance in HCC to inform the formulation of more efficacious treatment strategies.
In recent decades, researchers have dedicated their efforts to the development of in vitro models for the purpose of studying HCC3,4. Despite some advancements, limitations persist. These models encompass a range of techniques, such as the utilization of cell lines, primary cells, and patient-derived xenografts (PDX). Cell lines serve as in vitro models for long-term culture of tumor cells obtained from HCC patients, offering the benefits of convenience and facile expansion. Primary cell models involve direct isolation and culture of primary tumor cells from patient tumor tissues, thereby providing a representation of biological characteristics that closely resemble those of the patients themselves. PDX models entail the transplantation of patient tumor tissues into mice, with the aim of more faithfully simulating tumor growth and response. These models have been instrumental in HCC research, yet they possess certain limitations, including the heterogeneity of cell lines and the inability to fully replicate in vivo conditions. Furthermore, prolonged in vitro cultivation may result in the deterioration of the cells&#39; original characteristics and functionalities, posing challenges in accurately representing the biological properties of HCC. Additionally, the utilization of PDX models is both time-consuming and costly3.
To address these limitations and more accurately replicate the physiological attributes of HCC, the utilization of organoid technology has been introduced as a promising research platform capable of surpassing previous constraints. Organoids, which are three-dimensional cell models cultured in vitro, have the ability to replicate the structure and functionality of actual organs. However, in the context of HCC, there exist certain challenges in establishing organoid models. These challenges include insufficiently detailed descriptions of HCC organoid construction procedures, a lack of comprehensive protocols for the entire process of HCC organoid construction, and the typically small size of cultured organoids6,7,8. In light of the typically limited dimensions of cultured organoids, we endeavored to tackle these challenges through the development of a comprehensive protocol encompassing the entirety of HCC organoid construction6. This protocol encompasses tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation. By optimizing the procedural steps and refining the composition of the culture medium, we have successfully established HCC organoid models capable of sustained growth and long-term passaging6,8. In the subsequent sections, a comprehensive account of the operational intricacies and pertinent factors involved in the construction of HCC organoids will be presented.

<table><tbody><tr><td>[Leu15]-gastrin I human</td><td>Merck</td><td>G9145</td><td></td></tr><tr><td>1.5 mL Microtubes</td><td>Merck</td><td>AXYMCT150LC</td><td></td></tr><tr><td>A8301 (TGF&amp;beta; inhibitor)</td><td>Tocris Bioscience</td><td>2939</td><td></td></tr><tr><td>B27 Supplement (503), minus vitamin A</td><td>Thermo Fisher Scientific</td><td>12587010</td><td></td></tr><tr><td>B-27 Supplement (503), serum-free</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>BMP7</td><td>Peprotech</td><td>120-03P</td><td></td></tr><tr><td>Cell strainer size 100 &amp;mu;m</td><td>Merck</td><td>CLS352360</td><td></td></tr><tr><td>CHIR99021</td><td>Merck</td><td>SML1046</td><td></td></tr><tr><td>Collagenase D</td><td>Merck</td><td>11088858001</td><td></td></tr><tr><td>Corning&amp;nbsp;Costar Ultra-Low</td><td>Merck</td><td>CLS3473</td><td></td></tr><tr><td>Costar 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Individually Wrapped, Sterile</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>Costar 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Individually Wrapped, Sterile</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Cultrex Organoid Harvesting Solution</td><td>R&amp;amp;D SYSTEMS</td><td>3700-100-01</td><td>Organoid harvesting solution</td></tr><tr><td>Cultrex Reduced Growth Factor BME, Type 2 PathClear (BME)</td><td>Merck</td><td>3533-005-02</td><td></td></tr><tr><td>DAPT</td><td>Merck</td><td>D5942</td><td></td></tr><tr><td>Dexamethasone</td><td>Merck</td><td>D4902</td><td></td></tr><tr><td>DMSO</td><td>Merck</td><td>C6164</td><td></td></tr><tr><td>DNaseI</td><td>Merck</td><td>DN25</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Ham&#039;s F-12</td><td>Thermo Fisher Scientific</td><td>12634028</td><td>Advanced DMEM/F-12</td></tr><tr><td>Earle&amp;rsquo;s balanced salt solution (EBSS)</td><td>Thermo Fisher Scientific</td><td>24010043</td><td></td></tr><tr><td>Forceps</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Forskolin</td><td>Tocris Bioscience</td><td>1099</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>HEPES, 1 M</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Leica DM6 B Fluorescence Motorized Microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>N2 supplement (1003)</td><td>Thermo Fisher Scientific</td><td>17502048</td><td></td></tr><tr><td>N-acetylcysteine</td><td>Merck</td><td>A0737-5MG</td><td></td></tr><tr><td>Nicotinamide</td><td>Merck</td><td>N0636</td><td></td></tr><tr><td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339651</td><td></td></tr><tr><td>Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339653</td><td></td></tr><tr><td>Penicillin/streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Recombinant human EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Recombinant human FGF10</td><td>Peprotech</td><td>100-26</td><td></td></tr><tr><td>Recombinant human FGF19</td><td>Peprotech</td><td>100-32</td><td></td></tr><tr><td>Recombinant human HGF</td><td>Peprotech</td><td>100-39</td><td></td></tr><tr><td>Recombinant human Noggin</td><td>Peprotech</td><td>120-10C</td><td></td></tr><tr><td>Rho kinase inhibitor Y-27632 dihydrochloride</td><td>Merck</td><td>Y0503</td><td></td></tr><tr><td>R-spodin1-conditioned medium</td><td>(Broutier et al.)</td><td>N/A</td><td>Secretion of cell lines</td></tr><tr><td>Surgical scissors</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Surgical specimen of tumor removed from HCC patients</td><td>Affiliated Cancer Hospital and Institute of Guangzhou Medical University</td><td>N/A</td><td></td></tr><tr><td>TNF&amp;alpha;</td><td>Peprotech</td><td>315-01A</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), no phenol red</td><td>Thermo Fisher Scientific</td><td>12604013</td><td>Trypsin substitute</td></tr><tr><td>Wnt-3a-conditioned medium</td><td>(Broutier et al.)</td><td>N/A</td><td>Secretion of cell lines</td></tr></tbody></table>

development and optimization of a human hepatocellular carcinoma patient-derived organoid model for potential target identification and drug discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Upon implementing the aforementioned procedure, the emergence of HCC organoid spheroids is typically observable within a span of 3 days (Figure 1). Figure 1A,B show the established HCC organoid, which promptly develops compact spheroids characterized by rounded edges and permeable cytosol on the initial day of establishment. During the growth of HCC organoids, the use of different concentrations of BME had different effects on the growth rate of...

Watch this Scientific Journal Video about Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids at JoVE.com

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

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

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

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1.5K Views.  Affiliated Cancer Hospital and Institute of Guangzhou Medical University. Ons lab heeft tot doel de moleculaire pathogenese van leverkanker te onderzoeken en nieuwe therapeutische doelen voor de ontwikkeling van geneesmiddelen te identificeren. We willen graag antwoord geven op de vraag hoe kanker ontstaat, uitzaait en vordert naar medicijntolerantie met behulp van nieuwe ziektemodellen zoals van patiënten afgeleide organoïden. De huidige experimentele uitdagingen liggen in twee aspecten.Ten eerste is er een gebrek aan een volledige set protocollen voor he...

Video: Auteur in de schijnwerpers: Onderzoek naar de pathogenese van leverkanker met behulp van van patiënten afgeleide organoïden 

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Developmental Biology

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

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

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