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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+evolution+in+hepatocellular+carcinoma.">Tumour evolution in hepatocellular carcinoma.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 17 (3), 139-152 (2020).</li><li>Yang, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+view+of+hepatocellular+carcinoma:+trends,+risk,+prevention+and+management.">A global view of hepatocellular carcinoma: trends, risk, prevention and management.</a> Nature Reviews. Gastroenterology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+therapy+for+hepatocellular+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+and+Integrative+Genomic+Characterization+of+Hepatocellular+Carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+liver+cancer-derived+organoid+cultures+for+disease+modeling+and+drug+screening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+patient-derived+cancer+organoids+for+drug-screening+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+organoids+and+cell+transplantation:+What+the+future+holds.">Hepatocyte organoids and cell transplantation: What the future holds.</a> Experimental &amp; Molecular Medicine. 53 (10), 1512-1528 (2021).</li><li>Nuciforo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+liver+cancers+derived+from+tumor+needle+biopsies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hepatocyte+differentiation+model+reveals+two+subtypes+of+liver+cancer+with+different+oncofetal+properties+and+therapeutic+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+tumor+lineage+plasticity+in+hepatocellular+carcinoma+using+an+anti-CLDN6+antibody-drug+conjugate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+functional+characterization+of+potential+oncofetal+targets+in+human+hepatocellular+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell-mediated+therapeutic+resistance+in+hepatocellular+carcinoma.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#946; 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 &#956;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&#160;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&#38;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&#39;s Modified Eagle Medium/Ham&#39;s F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634028</td>
			<td>Advanced DMEM/F-12</td>
		</tr>
		<tr>
			<td>Earle&#8217;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&#945;</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.

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

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 organoids culture 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 organoids with close to the original tissue characteristics. We may monitor the tumor evolution using the patient-derived organoids and try to identify novel molecular and therapeutic targets for HCC treatment.

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

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.

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

development-optimization-human-hepatocellular-carcinoma-patient

Research

JoVE Journal

Cancer Research

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture

To begin the tissue dissociation, collect one to two cubic centimeters of hepatocellular carcinoma or HTC 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.Thaw 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 pre-warmed 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. Re-suspend the pellet in 50 milliliters of cold basal medium to obtain the cell pellet for organoid plating.

Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis

To perform organoid plating, first, remove the supernatant from the centrifuge and washed hepatocellular carcinoma cells. Re-suspend 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. Re-suspend 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 organoids 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.

983 Views.  Affiliated Cancer Hospital and Institute of Guangzhou Medical University.  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.

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Summary