本研究得到了国家自然科学基金（82122048;82003773;82203380）和广东省基础与应用基础研究基金（2023A1515011416）的支持。

在广州医科大学附属肿瘤医院和研究所对各自患者进行人体活检组织，并征得患者知情同意书。有关本协议中使用的所有材料、试剂和仪器的详细信息 ，请参阅材料表 。
1. 从手术样本中建立患者来源的 HCC 类器官
注：HCC类器官的建立包括组织解离、类器官接种、培养、传代、冷冻保存和复苏等各个阶段。组织解离过程需要持续2小时，而将类器官接种到平板上大约需要40分钟。在此之后，使用HCC分离培养基对第一代HCC类器官进行10-14天的培养期。一旦达到令人满意的密度，就会进行类器官传代，这需要1小时。然后使用HCC扩增培养基维持类器官的后续培养物7-10天，这可能因类器官的生长速率和条件而异。
<ol><li>组织解离<ol><li>准备用品和材料<ol><li>从手术前未接受过任何局部或全身治疗的患者中收集 1-2 cm3 的 HCC 组织。手术切除后，将组织保持在组织保存溶液（表1）中的4°C直至处理。 注意：高质量的新鲜组织样本对于成功建立类器官至关重要。及时处理样本很重要，最好在手术切除后 1-4 小时内处理，以保持组织活力。使用无菌材料和试剂在无菌环境中执行所有后续程序。</li>
			<li>在37°C的培养箱中预热超低附着表面细胞培养板（24孔）1小时。 将冷冻的基底膜提取物（BME）在4°C下解冻过夜，直到使用前。 注意：为确保BME完全解冻，必须将其放在冰上至少3小时。BME的完全熔解对于后续步骤中的成功类器官培养至关重要。</li>
			<li>使用前，确保将消解溶液（表1）加热至37°C的温度。 注意： 在无菌环境中新鲜制备消解溶液并及时使用。</li>
		</ol></li>
		<li>组织处理流程<ol><li>在层流柜中，使用培养皿上的手术剪刀将肿瘤组织切成小块（0.5-1mm3）。将夹紧的组织转移到 15 mL 锥形管中，并加入 5-10 mL 基础培养基（表 1）。 注意：基础培养基（表1）可以在4°C下储存长达1个月。</li>
			<li>使用气压移液管洗去尽可能多的血液，静置1-2分钟以去除一些上清液，包括任何血细胞和漂浮的脂肪凝块。重复此过程两次。</li>
			<li>在室温下以300× g 离心混合物5分钟，并小心除去上清液。向修剪的组织中加入 5 mL 预热的消化溶液（表 1）。</li>
			<li>在37°C下旋转试管进行消化。</li>
			<li>初始消化30分钟后，在显微镜下寻找小细胞簇。每 5-10 分钟检查一次，以避免过度消化。 注意：组织消化所需的时间取决于组织的大小和类型。组织消化不应超过90分钟。过度消化的组织在显微镜下会显示为一大片单个细胞。适当的消化水平表明，其中大多数是细胞簇，具有葡萄状外观。</li>
			<li>通过加入冷基础培养基（表1）停止消化，并用100μm细胞过滤器过滤到新的50mL管中。加入冷基础培养基至 50 mL 的体积。</li>
			<li>将细胞在8°C下以2× g 离心10分钟。 小心地除去上清液，并通过再加入 50 mL 冷基础培养基（表 1）重悬沉淀。重复此步骤两次。 注意：低速离心用于使小细胞簇沉降，而血细胞和细胞碎片仍悬浮在上清液中;该过程重复数次以获得相对纯净的组织细胞团。</li>
		</ol></li>
	</ol></li>
	<li>类器官电镀<ol><li>第二次洗涤后，尽可能多地除去上清液，并将所需数量的细胞（24孔板的每孔1,000-5,000个细胞）重悬于适当的BME中进行铺板。 注意：使用前始终将BME保持在4°C。</li>
		<li>加入 BME 并在 Advanced DMEM/F-12 中悬浮小细胞簇，方法是添加 BME 并轻轻上下移液，直到细胞聚集体完全悬浮。将BME的浓度控制在30%至50%之间。</li>
		<li>将 50 μL BME 液滴与细胞簇混合在 24 孔培养板的中心接种。 注意：尽可能不要让液滴扩散到孔板的侧壁。低吸附板的侧壁不处于低吸附状态，液滴与侧壁接触会导致粘附，不利于后续的孵育。</li>
		<li>让添加液滴的板在37°C下凝固20分钟。在孵育结束时，向每个孔中加入500μL预热培养基（表1），并在37°C的细胞培养箱中孵育。</li>
	</ol></li>
	<li>类器官培养<ol><li>每 2-3 天刷新一次培养基（表 1）。在培养开始时，在HCC分离培养基（表1）中培养HCC类器官2周。</li>
		<li>用HCC分离培养基孵育2周后，用HCC类器官扩增培养基（表1）替换培养基（在4°C下储存长达2周）。</li>
		<li>每 3 天刷新一次培养基。</li>
		<li>用 HCC 扩增培养基培养 7-10 天后，当类器官达到适当的密度或大小时，根据需要启动实验干预或传代或冻干类器官。</li>
	</ol></li>
	<li>类器官传代<ol><li>准备用品和材料<ol><li>在37°C的培养箱中预热超低附着表面细胞培养板（24孔）1小时。 在4°C下解冻冷冻BME过夜，直到使用前。</li>
			<li>将类器官收获溶液和胰蛋白酶替代品在37°C下预热30分钟。</li>
		</ol></li>
		<li>传代过程<ol><li>从孔板中取出培养基后，将类器官悬浮液转移到 15 mL 管中。</li>
			<li>使用 1,000 μL 移液枪上下刮擦和移液，根据 BME 的量（每 50 μL BME 500 μL 类器官收获溶液）加入类器官收获溶液。 注意：在整个过程中避免气泡，以防止类器官丢失，因为气泡的存在表明移液压力较高。</li>
			<li>在室温下孵育30分钟。</li>
			<li>用 1,000 μL 移液管轻轻吸出 BME，观察 BME 完全溶解。每10分钟观察一次，直到观察到透明的类器官细胞簇，然后在室温下以400× g 离心5分钟，并尽可能多地除去上清液。 注意：在这个阶段，类器官可以被冷冻和保存。</li>
			<li>对于类器官的酶消化，加入1-5mL胰蛋白酶替代品（根据类器官的数量）并在37°C孵育2分钟。在显微镜下观察酶消化的程度，以确定类器官是否解离成2-10个细胞的小簇;如果没有，请继续酶消化。 注意：过度消化将导致类器官细胞簇裂解成单个细胞，降低其活力并延长后续培养时间。</li>
			<li>加入适量的冷基础培养基（表1）以停止消化。</li>
			<li>将类器官在8°C下以400× g 离心5分钟;尽可能去除上清液。</li>
			<li>将所需数量的类器官（24孔板每孔1,000-5,000个类器官）重悬于适当的基质中进行铺板。按照第 1.2 节中的相同步骤操作。 注意：应根据类器官的生长速度和大小优化电镀密度。</li>
			<li>根据类器官生长的密度，每 10 天以 1：3 或 1：4 的比例传代类器官。</li>
		</ol></li>
	</ol></li>
	<li>类器官冷冻保存和复苏<ol><li>类器官的冷冻保存<ol><li>准备冻干管，每个管与含有类器官的 24 孔板的两个孔汇合。</li>
			<li>按照步骤1.4.2.1至1.4.2.4进行类器官传代以获得不含BME的类器官，并通过向24孔板中加入500μL/孔类器官冻干溶液来轻轻重悬类器官。 注意：根据类器官的生长状态和大小，建议将类器官冷冻保存在培养物中，直到第三代。</li>
			<li>将悬浮液转移到冻干管中，并将其置于-80°C的梯度冷却箱中。 在-80°C梯度冷却至少24小时后，将管转移到液氮中进行长期储存。</li>
		</ol></li>
		<li>类器官复苏<ol><li>在37°C水浴中孵育冻干管，当只剩下一小块冰时停止孵育。</li>
			<li>在8°C下以400× g 离心5分钟以完全除去上清液。</li>
			<li>按照第 1.2 节和第 1.3 节进行后续操作。</li>
		</ol></li>
	</ol></li>
</ol>

优化 HCC 类器官形成，用于靶标识别和药物开发

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作者没有需要披露的利益冲突。

患者衍生类器官模型的一个显着好处在于它们能够忠实地复制肿瘤的生物学特征，包括组织结构和基因组景观。这些模型表现出非凡的准确性，并有效地反映了肿瘤的异质性和进展，即使在长时间的培养中也是如此6,8,9。通过利用这种改进的类器官培养方案，我们有效地建立了患者来源的 HCC 类器官模型，促进了体外的?...

肝细胞癌 （HCC） 是一种普遍存在且广泛多样化的肿瘤1，在医学界引起了相当大的关注。HCC中谱系可塑性和大量异质性的存在表明，来自不同患者的肿瘤细胞，甚至同一患者体内的不同病变都可能表现出不同的分子和表型特征，从而为创新治疗方法的发展带来了巨大的障碍2,3,4,5 .因此，迫切需要加强对肝癌耐药性的生物学特性和机制的理解，以便为制定更有效的治疗策略提供信息。
近几十年来，研究人员致力于开发体外模型，以研究HCC 3,4。尽管取得了一些进步，但局限性仍然存在。这些模型包含一系列技术，例如细胞系、原代细胞和患者来源的异种移植物 （PDX） 的利用。细胞系可作为从HCC患者获得的肿瘤细胞长期培养的体外模型，具有方便和易于扩增的优点。原代细胞模型涉及从患者肿瘤组织中直接分离和培养原代肿瘤细胞，从而提供与患者本身非常相似的生物学特征的表示。PDX模型需要将患者肿瘤组织移植到小鼠体内，目的是更忠实地模拟肿瘤的生长和反应。这些模型在HCC研究中发挥了重要作用，但它们具有一定的局限性，包括细胞系的异质性和无法完全复制体内条件。此外，长时间的体外培养可能导致细胞原有特征和功能的恶化，从而对准确表示HCC的生物学特性构成挑战。此外，PDX 模型的使用既费时又费钱3.
为了解决这些局限性并更准确地复制HCC的生理特性，类器官技术的利用已被引入，作为一个有前途的研究平台，能够超越以前的限制。类器官是在体外培养的三维细胞模型，具有复制实际器官结构和功能的能力。然而，在HCC的背景下，在建立类器官模型方面存在一定的挑战。这些挑战包括对 HCC 类器官构建程序的描述不够详细，缺乏针对 HCC 类器官构建整个过程的综合方案，以及培养的类器官通常尺寸较小 6,7,8。鉴于培养的类器官通常尺寸有限，我们努力通过开发涵盖整个 HCC 类器官构建的综合方案来应对这些挑战6。该方案包括组织解离、类器官铺板、培养、传代、冷冻保存和复苏。通过优化程序步骤和改进培养基的组成，我们成功建立了能够持续生长和长期传代的 HCC 类器官模型 6,8。在随后的章节中，将全面介绍HCC类器官构建所涉及的操作复杂性和相关因素。

<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

肝细胞癌 （HCC） 是全球范围内高度流行和致命的肿瘤，其发现较晚且缺乏有效的特异性治疗药物，因此需要对其发病机制和治疗进行进一步研究。类器官是一种与天然肿瘤组织非常相似且可以在 体外培养的新型模型，近年来引起了人们的极大兴趣，有许多关于肝癌类器官模型开发的报道。在这项研究中，我们成功地优化了程序并建立了培养方案，能够形成具有稳定传代和培养条件的更大尺寸的HCC类器官。本文全面概述了该过程的每个步骤，涵盖了HCC组织解离、类器官接种、培养、传代、冷冻保存和复苏的整个过程，并在本文中提供了详细的注意事项。这些类器官表现出与原始HCC组织的遗传相似性，可用于各种应用，包括识别肿瘤的潜在治疗靶点和随后的药物开发。

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.

作者聚焦：使用患者来源的类器官研究肝癌发病机制 

开发和优化用于潜在靶点识别和药物发现的人肝细胞癌患者来源类器官模型

在实施上述程序后，通常可以在 3 天内观察到 HCC 类器官球体的出现（图 1）。图1A，B显示了已建立的HCC类器官，该类器官在建立的最初一天迅速发育出具有圆润边缘和可渗透性胞质溶胶的致密球状体。在HCC类器官的生长过程中，使用不同浓度的BME对类器官的生长速率有不同的影响。我们在 10%、30%、50% 和 100% 的 BME 中培养两种患者来源的 HC...

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

我们对肝细胞癌（HCC）类器官形成的现有方案进行了全面概述和改进，涵盖了类器官培养的所有阶段。该系统可作为识别潜在治疗靶点和评估候选药物有效性的宝贵模型。

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

我们的实验室旨在研究肝癌的分子发病机制，并确定药物开发的新治疗靶点。我们想利用新的疾病模型（如患者来源的类器官）来回答癌症如何启动、转移和进展为药物耐受性。目前的实验挑战主要体现在两个方面。首先，缺乏一套完整的肝细胞癌类器官构建方案。其次，培养的HCC类器官的尺寸太小，无法进行后续的实验处理。在该协议中，我们提供了优化的类器官培养和随后的转移和冷冻程序的详细信息，以及在所有程序中应采取的预防措施。我们优化了类器官培养基的组成，使其具有接近原始组织特征的类器官。我们可以使用患者来源的类器官监测肿瘤的演变，并尝试确定用于HCC治疗的新分子和治疗靶点。

development-optimization-human-hepatocellular-carcinoma-patient

Research

JoVE Journal

Cancer Research

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

1.0K 次观看.  Affiliated Cancer Hospital and Institute of Guangzhou Medical University. 我们的实验室旨在研究肝癌的分子发病机制，并确定药物开发的新治疗靶点。我们想利用新的疾病模型（如患者来源的类器官）来回答癌症如何启动、转移和进展为药物耐受性。目前的实验挑战主要体现在两个方面。首先，缺乏一套完整的肝细胞癌类器官构建方案。其次，培养的HCC类器官的尺寸太小，无法进行后续的实验处理。在该协议中，我们提供了优化的类器官培?...

视频: 作者聚焦：使用患者来源的类器官研究肝癌发病机制 

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.

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.