该研究由德国癌症研究中心 DKTK 资助，合作伙伴网站慕尼黑，DKFZ 和慕尼黑大学医院 LMU 之间的合作伙伴关系。该研究还得到了德国癌症援助基金（#70113426和#70113433）的支持。组织和类器官的石蜡包埋已在慕尼黑慕尼黑大学医学院解剖学研究所的核心设施中进行。共聚焦成像已在生物医学中心 （BMC） 的核心设施生物成像中进行。作者要感谢 Simone Hofmann、Maria Fischer、Cornelia Herbst、Sabine Fink 和 Martina Rahmeh 提供的技术帮助。

收集卵巢癌手术的肿瘤组织标本，并按照 LMU 大学伦理委员会 （17-471） 的规定生成患者来源的类器官，并遵守现有的适用欧盟、国家和地方法规。参与研究的每位患者均以书面形式同意。处理新鲜组织样品时，需要获得生物安全 2 级安全许可和层流柜。鉴于组织样本具有潜在的传染性，由于缺乏对相关传染病的常规检测，不能排除这种传染性，因此有必要确保严格遵守机构生物安全法规，并为进行实验的人员提供足够的个人防护设备。
1. 准备工作
<ol><li>培养基制备<ol><li>每周新鲜制备一次2D和3D培养基，并将其储存在4°C。 注： 表1列出了每种培养基成分的确切成分。卵巢癌培养基 1 （OCM1） 和 OCM2 都可以额外补充 HER1ß（终浓度：50 ng/mL），从而产生四种不同的情况：OCM1、OCM1+HER1ß、OCM2、OCM2+HER1ß。</li>
		<li>创建生长因子试剂的储备溶液，并将其保持在20°C和A83-01和烟酰胺（储存在+4°C）的储备溶液。由于生长因子对温度的敏感性，请立即使用解冻的储备溶液进行培养基制备，并通过创建等分试样来避免重复解冻循环。 注意：培养基制备是需要严格控制的关键步骤。</li>
		<li>RSPO-1条件培养基的制备<ol><li>在37°C下快速解冻HA-R-Spondin1-Fc 293 T细胞（在-80°C下储存）。 用 10 mL 基础培养基洗涤它们，辅以 10% 胎牛血清 （FCS），以下称为基础培养基++。</li>
			<li>将细胞沉淀重悬于12mL基础培养基++中，并将其接种在T75烧瓶中。</li>
			<li>当细胞达到汇合时，用含有胰蛋白酶的解离试剂（在37°C下4分钟）以1：6的比例分裂细胞。将它们接种到新的 T75 烧瓶中。</li>
			<li>第二天，向培养基中加入绒霉素D1（1,25μL / mL）。</li>
			<li>当细胞达到汇合时，用胰蛋白酶以1：20的比例（在37°C下4分钟）分裂细胞。将细胞接种到补充有藻霉素 D1 的 30 mL 基础培养基++（含有 10% FCS）中的多个 T75 烧瓶（根据目标最终体积的烧瓶数量）。</li>
			<li>当细胞达到约50%的汇合度时，开始条件培养基生产：用补充有5%FCS的40 mL基础培养基代替培养基，不含藻霉素D1。</li>
			<li>3天后收集第一个上清液。为了去除碎片，在1,200× g下离心10分钟。将上清液保存在4°C的无菌容器中。</li>
			<li>向细胞中加入补充有 5% FCS 的新基础培养基。3-4天后收集第二个上清液。在1,200× g下离心10分钟。将第二上清液加入第一上清液中。</li>
			<li>使用0.2μm瓶顶过滤器过滤调节介质。</li>
			<li>为了进行质控和RSPO1定量，将产生的培养基加入293T细胞系（293T WntR，7xTcf-eGFP）14中，用携带Wnt报告基因18的GFP质粒稳定转导。 注意：根据GFP信号的数量和强度，通过Wnt报告细胞系的定量来测试RSPO1作为Wnt通路激动剂的活性。</li>
			<li>将15mL的等分试样保持在4°C下立即使用（1周内）。 对于更长的储存时间（6个月），将调节培养基保持在-20°C。</li>
		</ol></li>
	</ol></li>
</ol>2. 卵巢癌类器官培养的开始
<ol><li>原代组织分离<ol><li>最好在手术后立即运输新鲜和活的组织，并在收集后最多20小时内进行隔离。使用配备冷袋的运输箱在大约4°C下确保组织收集介质中的适当运输条件。</li>
		<li>在实验室中，准备必要的试剂和设备，以便及时处理样品：<ol><li>在冰上解冻基底膜提取物II型基质（BME 2基质）（约2小时）。</li>
			<li>准备一次性手术刀、灭菌解剖工具（剪刀和镊子）和 400 μm 大小的过滤器插入物，用于 50 mL 试管。</li>
			<li>准备一个装有少量液氮的容器进行快速冷冻，并在37°C下进行预热水浴。 注意：在细胞培养层流罩内工作。</li>
		</ol></li>
		<li>在培养皿中用磷酸盐缓冲盐水（PBS）（不含Ca++和 Mg++）彻底洗涤新鲜组织。在培养皿内，使用一次性手术刀和/或剪刀将组织切成 3-5 毫米大小的小块。</li>
		<li>将获得的组织分为三个部分，用于以下目的：<ol><li>在低温管中收集组织。将低温管转移到装满液氮的准备好的容器中进行冲击冷冻。</li>
			<li>将组织（2-3mm）收集到装有福尔马林的组织学标本容器中进行固定（24小时），并将其嵌入石蜡中以进行染色19,20。</li>
			<li>此外，在酶消化之前使组织均质化。用手术刀最大限度地进行机械解离，并将其转移到 50 mL 组织管中。 注意： 确保在均质化过程中始终将组织浸泡在 PBS 中，以避免组织变干。</li>
		</ol></li>
		<li>将匀浆组织与含有PBS（不含Ca++和 Mg++），胶原酶I（储备溶液5U / μL，终浓度1U / μL）和选择性ROCK1和2抑制剂（3μM终浓度）的消化混合物（ 表1中列出的组分）混合。在 50 mL 管中使用 15 mL 消化混合物，用于每 2 cm3 的肿瘤。 注意：有限的材料（例如活检样品）只需要少量（约 2-3 mL）消化混合物即可确保酶解离。</li>
		<li>对于酶解离，将管在37°C的水浴中孵育1.5小时。 进行零星和剧烈的涡旋（大约每 20 分钟一次，持续 10-15 秒）以支持机械解离。</li>
		<li>孵育后，向试管中加入 15 mL 冷基础培养基 ++。在300× g下离心5分钟。</li>
		<li>除去上清液后，加入 5 mL 新的基础培养基++。使用 400 μm 过滤器将细胞悬液过滤到新鲜的 50 mL 管中。</li>
		<li>在300× g下离心5分钟。除去上清液并保留细胞沉淀。</li>
		<li>为了去除红细胞，向细胞沉淀中加入 5 mL 红细胞裂解 （RBC） 缓冲液。在37°C的水浴中孵育5分钟。</li>
		<li>加入 5 mL 基础培养基++ 用于裂解灭活。在300× g下离心5分钟。向细胞沉淀中加入 3 mL 基础培养基++。</li>
		<li>用台盼蓝染色溶液（例如，10 μL 样品 + 10 μL 台盼蓝染色溶液）在自动细胞计数器中或在 Neubauer 室中手动稀释后，对活细胞进行计数。</li>
		<li>计算直接 3D 接种所需的细胞数量。对于每个肿瘤沉积物，将每个卵巢癌培养基至少 2-3 孔接种到 48 孔格式的培养皿中，这相当于接种基质中总共 8-12 个孔（OCM1、OCM1+HER1ß、OCM2、OCM2+HER1ß）。在 25 μL BME 2 型基质的液滴中计算每个孔的 30,000 个细胞。或者，选择含有 50 μL BME 2 基质和每孔 50,000 个接种细胞的 24 孔规格。</li>
		<li>在2D中播种剩余的细胞（参见步骤2.1.13）。</li>
		<li>将含有所需细胞总数的基础培养基++悬浮液的量移液到新管中，并以300× g离心5分钟。除去上清液并保留细胞沉淀。</li>
		<li>在冰上，将冷 BME 2 基质的总量（48 孔板中每个孔 25 μL;因此 4 孔 100 μL）添加到沉淀中并充分混合，通过轻轻上下移液沉淀来实现每个孔的细胞均匀分布不产生气泡。</li>
		<li>将细胞接种在 BME 2 基质 （25 μL） 的液滴中，接种到预热的空 48 孔板中。为了实现孔之间的均匀分布，在将BME 2基质液滴转移到每个孔中之前，在管内轻轻缓慢地上下移动移液器（3-4x），以防止分布过程中细胞沉淀。</li>
		<li>在37°C孵育板至少30分钟以巩固BME 2基质的液滴后，将四种卵巢癌培养基（OCM1，OCM1 + HER1ß，OCM2，OCM 2 + HER1ß）中的每一种250μL移液到相应的孔中。</li>
		<li>在直接 3D 接种程序的同时，通过开始 2D 培养来保留剩余的分离细胞。<ol><li>在300× g离心5分钟后，将沉淀加入2D培养基中。选择格式（T75 培养瓶或 T25 培养瓶），具体取决于 3D 接种后可用的细胞数量。</li>
			<li>将烧瓶在37°C孵育3-5天以进行细胞粘附。在前72小时内保持相同的培养基。</li>
			<li>当细胞达到汇合时，将培养物转移到 BME 2 基质中。不要通过分裂来扩增 2D 培养物中的原代分离株，因为在 2D 上长期繁殖不利于干性潜力。</li>
		</ol></li>
	</ol></li>
	<li>从 2D 培养到 3D 培养的转移<ol><li>在 T75 或 T25 烧瓶中，用 5 mL PBS 洗涤单层 2 次，以将细胞从底表面分离。在37°C下与1mL胰蛋白酶孵育10分钟。</li>
		<li>加入 5 mL 冷基础培养基++ 以灭活解离试剂。在300× g下离心5分钟。</li>
		<li>向沉淀中加入 3 mL 基础培养基 ++。按照步骤 2.1.12-2.1.13 中的说明对单元格进行计数。不同介质组合物的种子（见 图1），如步骤2.1.16-2.1.18中所述。</li>
	</ol></li>
	<li>类器官生长的评估<ol><li>每周至少对孔进行一次成像，以记录类器官的生长。通过相差显微镜制作高质量的 2D/3D 培养物和直接接种板的可比图像。注意放大倍率的一致性，并使用 4 倍来概述孔（定量），并使用 10 倍和 20 倍来记录类器官的形态。</li>
		<li>将图片存储在专用于单个行的文件夹中。</li>
		<li>通过比较评估不同接种方式（2D 后 3D 接种与直接 3D 接种）和不同培养基组成（OCM1、OCM1+ HER1ß、OCM2 和 OCM2+ HER1ß）的类器官形成，选择最佳的类器官系进行长期培养。<ol><li>平均而言，在分离后 14-21 天，评估类器官形成潜力（数量）以及在不同条件下生长的类器官的大小和细胞表型。寻找以下特征：没有液泡、膜起泡或失去粘附，以及细胞四舍五入。</li>
		</ol></li>
	</ol></li>
</ol>3.长期类器官培养
<ol><li>类器官传代<ol><li>避免在增殖阶段过于频繁地分裂类器官。进行间隔超过 10 天的机械和酶消化程序。</li>
		<li>要破坏每个 BME 2 基质液滴，加入 250 μL（48 孔形式）或 500 μL（24 孔形式）冰冷基础培养基++。确保液滴和移液管间歇性地上下完全溶解，并刮擦板底部。将细胞悬液转移到 15 mL 试管中并将其置于冰上。加入 1 mL 冰冷基础培养基++。 注：BME 2基质的去除效率很大程度上取决于介质温度，因为在4°C以下可实现最佳溶解。</li>
		<li>池2-3技术重复井，均匀扩容。在300× g下离心5分钟。对光源检查悬浮液的内容物，看看BME 2凝胶是否仍然可见。除去上清液，如果BME 2基质仍然可见，则用冰冷培养基重复洗涤。</li>
		<li>在37°C的水浴中与1mL胰蛋白酶（储存在20-25°C）一起孵育7-10分钟。零星涡旋 10 秒以增强解离。定期目视检查试管，看看解离是否在进行。</li>
		<li>用注射器通过大小范围为23G至27G的针头上下拉动细胞悬浮液，检查是否仍然存在大细胞团块，并在必要时重复该过程。 注意：通过注射器碎裂是可选的，针头尺寸取决于所需的解离效果。均匀的细胞悬液导致孔之间的均匀分布。</li>
		<li>加入 1 mL 冷基础培养基++ 以灭活反应。</li>
		<li>可选：按照步骤 2.1.12-2.1.13 中的描述对细胞进行计数，并确定与亲本传代的分裂比例（例如，1：3 孔）。</li>
		<li>在300× g下离心5分钟。除去上清液。</li>
		<li>按照步骤2.1.16-2.1.18中所述进行接种，并根据已经建立的长期培养物应用相应的最佳卵巢癌类器官培养基。每 3-4 天更换一次卵巢癌培养基。</li>
		<li>如果在培养基变化过程中 BME 2 基质液滴被破坏，请考虑在不进行酶消化的情况下重新接种。为避免液滴中断，请小心地进行介质更换程序，不要直接移液到BME 2基质液滴上。此外，在步骤2.1.16中使用BME 2基质稀释之前，避免在沉淀上留下过量的残留基础培养基++，因为它可能会影响液滴的脆弱性。</li>
	</ol></li>
	<li>类器官的冷冻保存 注意：在传代后第一周内的增殖阶段对类器官进行冷冻保存。<ol><li>根据步骤 2.1.2，用冰冷的基础培养基++破坏BME 2基质液滴。如步骤3.1.3所述离心并弃去上清液后，在冰上工作并将细胞沉淀重悬于1mL冰冷的冻存培养基中。将细胞悬液转移到预先标记的1.8ml低温管中。</li>
		<li>将试管储存在预冷的含有异丙醇的冷冻容器中，并将其置于-80°C过夜。</li>
		<li>将储备管保持在-80°C最多2个月。转为液氮进行长期稳定储存。</li>
	</ol></li>
	<li>类器官储备的解冻<ol><li>在标记的 15 mL 试管中的 37 °C 水浴中预热 9 mL 基础培养基++。</li>
		<li>将所需的储备瓶从-80°C的储存或液氮转移到充满液氮的运输容器中。</li>
		<li>将冷冻管转移到37°C的水浴中，并轻轻搅拌，直到管壁附近的冷冻材料开始解冻。</li>
		<li>将类器官悬浮液缓慢分布到装有 9 mL 培养基的标记管中。轻轻摇晃管子。在300× g 下离心5分钟，除去上清液。</li>
		<li>按照步骤2.1.16-2.1.18中所述进行接种，并根据已经确定的单个品系的长期培养条件施用相应的最佳卵巢癌类器官培养基。</li>
	</ol></li>
	<li>类器官的固定<ol><li>按照步骤3.1.2-3.1.3中所述进行类器官收集。</li>
		<li>加入 3 mL 4% 多聚甲醛 （PFA） 的 PBS （pH 7.4） 溶液，并在室温下孵育 1 小时。</li>
		<li>用 5 mL PBS 洗涤 2 次，以 300 × g 离心 3 分钟，并除去上清液。向沉淀中加入4mL新鲜PBS，并将其保持在4°C直至嵌入。 注意：固定类器官是稳定的，在嵌入前可以在4°C下储存1个月。</li>
		<li>在65°C下加热组织学凝胶（储存在-20°C）直至液化。</li>
		<li>检测固定的类器官沉降并在管底部可见。除去上清液。如果细胞沉淀被破坏，则在300× g 下离心3分钟，并弃去上清液PBS。</li>
		<li>通过轻轻上下移液，将沉淀悬浮在 100 μL 温凝胶中。将液滴转移到一块密封膜上，并在室温下~15分钟后凝固。</li>
		<li>将固定凝胶液滴移至组织石蜡盒中。进行标准组织包埋方案19,20。</li>
		<li>用微切片切割工具切割 5-10 μm 厚的切片。将切口转移到组织学载玻片上。将载玻片在65°C下干燥1小时;将它们放在干燥的地方。</li>
	</ol></li>
	<li>类器官切片的免疫荧光染色<ol><li>使用以下稀释系列将制备的载玻片移过玻璃托盘：2×15分钟用于组织学的透明剂;15分钟100%乙醇;1分钟100%乙醇;10分钟96%乙醇;5分钟70%乙醇;5分钟50%乙醇。</li>
		<li>加入 PBS 并以 100 rpm 的速度在振动台上保持 5 分钟。重复洗涤 2 次。</li>
		<li>将抗原修复溶液（三（羟甲基）氨基甲烷 - 乙二胺四乙酸（EDTA）-缓冲液[pH 9.0]或柠檬酸盐[pH 6.0]）添加到放置在热稳定室中的载玻片中。在蒸锅中，将装有载玻片的容器保持 30 分钟，然后冷却至室温。</li>
		<li>重复步骤 3.5.2。</li>
		<li>将含有载玻片的腔室转移到1%透化洗涤剂溶液（PBS中）中15分钟。</li>
		<li>重复步骤 3.5.2。</li>
		<li>在以下步骤中，用免疫染色蜡笔勾勒出载玻片上类器官的位置，以保持液滴。</li>
		<li>在每张载玻片上加入 100 μL 10% 血清稀释培养基，具体取决于二抗的种类。</li>
		<li>重复步骤 3.5.2。</li>
		<li>在培养基中稀释一抗，最终体积为 100 μL。 在培养盘/湿室中保持在4°C至少16小时。</li>
		<li>重复步骤 3.5.2。</li>
		<li>在培养基中稀释二抗，产生 100 μL 液滴，并在室温下在培养盘中保存 2 小时。</li>
		<li>重复步骤 3.5.2。</li>
		<li>加入核复染液DAPI（4'，6-二脒-2-苯吲哚，二盐酸盐）在稀释介质中1：1,000稀释。在室温下保存10分钟进行孵育。</li>
		<li>重复步骤 3.5.2。</li>
		<li>添加安装介质并用盖玻片盖住。干燥后（大约8-12小时后），用透明指甲油固定载玻片。</li>
		<li>使用共聚焦显微镜或其他荧光显微镜拍摄图像。制作概述图片以及亚细胞结构的详细图像，以捕捉类器官形态的主要特征。</li>
	</ol></li>
</ol>

标准化卵巢癌类器官生物样本库

<ol>
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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+pan-cancer+organoid+platform+for+precision+medicine.">A pan-cancer organoid platform for precision medicine.</a> Cell Rep. 36 (4), 109429 (2021).</li><li>Bartfeld, S., 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=Stem+cell-derived+organoids+and+their+application+for+medical+research+and+patient+treatment.">Stem cell-derived organoids and their application for medical research and patient treatment.</a> J Mol Med (Berl). 95 (7), 729-738 (2017).</li><li>Larsen, B. M., Cancino, A., Shaxted, J. M., Salahudeen, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+drug+screening+of+patient-derived+tumor+organoids+using+high-content+fluorescent+imaging.">Protocol for drug screening of patient-derived tumor organoids using high-content fluorescent imaging.</a> STAR Protoc. 3 (2), 101407 (2022).</li><li>Senkowski, W., 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+platform+for+efficient+establishment+and+drug-response+profiling+of+high-grade+serous+ovarian+cancer+organoids.">A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids.</a> Dev Cell. 58 (12), 1106-1121 (2023).</li><li>LeSavage, B. L., Suhar, R. A., Broguiere, N., Lutolf, M. P., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+cancer+organoids.">Next-generation cancer organoids.</a> Nat Mater. 21 (2), 143-159 (2022).</li><li>Hoffmann, K., 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=Stable+expansion+of+high-grade+serous+ovarian+cancer+organoids+requires+a+low-Wnt+environment.">Stable expansion of high-grade serous ovarian cancer organoids requires a low-Wnt environment.</a> EMBO J. 39 (6), e104013 (2020).</li><li>Kessler, 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=The+Notch+and+Wnt+pathways+regulate+stemness+and+differentiation+in+human+fallopian+tube+organoids.">The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids.</a> Nat Commun. 6, 8989 (2015).</li><li>Trillsch, F., 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=Protocol+to+optimize+the+biobanking+of+ovarian+cancer+organoids+by+accommodating+patient-specific+differences+in+stemness+potential.">Protocol to optimize the biobanking of ovarian cancer organoids by accommodating patient-specific differences in stemness potential.</a> STAR Protoc. 4 (3), 102484 (2023).</li><li>Maenhoudt, N., 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=Developing+organoids+from+ovarian+cancer+as+experimental+and+preclinical+models.">Developing organoids from ovarian cancer as experimental and preclinical models.</a> Stem Cell Reports. 14 (4), 717-729 (2020).</li><li>Fuerer, C., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+to+probe+and+manipulate+the+Wnt+signaling+pathway.">Lentiviral vectors to probe and manipulate the Wnt signaling pathway.</a> PLoS One. 5 (2), e9370 (2010).</li><li>. 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M.K.被列为与卵巢癌类器官培养基相关的专利的发明人。F.T.获得了阿斯利康、克洛维斯、卫材、ImmunoGen、Medac、默沙东、PharmaMar、罗氏、SAGA diagnostics和Tesaro/GSK的研究经费、顾问委员会、酬金和差旅费。S.M.获得了研究经费、顾问委员会、荣誉或差旅费：艾伯维、阿斯利康、克洛维斯、卫材、葛兰素史克、Hubro、Medac、默沙东、诺华、尼科德、奥林巴斯、PharmaMar、辉瑞、罗氏、Sensor Kinesis、Teva、Tesaro。

所设计的方案解决了卵巢癌类器官生物样本库在类器官形成和长期传代潜力方面的先前挑战，并确保从大多数实体瘤沉积物中产生完全可扩展的细胞系。用于类器官生成的肿瘤样本的手术采集过程显着影响产量和扩增潜力。肿瘤组织样本可以在各种手术中获得，包括多脏器手术、诊断性腹腔镜检查或活检。有经验的妇科肿瘤外科医生应优先从腹膜位置获取干净的肿瘤样本。值得注意的是，已经观?...

由于上皮性卵巢癌晚期临床表现异质性和高复发率，其临床管理仍然具有挑战性1。提高我们对卵巢癌发展和生物学行为的理解需要研究方法，以解决疾病过程中患者的特异性变异性、治疗反应以及组织病理学和分子特征2。
生物样本库的特点是系统地收集和长期保存卵巢癌患者的肿瘤样本及其临床信息，为处于不同疾病阶段的大量患者群体提供保存，包括来自初次减瘤手术、新辅助化疗后和复发性疾病的肿瘤样本。它具有推进癌症研究的宝贵潜力，可作为有前途的预后生物标志物和治疗靶点的资源3。然而，传统的生物样本库方法，如福尔马林固定和冷冻，由于活力的丧失和天然三维组织结构的破坏，不适合对原始肿瘤样本进行功能研究4,5。
肿瘤学及其他领域的分子机制研究在很大程度上取决于使用适当的实验模型，这些模型忠实地反映了疾病的生物学特性，并保持了体内观察到的组织的体外特性。基于保留更新潜力的患者来源类器官，在实验室中复制上皮细胞的原始结构和功能，并允许在患者特定环境中进行测试。因此，它们已成为癌症研究和个性化医疗的非常有前途的工具，弥合了临床多样性和实验室研究之间的差距6,7,8,9。基于类器官系的个体药物反应和分子图谱的功能相关性测试的定制治疗策略，可以潜在地直接应用于患者护理10,11。长期培养的可能性，包括患者特异性特征，以及随着时间的推移收集相关的前瞻性临床数据，对于确定与疾病进展和耐药机制有关的新的预后和预测因素具有很大的希望3,9。
尽管如此，建立一个包含来自不同肿瘤样本的类器官的生物库需要严格遵守复杂的方法，并建立易于维护的方案12。流程标准化确保即使在高流动率下，训练有素的员工也能有效地建立和维护生物样本库，同时遵守最高质量标准13.几项研究报告了与原始肿瘤的突变和表型特征相对应的稳定卵巢癌类器官系的成功生成，且效率各不相同。尽管如此，常规生物样本库在实践中仍然具有挑战性，特别是对于细胞系的长期稳定生长，这是大规模扩增或成功基因组编辑的先决条件。
特别是，可扩展性问题在该领域仍然定义模糊，因为显示出缓慢和有限的生长潜力的类器官偶尔被算作已建立的品系。正如 Hoffmann 等人最初证明的那样，该研究的主要发现为这一进一步发展的方案提供了基础，卵巢癌组织的最佳处理需要一种独特的策略来适应异质性14。通过成熟培养物的 DNA 测序和转录组学分析（培养 4-10 个月）证实了通过该方法获得的类器官的表型表征以及与亲本肿瘤组织的密切相似性，证明了模型的稳定性 8、9、12、14。
与调节健康输卵管稳态的旁分泌环境相比，上皮层可能产生高级别浆液性卵巢癌 （HGSOC）、癌症再生潜力和类器官形成能力，对外源性 Wnt 补充的依赖性较小。此外，以类器官培养基中不存在 Noggin 为特征的活性骨形态发生蛋白 （BMP） 信号转导被证明有利于从卵巢癌实体组织沉积物中建立长期培养物14,15。在卵巢癌固体沉积物的系统生物样本库期间，我们已经证实了这些发现并建立了管道，该协议中概述了详细信息，以确保在大多数情况下持续长期扩展。我们发现，在使用原代分离株时，对不同培养基组成和接种方式进行平行测试对于改善长期稳定的类器官系的建立和提高产量至关重要，从而能够稳健地繁殖和扩展到下游实验所需的多孔形式16。
此外，手术过程中收集的样本的纯度和质量对于卵巢癌类器官在基础研究和分子诊断中的转化潜力至关重要。HGSOC 临床表现的复杂性要求外科医生、肿瘤学家和实验室科学家之间的密切合作，以确保正确识别相关材料，保持运输条件恒定，并高效生成类器官系，代表每个患者疾病的最重要特征。考虑到卵巢癌的异质性，该协议提供了一个标准化但适应性强的框架，以捕获卵巢癌类器官的全部潜力16,17。值得注意的是，该协议能够对广泛的卵巢癌临床表现进行可靠的生物样本库，包括不同的组织学类型（高级别和低级别卵巢癌，LGSOC），来自同一患者的不同沉积物，这些患者在干性调节方面表现出差异，来自新辅助治疗后手术的组织，活检材料，以及来自疾病进展复发期的手术样本。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 Sterican 26 G</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>100 Sterican 27 G</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4657705</td>
			<td></td>
		</tr>
		<tr>
			<td>293T HA Rspo1-Fc</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>3710-001-01</td>
			<td>Alternative: R-Spondin1 expressing Cell line, Sigma-Aldrich, SC111</td>
		</tr>
		<tr>
			<td>A-83-01 (TGF-b RI Kinase inhibitor IV)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>616454</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12 Medium&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p53 antibody (DO1)</td>
			<td>Santa Cruz Biotechnology, Texas, USA</td>
			<td>sc-126</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAX8 antibody</td>
			<td>Proteintech, Manchester, UK&#160;</td>
			<td>10336-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle-top vacuum filter 0.2 &#181;m</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>430049</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 175 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>661175</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 25 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>690160</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 75 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 48-well Clear TC-treated&#160;</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo SFM</td>
			<td>PromoCell &#8211; Human Centered Science, Heidelberg, Germany</td>
			<td>C-29912</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>3533-005-02</td>
			<td>Alternative: Matrigel, Growth Factor Reduced Basement membrane matrix&#160; Corning, 356231&#160;</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Mouse IgG</td>
			<td>Jackson Immuno</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO&#160; Citrate Buffer, pH 6.0, 10x Antigen Retriever</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>C9999-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti rabbit Alexa Fluor Plus 555</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>A32794</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG Alexa Fluor Plus 488</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>A32814</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Phosphate-Buffered Saline&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia Richard-Allan Scientific HistoGel</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>Epredia HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Polystyrene&#160;</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>351447</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather scalpel&#160;</td>
			<td>Pfm medical, Cologne, Germany</td>
			<td>200130010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin 37% acid free, stabilized</td>
			<td>Morphisto, Offenbach am Main, Germany</td>
			<td>1019205000</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>156630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EpCAM/TROP-1 Antibody</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>AF960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF10</td>
			<td>Peprotech, NJ, USA</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant BMP2</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>PHC7146</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant EGF</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>PHG0311L</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant Heregulin beta-1</td>
			<td>Peprotech, NJ, USA</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X core Software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>https://webshare.leica-microsystems.com/latest/core/widefield/</td>
		</tr>
		<tr>
			<td>Leica TCS SP8 X&#160;White Light Laser Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix 1 mL</td>
			<td>Braun, Melsungen, Germany</td>
			<td>3570519</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Omnilab, Munich, Germany</td>
			<td>5170002</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyd&#160;</td>
			<td>Morphisto, Offenbach am Main, Germany</td>
			<td>1176201000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>P4333-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PluriStrainer 400 &#181;m</td>
			<td>PluriSelect, Leipzig, Germany</td>
			<td>43-50400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, Toulouse, France</td>
			<td>ant-pm-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>Roticlear</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>A538.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgipath Paraplast</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>39602012</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cryovials</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>375418PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>PanReac AppliChem, Darmstadt, Germany</td>
			<td>A4974-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>TOCRIS biotechne, Wiesbaden, Germany</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Invitrogen, Thermo Scientific, Waltham, USA</td>
			<td>R25001</td>
			<td></td>
		</tr>
	</tbody>
</table>

strategy for biobanking of ovarian cancer organoids: addressing the interpatient heterogeneity across histological subtypes and disease stages

虽然从患者来源的类器官及其临床背景信息中建立卵巢癌生物库有望在研究和患者护理方面取得进展，但由于这种致命恶性肿瘤的异质性，加上类器官技术固有的复杂性，标准化仍然是一个挑战。这种适应性强的方案提供了一个系统框架，考虑到祖细胞的患者特异性变异性，以实现卵巢癌类器官的全部潜力。通过实施结构化的实验工作流程来选择最佳培养条件和接种方法，并同时测试直接 3D 接种与 2D/3D 路线，在大多数情况下，我们获得了适用于各种下游应用的稳健的长期膨胀系。 
值得注意的是，该方案已在大量高度异质的起始材料病例 （N = 120） 中经过测试并证明有效，包括高级别和低级别卵巢癌以及原发性减瘤、复发性疾病和新辅助手术标本的疾病阶段。在低 Wnt、高 BMP 的外源信号转导环境中，我们观察到祖细胞对 Heregulin 1 ß （HERß-1） 通路的激活具有不同的敏感性，其中 HERß-1 促进某些类器官的形成，而抑制另一些类器官的形成。对于患者样本的一部分，最佳的类器官形成和长期生长需要向培养基中添加成纤维细胞生长因子 10 和 R-Spondin 1。 
此外，我们强调了组织消化和祖细胞分离的关键步骤，并指出了在塑料上进行 2D 短暂培养有利于基底膜提取物 2 型基质中后续类器官形成的例子。总体而言，最佳生物样本库需要同时对所有主要条件进行系统测试，以确定单个品系的适当生长环境。该协议还描述了有效包埋、切片和染色以获得类器官的高分辨率图像的处理程序，这是综合表型分析所必需的。

Clinical management of patients with epithelial ovarian cancer remains challenging due to its heterogeneous clinical presentation at advanced stages and high recurrence rates1. Improving our understanding of ovarian cancer development and biological behavior requires research approaches that address the patient-specific variability during the course of the disease, treatment response, and histopathological as well as molecular features2.
Biobanking, characterized by the systematic collection and long-term preservation of tumor samples derived from ovarian cancer patients along with their clinical information offers the preservation of a large patient cohort in different disease stages, including tumor samples from primary debulking surgeries, after neoadjuvant chemotherapy and from recurrent disease. It holds valuable potential for advancing cancer research serving as a resource of promising prognostic biomarkers and therapeutic targets3. However, conventional biobanking methods, such as formalin fixation and freezing, are not amenable to conducting functional studies on the original tumor samples due to the loss of viability and the disruption of the native three-dimensional tissue architecture4,5.
Studies of molecular mechanisms, in oncology and beyond, crucially depend on the use of appropriate experimental models that faithfully reflect the biology of the disease and maintain in vitro properties of the tissue observed in vivo. Patient-derived organoids, based on the preservation of the renewal potential, reproduce in the lab the original structure and function of the epithelium and allow testing in a patient-specific context. Therefore, they have emerged as highly promising tools for cancer research and personalized medicine, bridging the gap between clinical diversity and laboratory research6,7,8,9. Tailored therapeutic strategies based on individual drug responses of organoid lines and testing of the functional relevance of molecular profiles, can potentially be directly applied to patient care10,11. The possibility of long-term cultivation including patient-specific characteristics and the collection of relevant prospective clinical data over time holds great promise to identify novel prognostic and predictive factors involved in disease progression and resistance mechanisms3,9.
Nonetheless, building a biobank that includes organoids from different tumor samples requires a combination of strict adherence to complex methodology and setting up protocols for easy maintenance12. Process standardization ensures that the biobank can be established and maintained efficiently by trained staff even at high turnover, while at the same time adhering to the highest quality standards13. Several studies reported the successful generation of stable ovarian cancer organoid lines corresponding to the mutational and phenotypical profile of the original tumor with varying efficiency rates. Still, routine bio banking remains challenging in practice, particularly for long-term stable growth of lines, which is a prerequisite for large-scale expansion or successful genomic editing.
In particular, the issue of expandability remains vaguely defined in the field as organoids that show slow and limited growth potential are occasionally counted as established lines. As initially demonstrated by Hoffmann et al., a study whose principal findings provided the basis for this further developed protocol, optimal handling of ovarian cancer tissue requires a unique strategy to accommodate heterogeneity14. Phenotypic characterization of the organoids obtained by this method and close similarity with parental tumor tissue were confirmed by panel DNA sequencing and transcriptomics analysis of mature cultures (4-10 months of cultivation) demonstrating the stability of the model8,9,12,14.
In contrast to the paracrine environment that regulates the homeostasis in the healthy fallopian tubes, the epithelial layer, which likely yields high-grade serous ovarian cancer (HGSOC), cancer regeneration potential, and organoid formation capacity, is less dependent on exogenous Wnt supplementation. Moreover, active Bone Morphogenetic Protein (BMP) signaling, characterized by the absence of Noggin in organoid medium, proved to be beneficial for the establishment of long-term cultures from ovarian cancer solid tissue deposits14,15. During systematic biobanking of solid deposits of ovarian cancer, we have confirmed these findings and set up the pipeline, with details outlined in this protocol that ensures sustained long-term expansion in the majority of cases. We find that parallel testing of different media compositions and seeding modalities when working with primary isolates are essential to improve the establishment of long-term stable organoid lines and to increase yields enabling robust propagation and expansion to multi-well formats required for downstream experiments16.
Furthermore, the purity and quality of the samples collected during surgery are of crucial importance for the translational potential of ovarian cancer organoids in basic research and molecular diagnostics. The complexity of the clinical presentation of HGSOC requires close cooperation between the surgeons, oncologists, and the scientists in the lab to ensure that relevant material is correctly identified, transport conditions are kept constant, and organoid lines are generated with high efficiency representing the most important characteristics of the disease of each patient. This protocol provides a standardized but adaptable framework to capture the full potential of ovarian cancer organoids, considering the heterogeneity that characterizes ovarian cancer16,17. Notably, this protocol enables reliable biobanking of the broad spectrum of ovarian cancer clinical presentation, including different histological types (high-grade and low-grade ovarian cancer, LGSOC), different deposits from the same patients who exhibit differences in stemness regulation, tissues from surgeries in post neoadjuvant setting, biopsy material, and samples from surgeries in the recurrent phase of disease progression.

作者聚焦：推进卵巢癌的个性化医疗 

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

The best clinical management in cutting-edge translational research for patients with advanced ovarian cancer has highest priority at our institution. With the biobanking of patient-derived organoids, we have achieved major progress towards individually-tailored management. This enables us to evaluate patient-specific characteristics and to identify predictive biomarkers.We have made significant advances in recent years in improving radical multivisceral surgery, chemotherapy, anti-angiogenic therapy, and also PARP inhibitor therapy in patients with advanced ovarian cancer and improved survival rates even with that. Now, the next big step would be to better predict which patient would benefit from which specific treatment. Currently, clinical treatment decisions are based on general histopathological features and on information on homologous recommendation deficiency or tested in paraffin-embedded tumor tissue.Biologic models that evaluate specific responses to chemotherapy and to targeted treatments have not yet been implemented in clinical routine. We have identified key characteristics of ovarian cancer tissue, specifically the culture requirements necessary to sustain its renewal potential in vitro, and use this knowledge to achieve robust long-term growth of organoid lines and establish biobank from samples at various stages of the disease. Our day-to-day experience in biobanking of ovarian cancer organoids effectively shows a remarkable heterogeneity in the biology of this malignancy, which could pave the way to new classification or provide the basis for new therapeutic solutions targeting STEMI's potential.

在初始组织解离、过滤和计数后，如上所述，直接以 3D 形式平行接种细胞，并在烧瓶中悬浮液进行短暂的 2D 扩增。在某些情况下，瞬时 2D 扩增对类器官形成产生积极影响，并且通过该途径成功建立了长期品系，而相对平行的 3D 接种可导致生长停滞（图 1）。对于处理的每个供体组织，根据培养基基质测试细胞。按照这一策略，我们的生物样本库现在包含代表每种标准生长?...

Watch this Scientific Journal Video about Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer at JoVE.com

该协议为建立来自不同疾病阶段的卵巢癌类器官提供了一个系统框架，并解决了患者特异性变异性的挑战，以提高产量并为后续应用实现稳健的长期扩展。它包括组织处理、接种、调整培养基要求和免疫荧光染色的详细步骤。

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

对于晚期卵巢癌患者的前沿转化研究，最佳临床管理是我们机构的重中之重。通过患者来源类器官的生物样本库，我们在个性化管理方面取得了重大进展。这使我们能够评估患者的特异性特征并识别预测性生物标志物。近年来，我们在改善晚期卵巢癌患者的根治性多脏器手术、化疗、抗血管生成治疗以及 PARP 抑制剂治疗方面取得了重大进展，并提高了生存率。现在，下一个重要步骤是更好地预测哪些患者将从哪种特定治疗中受益。目前，临床治疗决策基于一般组织病理学特征和同源推荐缺陷的信息或在石蜡包埋的肿瘤组织中测试的信息。评估对化疗和靶向治疗的特异性反应的生物模型尚未在临床常规中实施。我们已经确定了卵巢癌组织的关键特征，特别是维持其体外更新潜力所需的培养要求，并利用这些知识来实现类器官系的稳健长期生长，并从疾病各个阶段的样本中建立生物库。我们在卵巢癌类器官生物样本库方面的日常经验有效地表明了这种恶性肿瘤的生物学存在显着的异质性，这可能为新的分类铺平道路，或为针对STEMI潜力的新治疗解决方案提供基础。

strategy-for-biobanking-ovarian-cancer-organoids-addressing

Research

JoVE Journal

Cancer Research

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

1.1K 次观看.  University Hospital, Ludwig-Maximilian-University (LMU) Munich. 对于晚期卵巢癌患者的前沿转化研究，最佳临床管理是我们机构的重中之重。通过患者来源类器官的生物样本库，我们在个性化管理方面取得了重大进展。这使我们能够评估患者的特异性特征并识别预测性生物标志物。近年来，我们在改善晚期卵巢癌患者的根治性多脏器手术、化疗、抗血管生成治疗以及 PARP 抑制剂治疗方面取得了重大进展，并提高了生存率。现在，下?...

视频: 作者聚焦：推进卵巢癌的个性化医疗 

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.