CN106574242B - 源自单细胞的类器官 - Google Patents
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Abstract
本发明涉及源自单细胞例如前列腺癌细胞的类器官，以及与其生产和使用相关的方法和组合物，包括用于生产类器官的细胞培养基和用于前列腺癌的个性化治疗方法。本发明还提供了包含源自患者的前列腺细胞的前列腺类器官的人源化小鼠。
Description
相关申请的交叉引用
本申请要求于2014年6月20日提交的美国临时申请号62/015,020的权益，其公开内容通过引用整体并入本文。
发明背景
不能增殖初生组织成为理解成年生物体中的再生机制和分化细胞与干细胞的平衡的主要障碍。对于更好地理解原发性人类病理学病症例如肿瘤的发展存在需求。对于癌症研究，目前的癌症模型不能充分代表人类癌症的分子和细胞的多样性。现有的人癌细胞系缺少关于癌症的临床表现的明确和详细的信息，并且对于治疗抗性机制的解释具有固有局限性。因此，需要用于维持癌症初生组织的新方法和用于治疗癌症的新型药物开发方法。
维持正常分化细胞和祖细胞或干细胞之间的平衡是复杂的。成体干细胞通过响应调节增殖、分化和凋亡之间的平衡的线索来提供不同组织、器官的再生或赘生物生长。独立于遗传指令但可在每个细胞分裂可遗传的外遗传改变可能是疾病引发或进展的驱动力。组织干细胞在增殖、自我更新和分化方面的能力是多样的，并且它们可以在应激条件下在不同亚型之间可逆地转换。组织干细胞容纳具有多谱系分化倾向的多种亚型。例如，造血干细胞(HSC)可以可逆地获得三种增殖状态：细胞处于细胞周期的静止期的休眠状态、细胞偶尔循环以维持组织分化的内稳态、以及细胞连续循环的活化状态。许多成体干细胞池(cellpool)的生长和再生受到对来自通过生态位相互作用分泌的生长因子和细胞因子的调节信号和基质反馈信号的这些遗传和/或外遗传响应的严格控制。
前列腺癌是最常见的恶性肿瘤，并且是西方男性癌症死亡的第二大常见病因。虽然其流行，但是已证明前列腺癌非常难以在体外繁殖。前列腺癌代表复杂的器官样多细胞结构，其存活和功能依赖于前列腺癌细胞与薄壁组织的成纤维细胞、内皮细胞、免疫细胞和细胞外基质(ECM)的组分的动态相互作用。概括前列腺癌的ECM和细胞多样性的前列腺癌模型的缺乏阻碍了对理解疾病进展和缺乏治疗响应的发展。例如，生长在单层中的细胞系和遗传工程小鼠模型不能模拟前列腺癌微环境的复杂性或再现治疗抗性的多种机制。
发明概述
在一个实施方案中，本发明提供了一种在体外从哺乳动物组织制备类器官的方法，该方法包括：从哺乳动物组织中分离细胞以提供分离的细胞；在分化培养基中培养分离的细胞，持续足以富集干细胞并诱导分化的时间；并通过在类器官培养基中的细胞外基质中培养足以产生类器官的时间来扩增细胞。
在另一个实施方案中，本发明提供了包含上皮细胞和间充质细胞的体外前列腺类器官。
在另一个实施方案中，本发明提供了源自原发性前列腺癌组织的体外前列腺类器官，其中类器官包含上皮细胞和间充质细胞并产生前列腺特异性抗原(PSA)。
在另一个实施方案中，本发明提供了补充有牛垂体提取物(BPE)和肾上腺素的细胞培养基。
在另一个实施方案中，本发明提供了补充有碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和氢化可的松的细胞培养基。
在另一个实施方案中，本发明提供了试剂盒，其包含补充有BPE和肾上腺素的细胞培养基和补充有bFGF、EGF和氢化可的松的细胞培养基。
在另一个实施方案中，本发明提供了一种用于鉴定对前列腺癌细胞具有抗癌活性的药剂的方法，该方法包括选择至少一种测试药剂，使源自患者的前列腺癌细胞的多种患者特异性前列腺类器官与测试药剂接触，测定在存在测试药剂和不存在测试药剂的情况下前列腺类器官的数量，并且如果类器官的数量在存在药剂的情况下少于不存在药剂的情况，则鉴定出具有抗癌活性的药剂。在另一个实施方案中，该方法提供用被鉴定为对患者特异性类器官具有抗癌活性但对于正常类器官不具有抗癌活性的药剂治疗患者的步骤。
在另一个实施方案中，本发明提供了植入患者特异性前列腺类器官的免疫人源化小鼠，以及使用此类小鼠鉴定前列腺癌的个性化治疗的方法。
发明详述
在某些实施方案中，本发明提供源自体外正常和癌性组织的前列腺类器官，制备和使用这样的类器官的方法，以及细胞培养基和试剂盒。如本文的一个实施方案中所公开的，在三维培养装置中在包含细胞外基质分子的体外环境中的某些生长因子可以用于制备类器官。
类器官是组织的微型形式，其在体外产生并显示内源性三维器官结构。参见例如Cantrell and Kuo(2015)Genome Medicine 7:32-34。本发明的类器官可用于例如：a)在临床前和临床试验中确定肿瘤内的基因组靶点和预测治疗响应；b)通过检查治疗后存活的类器官数量来检测抗癌剂的活性；c)通过测定每种类器官中增殖细胞的数量和测定相关途径的基因表达谱图来检测增殖剂的活性；d)检查药剂靶向类器官内的不同细胞类型的特异性；e)确定化疗和放射的效果；f)通过体内植入类器官创建小鼠模型；g)创建用于在体外和体内检查治疗响应和药物发现的临床前模型；和h)确定克隆靶向抗癌疗法。
因此，在一个实施方案中，本发明提供了在体外从哺乳动物组织制备类器官的方法，该方法包括：从哺乳动物组织分离细胞以提供分离的细胞；在分化培养基中培养分离的细胞，持续足以富集干细胞并诱导分化的时间；并通过在类器官培养基中的细胞外基质中培养足以产生类器官的时间来扩增细胞。本领域普通技术人员可以通过检查与分化相关的形态学变化来确定足以诱导分化的时间。在一个优选的实施方案中，足以诱导分化的时间为约5至约15天。在另一个优选的实施方案中，足以诱导分化的时间为约14天。在一个优选的实施方案中，足以产生类器物的时间为约5至约15天。在另一个优选的实施方案中，足以产生类器官的时间为约14天。
在一个优选的实施方案中，分化培养基包括牛垂体提取物(BPE)和肾上腺素。存在于分化培养基中的BPE的浓度可以为约0.1-100mg/L。存在于分化培养基中的肾上腺素的浓度可以为约0.1-100μM。在另一个实施方案中，分化培养基包含以下一种或多种：胰岛素(1-10mg/L)、氢化可的松(0.1-10μM)、庆大霉素(5-50μg/L)、两性霉素(1-30μg/L)、转铁蛋白(0.5-25mg/L)、三碘甲状腺原氨酸(0.1-10μM)、表皮生长因子(EGF)(5-50ng/mL)和视黄酸(0.1-10μM)。在最优选的实施方案中，分化培养基包含以下浓度：前列腺上皮细胞生长培养基(PrEBMTM，Lonza)(约1X)、牛垂体提取物(约52mg/L)、胰岛素(约5mg/L)、氢化可的松(约1μM)、庆大霉素(约30μg/L)、两性霉素(约15μg/L)、转铁蛋白(约5mg/L)、三碘甲状腺原氨酸(约0.1-10μM)、肾上腺素(约1X)、rhEGF(约20ng/mL)、视黄酸(0.1-10μM)。分化培养基可以进一步包括以下物质或用以下物质代替：本领域已知的具有类似性质的合成或天然的其他补充物、生长因子、抗生素、维生素代谢物和激素。在优选的实施方案中，分化培养基是补充有上述组分的市售的细胞培养物，例如达尔伯克氏改良伊格尔培养基(Dulbecco'sModified Eagle Mediun，DMEM；Life Technologies)、高等DMEM(advanced-DMEM，LifeTechnologies)或无角化细胞血清培养基(KSFM)(Life Technologies)。在最优选的实施方案中，分化培养基是补充有上述组分的市售的前列腺上皮细胞生长培养基，例如PrEBMTM(Lonza)。
在优选的实施方案中，类器官培养基包括碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和氢化可的松。存在于类器官培养基中的bFGF的浓度可以为约0.1-10μM，存在于类器官培养基中的氢化可的松的浓度可以为约1-50ng/L。在另一个实施方案中，类器官培养基可以包括以下的一种或多种：BPE(10-100mg/L)、胰岛素(0.5-25mg/L)、氢化可的松(0.1-5μM)、庆大霉素(5-50μg/L)、两性霉素(1-30μg/L)、转铁蛋白(1-25mg/L)、三碘甲状腺原氨酸(0.1-10μM)、肾上腺素(0.1-5X)、EGF(1-50ng/L)和bFGF(1-50ng/mL)。在最优选的实施方案中，类器官培养基包含以下浓度：PrEBM培养基(约1X)、牛垂体提取物(约52mg/L)、胰岛素(约5mg/L)、氢化可的松(约1μM)、庆大霉素(约30μg/L)、两性霉素(约15μg/L)、转铁蛋白(约5mg/L)、三碘甲状腺原氨酸(0.1-10μM)、肾上腺素(约1X)、rhEGF(约20ng/mL)和bFGF(约20ng/mL)。每隔一天加入以下另外3种组分(例如在14天的培养期间加入7次)：氢化可的松(约1μM)、rhEGF(约20ng/mL)和bFGF(约20ng/mL)。类器官培养基可以进一步包括以下物质或用以下物质代替：本领域已知的具有类似性质的合成或天然的其他补充物、生长因子、抗生素、维生素代谢物和激素。在优选的实施方案中，类器官培养基是补充有上述组分的市售的前列腺上皮细胞生长培养基，例如PrEBMTM(Lonza)。
在某些实施方案中，细胞来自哺乳动物癌组织、人组织、人癌组织、人前列腺组织和人原发性前列腺癌组织。在某些实施方案中，可用于制备类器官的细胞是人前列腺干细胞。这样的细胞是本领域已知的，并且可以使用标记物例如前列腺整合素α2β1、CD44和CD133(Prominin-1)10和CK6a(细胞角蛋白6a)来鉴定和分离。
在一个实施方案中，细胞对选自由CD29、CD49b、CD49f、CD44、Erg和AMACR组成的组中的至少一种标记物是阳性的。在另一个实施方案中，细胞对CD29、CD49b和CD44是阳性的。在另一个实施方案中，细胞对CD29、CD49b、CD49f、CD44、Erg和AMACR是阳性的。在另一个实施方案中，细胞对Erg和AMACR是阳性的。这样的细胞可以通过本领域已知的细胞分选方法来鉴定和分离。例如，在一个实施方案中，可以使用SmartFlareTM探针方案(EMO Millipore)对细胞进行分选。
在一个优选的实施方案中，通过使手术切除组织经受机械解离、胶原酶处理和过滤而从该组织获得细胞。
通过执行所述方法并诱导分化以富集干细胞，然后使该过程逆转以通过调节干细胞维持的转录因子的响应来诱导干细胞途径的外遗传活化，源自干细胞的类器官可以在培养条件中保持延长的时间段。在某些实施方案中，分化步骤包括培养细胞约14天。在优选的实施方案中，分化培养基在七天后进行更换。在该方法的优选实施方案中，如本领域已知的，在缺氧条件下进行扩增步骤。在一些实施方案中，扩增步骤包括培养分离的细胞约14天。在某些实施方案中，每隔一天更换类器官培养基。
在某些实施方案中，前列腺癌细胞与基质细胞进行共培养。
在某些实施方案中，前列腺癌细胞与内皮细胞进行共培养。
在某些实施方案中，该方法用市售的细胞外基质如MatrigelTM进行。其它用于培养细胞的细胞外基质是本领域中已知的。通常，细胞外基质包含层粘连蛋白、巢蛋白和胶原。在优选的实施方案中，使用模拟用于培养细胞的体内环境的三维培养装置(室)进行该方法，其中优选细胞外基质在能够在缺氧条件下诱导干细胞增殖的板内形成。这样的三维装置在本领域中是已知的。这样的装置的一个实例由以下公开：Bansal,N.,et al.(2014)Prostate 74,187-200，其公开内容通过引用整体并入本文。根据本发明已经发现，在制造类器官的方法中使用三维培养装置具有令人惊讶的超过其他形式的优点，如表1所示。
表1.测试形式的优点和缺点
| 形式 | 类器官的一致性 | 重现性 | 效率 |
| Matrigel<sup>TM</sup>室中 | +++ | +++ | ++++ |
| Matrigel<sup>TM</sup>上 | + | --‐ | ++ |
| 悬挂滴板 | --‐ | --‐ | --‐ |
| 非粘附板 | + | --‐ | + |
在另一方面，本发明提供了前列腺类器官。前列腺是激素调节的腺体器官，由于雄激素对基质细胞和上皮细胞的作用，其在性成熟时加速生长。人前列腺是导管腺泡腺体，其被分成三个解剖区域：外周区域、过渡区域和中心区域，其被密集和连续的纤维肌肉基质包围。人前列腺基底细胞构成连续层并在彼此之间形成紧密连接。在组织学水平，人前列腺主要包含称为上皮细胞和基质细胞的两种类型的细胞。本发明的前列腺类器官类似于初生组织的结构。通过组织学和免疫荧光分析，本领域技术人员可以确定类器官由上皮细胞内层形成，其可表达上皮标志物E-钙粘蛋白和高分子量细胞角蛋白并可分泌PSA。类器官的外层由间充质系细胞制成，其可以表达波形蛋白，并且被厚的基底膜包围，这是前列腺组织的特征。
在另一方面，本发明提供体外源自原发性前列腺癌组织的前列腺类器官。最常见的前列腺癌是腺泡性腺癌(acinar adenocarcinoma)。通过绘制来自每个患者活检样品的诊断显性克隆和肿瘤亚克隆，产生来自每个克隆的类器官并限定每个克隆的遗传特征(genetic signature)，可以使用所述制备类器官的方法来有效地对肿瘤异质性建模。源自原发性前列腺癌组织的前列腺类器官一般将具有分泌功能并产生前列腺特异性抗原(PSA)；表达类似于前列腺的主要细胞群体(即基底细胞、腔细胞和基质细胞)的特异性表型标记物，可连续传代超过六个月并保持原始初生组织的遗传图谱和特征性融合序列。
在另一方面，本发明提供了体外从肿瘤的手术切除组织产生的前列腺类器官，其被鉴定为表达组织病理学组织特异性和肿瘤发生标记物。可以用非接触激光捕获显微切割或通过RNA分选，例如使用SmartFlareTM探针来分离来自这些组织的单个细胞，以产生具有已知表达特征的单细胞类器官。
在另一个实施方案中，本发明提供了一种鉴定对前列腺癌细胞具有抗癌活性的药剂的方法，该方法包括选择至少一种测试药剂，使源自患者的前列腺癌细胞的多种患者特异性前列腺类器官与测试药剂接触，测定在存在测试药剂和不存在测试药剂的情况下前列腺类器官的数量，并且如果类器官的数量在存在药剂的情况下少于不存在药剂的情况，则鉴定具有抗癌活性的药剂。在另一个实施方案中，该方法提供用被鉴定为对患者特异性类器官具有抗癌活性的药剂来治疗患者的步骤。
在另一个实施方案中，本发明提供了一种选择个性化治疗受试者中的前列腺癌的方法，该方法包括：选择至少一种形式的治疗，使多个前列腺类器官与该形式的治疗接触，其中类器官源自受试者的前列腺癌细胞，测定在存在治疗和不存在治疗的情况下的前列腺类器官的数量，并且如果在存在治疗的情况下前列腺类器官的数量少于不存在治疗的情况，则选择治疗。然后可以使用类器官来检查各种类型的治疗，以在开始之前确定治疗抗性，基于致癌驱动剂在类器官中的表达为每个个体患者定制治疗，以及进一步研究诱导的克隆选择过程，其是常见的复发原因。治疗的各种形式、组合和类型是本领域已知的，例如放射疗法、激素疗法、化疗、生物学疗法和双膦酸盐疗法。术语“受试者”是指任何动物(例如哺乳动物)，包括但不限于人、非人类灵长类动物、啮齿动物等，其将是特定治疗的受体。通常，术语“受试者”和“患者”在本文中在提及人受试者时可互换使用。诸如“治疗(treating)”或“治疗(treatment)”或“治疗(to treat)”或“缓解(alleviating)”或“缓解(alleviate)”的术语是指治愈、减缓、减轻诊断的病理状况的症状和/或停止其进展的治疗措施。
可以通过比较源自相同患者的癌细胞和正常细胞的类器官中的治疗效果来促进前述方法。例如，可以评估源自相同患者的细胞的正常类器官和癌症类器官来确定癌症特异性的遗传和外遗传突变以及基因表达谱，从而能够确定基因-药物缔合和优化治疗。这种比较还使得人们能够预测治疗响应并个性化在特定患者中的治疗。
在该方法的另一方面，克隆靶向治疗可以通过测试治疗剂对源自从患者的肿瘤组织中鉴定的前列腺癌细胞的多个不同克隆的多个类器官的效果并与治疗剂对源自相同患者的正常细胞的类器官的效果的比较来确定。
在另一方面，本发明提供了补充有BPE和肾上腺素的细胞培养基。在另一个实施方案中，本发明提供了补充有bFGF、EGF和氢化可的松的细胞培养基。在优选的实施方案中，培养基是市售的前列腺上皮细胞生长培养基，例如PrEBMTM(Lonza)。
在另一方面，本发明提供了从单细胞制备类器官的试剂盒。在一个实施方案中，试剂盒含有用于如前所述的分化培养基和类器官培养基的容器。容器还可含有用于分化培养基和类器官培养基的必需补充物(生长因子、抗生素、激素、维生素、氨基酸及其组合)。试剂盒可以进一步包含用于三维培养装置的必需组件，例如板，和/或用于细胞外基质的材料，例如MatrigelTM。试剂盒可以进一步包含一组说明书，以执行如前所述的从单个细胞制备类器官的方法。
在另一个实施方案中，本发明提供植入了患者特异性前列腺类器官的小鼠。在一个实施方案中，小鼠是人源化小鼠。在另一个实施方案中，小鼠是人免疫系统(HIS)-重构小鼠。在另一个实施方案中，小鼠是非肥胖糖尿病(NOD)-Rag(-)-γ链(-)(NRG)小鼠。
制备HIS-重构小鼠的方法是本领域已知的，并且例如公开在以下：Drake et al.(2012)Cell Mol Immunol 9:215-24和Harris et al.(2013)Clinical and Experimental Immunology 174:402-413。根据本发明的一个方面，将来自患者例如来自诊断性骨髓样品或HLA匹配的人干细胞移植到新生NRG小鼠中，以移植患者免疫系统的组成。随后使小鼠经受以下处理：用源自相同患者的前列腺细胞的前列腺类器官原位移植至前列腺中。小鼠可用于鉴定新的治疗，评估治疗响应和评价联合治疗。
以下非限制性实施例用于进一步说明本发明。
实施例1
由单一原发性前列腺癌细胞制备包含上皮和间充质组织的类器官。使用具有缺氧条件的MatrigelTM室以及在第一步骤中的1-2周使用促进自我更新的生长因子和同时诱导分化的因子，以富集干细胞群体并诱导分化。生长因子的浓度和加入生长因子的时间点决定了细胞对于自我更新或细胞分化程序的定型。第二步骤涉及促进自我更新的其他生长因子。在第二步骤培养的14天内，源自单细胞的类器官结构由多于14个患者源的样品形成。
具体地，使手术切除组织经受机械解离、胶原酶处理、过滤、然后在分化培养基中繁殖1-2周。在“分化期”之后，收集细胞、洗涤、并单独放入MatrigelTM室中；根据细胞密度确定腔室的尺寸。每隔一天通过从室中吸出培养基的上部而不干扰细胞结构来替换具有生长因子和细胞因子的类器官培养基。在2周内，形成源自原发性前列腺组织、正常组织和癌组织的类器官。这些类器官包含多种细胞类型，包括上皮和间充质组织。
所用的分化培养基包括以下市售组分和最终浓度：PrEBM培养基(1X)、牛垂体提取物(52mg/L)、胰岛素(5mg/L)、氢化可的松(1μM)、庆大霉素(30μg/L)、两性霉素(15μg/L)、转铁蛋白(5mg/L)、三碘甲状腺原氨酸(1X)、肾上腺素(1X)、rhEGF(20ng/mL)、视黄酸(0.1-10μM)。每两周更换具有这些组分的分化培养基。
在下一步骤/第二步骤中，将细胞在具有以下组分和浓度的类器官培养基中培养14天：PrEBM培养基(1X)、牛垂体提取物(52mg/L)、胰岛素(5mg/L)、氢化可的松(1μM)、庆大霉素(30μg/L)、两性霉素(15μg/L)、转铁蛋白(5mg/L)、三碘甲状腺原氨酸(1X)、肾上腺素(1X)、rhEGF(20ng/mL)和bFGF(20ng/mL)。每隔一天(在14天培养期间内7次)添加以下其他3种组分：氢化可的松(1μM)、rhEGF(20ng/mL)和bFGF(20ng/mL)。每隔一天更换具有这些组分的类器官培养基，基于这些生长因子的活性的相应寿命，类器官反映出具有更高的生长因子浓度。
实施例2
根据机构审查委员会(Institutional Review Board)批准的方法从前列腺切除术样本中分离前列腺癌组织。在手术后15分钟内获得反鉴定的组织(deidentifiedtissue)，使其解离并使用上述实施例1所述的条件在模拟正常前列腺的基底层的MatrigelTM的3D液滴室中使用该组织来制备类器官。这个过程产生了多种前列腺器官，其存活了几个月，并且可以再次驱动以再生一系列的类器官。使用上文实施例1中所述的条件，从24个尝试的前列腺癌样品中的21个中获取了类器官，效率为约87％。组织来自高风险前列腺癌，其利用前列腺切除术组织或淋巴结活检和正常邻近组织(NAT)细胞。
为了证明类器官从单个干细胞克隆获得而不是细胞聚集的结果，将患者源的前列腺癌细胞慢病毒工程化，以表达增强的绿色荧光蛋白(EGFP)并在3D板中分选为单细胞。4天后形成单细胞源的类器官，并在14天后获得良好形成的自组织化类器官。类器官形成能力的百分比在样品中变化2-3倍，并且在肿瘤中高于来自相同患者的NAT的百分比。
前列腺癌类器官形成圆形的明确的多细胞结构，其由厚的基底膜包围，如在成体前列腺中一样。与来自VCaP细胞系的细胞球体相反，源自单个初生细胞的类器官显示出其相应的初生组织的保留特征(例如内部上皮细胞中的E-Cad表达，外部间质细胞中的VIMENTIN)以及具有周围膜，如在镜像活检中一样。开发灵敏的Q-PCR分析以检测来自单一类器官的雄激素受体(AR)信号。AR表达和AR变体/靶体通过培养条件在24小时内进行调节。
其他人开发转移性前列腺癌组织的尝试仅低效率地生产由上皮细胞组成的类器官，但不是原发性前列腺癌类器官。Karthaus et al.(2014)Cell 159:163-75；Gao et al.(2014)Cell 159:176-87。
相比之下，前述实施例证实了单个干细胞源的良性前列腺增生和天然前列腺癌生成为由上皮组织和间充质组织组成的3D类器官。
实施例3
进行以下研究以确定与单个肿瘤细胞3D培养物相比，基质细胞共培养物是否能增强生长为3D类器官的前列腺癌细胞的细胞生长、迁移和侵入。上皮细胞和基质细胞用表达荧光素酶和荧光报道分子的不同慢病毒进行标记。研究了EGFP标记的前列腺癌细胞与用mCherry标记的正常骨髓源的基质细胞的单层共培养对肿瘤细胞的形态学、迁移和增殖的影响。同时，研究源自肿瘤起始细胞的标记的前列腺癌类器官与用mCherry标记的骨髓源的基质细胞的3D共培养对用于单层培养研究的相同参数的效果。细胞活力分析表明，与单培养对照相比，3D共培养将增殖和细胞存活率增加了约3倍，表明3D细胞对环境信号更敏感。
实施例4
使用用GFP-Luc慢病毒感染的细胞，将来自前述实施例的类器官植入小鼠前列腺并通过在活体小鼠中注射荧光素(150mg/kg)，使用
(Perkin Elmer)体内成像系统的生物发光成像(BLI)进行监测。
前述实施例和优选实施方案的描述应被视为示例性的，而不是用来限制本发明，本发明由权利要求所限定。如容易理解的，可以使用上述特征的许多变化和组合而不脱离如权利要求所阐述的本发明。不认为这样的变化偏离本发明的范围，并且所附权利要求的范围旨在包括所有这样的变化。本文引用的所有参考文献通过引用整体并入本文。
Claims (15)
1.一种从患有前列腺癌的人的原发性前列腺癌组织制备类器官的体外方法，所述方法包括：从人原发性前列腺癌组织中分离细胞以提供分离的细胞；在分化培养基中培养分离的细胞，持续足以富集干细胞并诱导分化的时间；并通过使用类器官培养基在三维培养装置中的细胞外基质中培养足以产生类器官的时间来扩增细胞，所述类器官培养基包含1-50ng/L的浓度的碱性成纤维细胞生长因子(bFGF)、1-50ng/L的浓度的表皮生长因子(EGF)和0.1-10μM的浓度的氢化可的松，所述类器官显示出内源性三维器官结构，其中分化培养基包含0.1-10μM的浓度的视黄酸(RA)、0.1-100mg/L的浓度的牛垂体提取物(BPE)和0.1-100μM的浓度的肾上腺素。
2.如权利要求1所述的方法，其中所述BPE以1-100mg/L的浓度存在。
3.如权利要求1所述的方法，其中所述分化培养基包含胰岛素、氢化可的松、庆大霉素、两性霉素、转铁蛋白、三碘甲状腺原氨酸和表皮生长因子(EGF)中的一种或多种。
4.如权利要求1所述的方法，其中所述类器官培养基包括BPE、胰岛素、氢化可的松、庆大霉素、两性霉素、转铁蛋白、三碘甲状腺原氨酸、肾上腺素、EGF和bFGF中的一种或多种。
5.如权利要求1所述的方法，其中所述组织是肿瘤组织。
6.如权利要求1所述的方法，其中所述类器官包含具有上皮和间充质表型标记物的细胞。
7.如权利要求1所述的方法，其中扩增细胞的步骤在缺氧条件下进行。
8.如权利要求1所述的方法，其中所述足以诱导分化的时间为约14天。
9.如权利要求8所述的方法，其中所述分化培养基在七天后进行更换。
10.如权利要求1所述的方法，其中所述足以产生类器官的时间为约14天。
11.如权利要求1所述的方法，其中所述类器官培养基每隔一天更换。
12.如权利要求1所述的方法，其中分选存在选自由CD29、CD49b、CD49f、CD44、Erg和AMACR组成的组中的至少一种标记物的分离的细胞。
13.一种前列腺类器官，其由前述权利要求中任一项所述的方法获得。
14.一种用于鉴定对前列腺癌细胞具有抗癌活性的药剂的方法，该方法包括选择至少一种测试药剂，使源自人前列腺癌细胞的多个人前列腺类器官与测试药剂接触，测定在存在所述测试药剂和不存在所述测试药剂的情况下前列腺类器官的数量，并且如果源自人前列腺癌细胞的类器官的数量在存在所述药剂的情况下少于不存在所述药剂的情况，则鉴定为具有抗癌活性的药剂，所述多个人前列腺类器官根据权利要求1所述的方法制备。
15.权利要求13所述的前列腺类器官在制备用于鉴定用于个性化治疗受试者的前列腺癌的抗癌活性药剂的制品中的用途。
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| CN116790476A (zh) | 2016-05-05 | 2023-09-22 | 儿童医院医疗中心 | 用于体外制造胃底组织的方法和与其相关的组合物 |
| CN111349604B (zh) | 2016-05-18 | 2023-12-12 | 学校法人庆应义塾 | 类器官培养用细胞培养基、培养方法及类器官 |
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| CN110373388A (zh) * | 2019-04-24 | 2019-10-25 | 博奥生物集团有限公司 | 一种用于类器官培养的培养基及类器官培养方法 |
| CN111004771B (zh) * | 2019-09-25 | 2022-04-29 | 中国人民解放军军事科学院军事医学研究院 | 建立体外模拟肝病模型的方法及其专用三维培养基 |
| KR102292457B1 (ko) * | 2019-10-11 | 2021-08-23 | 오가노이드사이언스 주식회사 | 편도 오가노이드의 제조방법 및 이의 용도 |
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