从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代
1Department of Internal Medicine, Division of Hematology and Oncology,University of Texas Southwestern Medical Center, 2Harold C. Simmons Comprehensive Cancer Center,University of Texas Southwestern Medical Center, 3Department of Molecular Biology,University of Texas Southwestern Medical Center, 4Hamon Center for Regenerative Science and Medicine,University of Texas Southwestern Medical Center, 5Department of Cell Biology,University of Texas Southwestern Medical Center, 6Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine,University of Southern California
Summary
该协议讨论了通过差异离心从原发性正常和肿瘤乳腺组织产生上皮类器官的方法。此外，还包括用于三维培养以及嵌入类器官的免疫荧光成像的说明。
Abstract
类器官是模拟器官组织的可靠方法，因为它们具有自组织特性，并且在从原代组织或干细胞繁殖后保留了功能和结构。这种类器官生成方法放弃了通过多次传代的单细胞分化，而是使用差异离心从机械和酶解离组织中分离乳腺上皮类器官。除了在胶原蛋白和基底细胞外基质中嵌入类器官的技术外，该协议提供了一种简化的技术，用于从小鼠和人类乳腺组织快速生产小型和大型上皮类器官。此外，还提供了凝胶内固定和免疫荧光染色的说明，以可视化类器官形态和密度。这些方法适用于无数的下游分析，例如与免疫细胞共培养和通过胶原侵袭测定进行离体转移建模。这些分析有助于更好地阐明细胞间行为，并更全面地了解肿瘤微环境中的相互作用。
Introduction
在体外模拟上皮细胞的能力一直是现代生物医学研究的基础，因为它捕获了体内无法获得的细胞特征。例如，在二维平面上生长上皮细胞系可以评估增殖过程中上皮细胞中发生的分子变化1。此外，测量信号传导和基因表达之间的动态调控在体内系统中受到限制2。在癌症研究中，癌症上皮细胞系建模能够识别疾病进展的分子驱动因素和潜在的药物靶点3。然而，在二维平面上生长癌症上皮细胞系具有局限性，因为大多数是遗传永生化和修饰的，通常是克隆性质的，选择因为它们在非生理条件下生长的能力，在评估三维（3D）肿瘤组织结构方面受到限制，并且不能充分模拟现实组织环境中的微环境相互作用4。这些限制在模拟转移时尤其明显，在体内包括几个不同的生物学阶段，包括远处器官部位的侵袭、播散、循环和定植5。
癌症上皮类器官已被开发出来，以更好地概括肿瘤的3D环境和行为6，7，8。类器官首先由单个LRG5 +肠隐窝细胞开发而成，并分化以代表在体外维持小肠分层结构的隐窝绒毛单位的3D结构9。这种方法允许在稳态和压力条件下实时可视化和表征自组织组织结构。作为一种自然延伸，癌症上皮类器官被开发用于模拟许多不同的癌症类型，包括结直肠癌10，胰腺11，乳腺癌12，肝脏13，肺癌14，脑癌15和胃癌16。癌症上皮类器官已被用于表征癌症进化17，18和转移时空行为19，20，询问肿瘤异质性21，并测试化学疗法22。在正在进行的临床试验中，癌症上皮类器官也被分离和收集，以预测患者对抗癌药物和离体放射治疗的反应8，23，24，25。此外，结合癌症上皮类器官的系统可以与其他非癌细胞（如免疫细胞）相结合，形成更全面的肿瘤微环境模型，以实时可视化相互作用，揭示癌症上皮细胞如何改变细胞毒性效应免疫细胞（如自然杀伤细胞）的基本性质，并测试潜在的免疫疗法和抗体药物依赖性细胞毒性活性26， 27，28.本文展示了一种无需传代和嵌入胶原蛋白和基底细胞外基质 （ECM） 即可生成上皮类器官的方法。此外，还分享了分离类器官的下游成像技术。
Protocol
本手稿中使用的所有小鼠组织均根据德克萨斯大学西南医学中心的机构动物护理和使用委员会 （IACUC） 规定和指南进行道德收集。同样，所有患者在机构审查委员会（IRB）的监督下在组织捐赠之前都同意，并且样本被去识别化。 注意：本协议描述了从原代组织产生类器官。 1. 过夜准备材料 为了包埋在基底细胞外基质（BECM）中，将其…
Representative Results
图1中的图像提供了来自人类和小鼠组织的野生型和肿瘤性乳腺上皮类器官的示例。图1A中的卡通工作流程中提供了通过差速离心分离上皮类器官的方法的概览图示，表明来自不同物种的原代组织可以以几乎相同的方式进行处理，同时产生上皮组织，如明场图像所示（图1B）).此外，这些种间组织组成相似性可以在图…
Discussion
文献中已经描述了生成肿瘤类器官的不同方法。该协议强调了一种直接从肿瘤生成肿瘤类器官而无需传代的方法。使用这种方法，肿瘤类器官可在启动手术后数小时内生产，并产生接近100%的活类器官，而文献中报道的这一比例为70%31%。相比之下，其他方法需要在几周内将细胞连续传代到类器官中。因此，下游应用，例如确定和可视化免疫细胞与来自同一宿主的匹配类器官和免疫…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了METAvivor，Peter Carlson Trust，Theresa研究基金会和NCI / UTSW西蒙斯癌症中心P30 CA142543的资金支持。我们感谢德克萨斯大学西南组织管理共享资源的帮助，这是西蒙斯综合癌症中心的共享资源，部分由国家癌症研究所支持，奖励号为P30 CA142543。特别感谢陈氏实验室的所有成员。
Materials
| 10 mM HEPES Buffer | Gibco | 15630080 | |
| 100x Antibiotic-Antimycotic | Gibco | 15240-096 | |
| 100x Glutamax | Life Technologies | 35050-061 | Glutamine supplement |
| 100x Insulin-Transferrin-Selenium (ITS) | Life Technologies | 51500-056 | |
| 100x Penicillin/Streptomycin (Pen/Strep) | Sigma | P4333 | |
| 10x DMEM | Sigma | D2429 | |
| 50 mL/0.2 µm filter flask | Fisher | #564-0020 | |
| Amphotericin B | Life Technologies | 15290-018 | |
| bFGF | Sigma | F0291 | |
| BSA Solution (32%) | Sigma | #A9576 | |
| Cholera Toxin | Sigma | C8052 | |
| CO2-Independent Medium | Gibco | 18045-088 | |
| Collagenase A | Sigma | C2139 | |
| Deoxyribonuclease I from bovine pancreas (DNase) | Sigma | D4263 | |
| DMEM with 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture | Sigma | D6546 | Common basal medium |
| D-MEM/F12 | Life Technologies | #10565-018 | Basal cell medium |
| Dulbecco's Phosphate Buffered Saline (D-PBS) | Sigma | #D8662 | PBS |
| Fetal bovine serum (FBS) | Sigma | #F0926 | |
| Gentamicin | Life Technologies | #15750-060 | |
| Human epidermal growth factor (EGF) | Sigma | E9644 | |
| Hydrocortisone | Sigma | H0396 | |
| Insulin | Sigma | #I9278 | |
| Matrigel | Corning | #354230 | Basement Extracellular Matrix (BECM) |
| NaOH (1 N) | Sigma | S2770 | |
| Rat Tail Collagen I | Corning | 354236 | |
| RPMI-1640 media | Fisher | SH3002701 | |
| Trypsin | Life Technologies | 27250-018 |
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Citer Cet Article
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