食管腺癌类器官的传代培养和冷冻保存：单细胞消化的利弊
Summary
该协议描述了食管腺癌类器官的传代培养和冷冻保存方法，有和没有单细胞消化，使研究人员能够根据其实验设计选择适当的策略。
Abstract
缺乏反映原发病的合适的转化研究模型来探索肿瘤发生和治疗策略是食管腺癌（EAC）的主要障碍。患者来源的类器官（PDO）最近已成为各种癌症中一种非凡的临床前模型。但是，可用于开发 EAC PDO 的协议仍然有限。一旦建立了PDO，繁殖和低温保存对于进一步的下游分析至关重要。在这里，两种不同的方法已经标准化为EAC PDO传代培养和冷冻保存，即有和没有单细胞消化。这两种方法都可以可靠地获得适当的细胞活力，并适用于不同的实验设置。目前的研究表明，用单细胞消化传代培养EAC PDO适用于大多数需要细胞数量控制，均匀密度和有利于尺寸跟踪的空心结构的实验。然而，基于单细胞的方法在培养物中以及在从冷冻库存重新培养后显示出较慢的生长。此外，单细胞消化的传代培养的特点是形成具有空心核心的中空结构。相反，在不进行单细胞消化的情况下处理EAC PDO有利于冷冻保存，扩增和组织学表征。在该协议中，描述了具有和不具有单细胞消化的EAC PDO的传代培养和冷冻保存的优缺点，以使研究人员能够选择适当的方法来处理和研究其类器官。
Introduction
食管癌 （EC） 是全球第十大常见和第六大癌症死因1.食管腺癌（EAC）是EC的主要组织学亚型之一，主要发生在西方国家2。近十年来，包括德国在内的许多发达国家的 EAC 发病率显著增加3。由于癌症的侵袭性和肿瘤发展早期缺乏症状，EAC患者的整体预后较差，5年生存率约为20%2，4，5。
自二十世纪末以来，已经为EAC的生物医学研究建立了几种模型。20世纪90年代建立的经典人类EAC细胞系6，扩展了我们对EAC肿瘤生物学，肿瘤遗传学以及抗肿瘤策略的知识，并广泛用于EAC研究。此外，一些研究小组通过手术或炎症方法将动物暴露于已知的风险因素（例如胃食管反流）来成功开发了EAC或Barrett食道的动物模型7，8，9。此外，开发了患者源性异种移植（PDX）模型，该模型将EAC原发性癌组织皮下或原位植入免疫缺陷小鼠中，以模拟人EAC肿瘤生物学行为和肿瘤环境10，11，12。然而，尽管这些模型改善了临床应用，并且我们对EAC肿瘤发生和进展背后的分子机制的理解，但将这些研究模型的结果外推到人类仍然存在重大挑战。
患者来源的肿瘤类器官（PDO）在3D培养系统中生长，该系统在体外模仿人类发育和器官再生。PDO由患者的原代组织产生，概括了人类肿瘤的分子和表型特征，并在药物开发和个性化癌症治疗中显示出有希望的应用13，14。通过比较10例EAC PDO与其配对的肿瘤组织，据报道EAC PDO与原发肿瘤具有相似的组织病理学特征和基因组学景观，保留了肿瘤内的异质性并促进了体外有效的药物筛选15。EAC PDO还用于研究EAC肿瘤细胞与患者来源的癌症相关成纤维细胞（CAFs）的相互作用，表明在肿瘤微环境研究领域的强大应用16。不幸的是，可用于开发和传播 EAC PDO 的协议有限。在这里，详细描述了两种不同的传代培养和保存EAC PDO的方法：有和没有单细胞消化。维护EAC PDO及其应用的标准化方法可以支持研究人员在EAC PDO研究中为不同目的选择适当的方法。
Protocol
成熟且生长良好的PDO培养物是该协议中描述的成功传代培养和冷冻保存的基础。在这里，EAC PDO是使用Karakasheva T. A.等人17描述的方案从EAC患者的原发肿瘤组织中产生的。EAC组织是在BioMaSOTA的批准下从生物库中收集的（由科隆大学伦理委员会批准，ID：13-091）。 注意：EAC PDO已在37°C和5%CO2的加湿培养箱中使用PDO培养基进行培养（表1）。在以…
Representative Results
该协议介绍了包括具有和不具有单细胞消化的EAC PDO的传代培养和冷冻保存的程序。 图1 显示了两种不同亚文化策略的代表性相差图片。EAC PDO达到适当的传代培养密度（图1，左）。没有单细胞消化的传代培养需要更少的时间即可达到可比的密度，并且主要导致结构紧凑（图1，顶行）。相比之下，单细胞消化?…
Discussion
在该协议中，描述了EAC PDO的两种不同的传代培养和冷冻保存方法，即具有和不具有单细胞消化。几项研究建议用单细胞消化15，17传代EAC PDO，这对于大多数需要细胞数量控制，均匀密度和促进大小跟踪的中空结构的实验是有益的。然而，基于单细胞的方法的特征在于从冷冻储备中重新培养后生长较慢，并且在培养期间形态不太紧凑。经验表明，基于单?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了科隆大学科隆财富计划/医学院的支持。我们感谢Susanne Neiss，Michaela Heitmann和Anke Wienand-Dorweiler的技术援助。宁波范由广州市精英奖学金委员会（GESC）资助。作者感谢Joshua D’Rozario博士在语言编辑方面的帮助。
Materials
| Equipment | |||
| -20°C Freezer | Bosch | Economic | |
| -80°C Freezer | Panasonic | MDF DU500VH-PE | |
| Automated Cell counter | Thermo Fisher | AMQAX1000 | Countess II |
| Biological Safety Cabinet Class II | Thermo Scientific | 51022482 | Herasafe KS12 |
| Centrifuge | Heraeus | 75003060 | Megafuge 1.0R |
| CO2 Incubator | Thermo Scientific | 50116048 | Heracell 150i |
| Inverted automated fluorescence microscope | Olympus | IX83 | |
| Inverted light microscope | Leica | DMIL LED Fluo | |
| Pipette 1000 µL | Eppendorf | 3123000063 | Research Plus |
| Pipette 200 µL | Eppendorf | 3123000039 | Research Plus |
| Rotating Incubator | Scientific Industries, sc. | SI-1200 | Enviro-genie |
| Shaker | Eppendorf | 5355 000.011 | Thermomixer Comfort |
| Vacuum pump | Vacuubrand | 20727200 | BVC control |
| Waterbath | Medingen | p2725 | W22 |
| Material | |||
| 15 mL tube | Sarstedt | 62.554.502 | Inc Screw cap tube PP 15 mL |
| Cryo vial 2 mL | Sarstedt | 72.379 | CryoPure 2.0 mL tube |
| Low bind tube 1.5 mL | Sarstedt | 72.706.600 | Micro tube 1.5 mL protein LB |
| Low bind tube 5 mL | Eppendorf | 0030 108.302 | Protein LoBind Tube 5.0 mL |
| Pipette tip 200 µL | Starlab | E1011-8000 | 200 µL Graduated tip, wide orifice |
| Pipette tip 1000 µL | Starlab | E1011-9000 | 1000 µL Graduated tip, wide orifice |
| Pipette tip 1000 µL | Sarstedt | 70.3050 | Pipette tip 1000 µL |
| Sterile filter 0.2 µm | Sarstedt | 83.1826.001 | Filtropur 0.2 µm sterile filter |
| Tissue culture plate | Sarstedt | 83.3921 | 12 well-plate |
| Reagent/Chemical | |||
| A83-01 | Tocris | 2939 | |
| Advanced DMEM/F-12 | Thermo Fisher Scientific | 12634010 | |
| Amphotericin B | Thermo Fisher Scientific | 15290026 | |
| B-27 | Thermo Fisher Scientific | 17504001 | |
| Cell Recovery Solution | Corning | 354253 | |
| CHIR-99021 | MedChemExpress | HY-10182/CS-0181 | |
| DNase I grade II, from bovine pancreas | Sigma-Aldrich | 10104159001 | |
| Dulbecco's phosphate-buffered saline (DPBS) | Thermo Fisher Scientific | 14190094 | |
| Extracellular matrix (ECM) gel: Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 356231 | |
| FGF-10a | Peprotech | 100-26-100 | |
| Freezing medium: Recovery Cell Freezing Medium | Thermo Fisher Scientific | 12648010 | |
| Gastrin | Sigma | G9020 | |
| Gentamicin-25 (25 mg/ 500 µL) | PromoCell | C-36030 | |
| HEPES (1 M) | Thermo Fisher Scientific | 15630080 | |
| L-Glutamine 200 mM (100X) | Thermo Fisher Scientific | 25030024 | |
| N-2 | Thermo Fisher Scientific | 17502-048 | |
| N-Acetylcysteine | Sigma | A9165 | |
| Nicotinamide | Sigma | N0636-100 | |
| Noggin | Peprotech | 120-10C-50 | |
| Penicillin-Streptomycin 10,000 U/ mL (100X) | Thermo Fisher Scientific | 15140122 | |
| Recombinant human epidermal growth factor (EGF) | Peprotech | AF-100-15 | |
| R-Spondin1 conditioned medium from Cultrex R-Spondin Cells | Biotechne | 3710-001-01 | |
| SB202190 | MedChemExpress | 152121-30-7 | |
| Trypsin inhibitor from Glycine max (soybean) | Sigma-Aldrich | 93620-1G | |
| Trypsin-EDTA (0.25 %), phenol red | Thermo Fisher Scientific | 25200056 | |
| Wnt-3A conditioned medium | Wnt-3A expressing cell line was kindly provided by Prof. Hans Clevers' group | ||
| Y-27632 | Sigma | Y0503 |
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Cite This Article
Fan, N., Raatz, L., Chon, S., Quaas, A., Bruns, C., Zhao, Y. Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion. J. Vis. Exp. (185), e63281, doi:10.3791/63281 (2022).