小鼠肝细胞类器官的建立与遗传操作
1Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, School of Basic Medical Sciences, Stem Cell and Regenerative Medicine Research Center, Cheeloo College of Medicine,Shandong University, 2The Research Center of Stem Cell and Regenerative Medicine,Shandong University Cheeloo Medical College, School of Basic Medical Sciences, Shandong University
Summary
从小鼠肝细胞建立了长期 的体外 3D类器官培养系统。这些类器官可以通过shRNA/异位构建的慢病毒感染，siRNA转染和CRISPR-Cas9工程进行遗传和遗传操纵。
Abstract
肝脏是哺乳动物最大的器官。它在葡萄糖储存，蛋白质分泌，代谢和解毒中起重要作用。作为大多数肝功能的执行者，原代肝细胞的增殖能力有限。这就需要建立 离体 肝细胞扩增模型，用于肝脏生理和病理研究。在这里，我们通过两步胶原酶灌注分离小鼠肝细胞，并建立了3D类器官培养物作为”迷你肝脏”，以概括细胞 – 细胞相互作用和物理功能。类器官由异质细胞群组成，包括祖细胞和成熟肝细胞。我们详细介绍了分离和培养小鼠肝细胞或胎儿肝细胞在2-3周内形成类器官的过程，并展示了如何通过机械上下移液来通过它们。此外，我们还将介绍如何将类器官消化成单细胞，用于shRNA/异位构建的慢病毒感染，siRNA转染和CRISPR-Cas9工程。类器官可用于药物筛选，疾病建模和基础肝脏研究，通过模拟肝脏生物学和病理生物学。
Introduction
类器官是自组织的三维（3D） 体外 簇，包括自我更新的干细胞和多系分化细胞1，2。许多器官的类器官已经通过明确定义的利基因素从多能或成体干细胞中建立，包括肠道，大脑，结肠，肾脏，肝脏，胰腺，甲状腺，胃，皮肤和肺3，4，5，6，7.类器官通过模仿发育（源自胚胎或诱导多能干细胞，PSC）或稳态/再生进展（源自成体干细胞，ASCs）来概括物理细胞功能，这为疾病研究和治疗开辟了新的途径8，9。
作为哺乳动物最大的器官，肝脏主要负责储存，代谢和解毒。两种上皮细胞类型，肝细胞和胆管细胞，构建肝小叶的基本单位。肝细胞负责70-80%的肝功能10。虽然肝脏具有显着的再生能力，但在传统的单层培养过程中，肝细胞特征的快速丧失发生在失调的细胞极化和去分化，这增加了研究人员和临床医生在培养皿中建立”间隙桥接”肝脏模型的需求。然而，直到最近，来自原代肝细胞的 离体 扩增模型尚未得到很好的建立11，12，13，14，15。肝脏类器官可以从胚胎/诱导多能干细胞、成纤维细胞转化为肝细胞样细胞和组织衍生细胞中建立。肝脏类器官的发展促进了体外模型在药物筛选和肝毒性测定中的应用16，17。
在这里，我们描述了从小鼠原发性肝细胞建立肝脏类器官的详细方案。通过使用该协议，我们建立了具有两种胶原酶灌注的肝细胞类器官的 体外 培养系统。这些类器官可以长期扩增数月。它们的生理功能与肝细胞高度一致。此外，我们还详细介绍了如何使用类器官进行遗传操作，例如慢病毒感染，siRNA转导和CRISPR-Cas9工程。肝细胞类器官的繁殖揭示了使用类器官来理解肝脏生物学并开发个性化和转化医学方法的可能性。
Protocol
所有小鼠实验均经山东大学基础医学院动物护理与使用委员会批准，并根据内政部指南和规定（No.ECSBMSSDU2019-2-079）。 注意：该方案主要用于从分离的原代肝细胞中培养3D类器官。 1. 建立小鼠肝细胞类器官培养 注意：细胞外基质（如基质胶或BME）用作培养类器官的基底基质。将细胞外基质储存在-20°C，并在4°C或冰上预解冻。在实验过?…
Representative Results
来自女性Alb-Cre的肝细胞类器官;在播种后五天观察Rosa26-GFP小鼠（8周）（图1A）。类器官随着Ki67阳性染色而增殖（图1B）。通过qRT-PCR和蛋白质印迹检查通过siRNA转染或慢病毒感染在肝细胞类器官中代表性基因的干扰表达。结果如图 2A 和 2B所示。 图2C 显示了用RSL-3和CRISPR / Cas9基因组文库筛选治疗14?…
Discussion
长期培养成熟肝细胞的能力是研究肝脏基础科学，药物毒理学和嗜肝性宿主微生物学感染（如疟疾和肝炎病毒）的基础。通过明确定义的生态位，这里的方案为肝细胞建立了一个培养系统。该协议驱动成熟肝细胞在3D培养物中扩增，具有异质性，概括了细胞 – 细胞相互作用，大多数肝细胞功能和遗传修饰，允许设计基因功能研究和新的治疗途径进行再生治疗。
高质量的肝细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Hans Clevers教授慷慨地提供了细胞系来生产重组R-spondin1。这项工作得到了中国国家重点研发计划（2019YFA0111400）、国家自然科学基金（31970779）和山东大学青年交叉学科创新研究组（2020QNQT003）对H.H.的资助。
Materials
| Perfusion Buffer | |||
| EGTA | Sangon Bitech | A600077-0100 | 3.50 g/L |
| Glucose | Sangon Bitech | A100188-0500 | 9.00 g/L |
| KCl | Sangon Bitech | A100395-0500 | 4.00 g/L |
| Na2HPO4·12H2O | Sangon Bitech | A501727-0500 | 1.51 g/L |
| NaCl | Sangon Bitech | A501218-0001 | 80.0 g/L |
| NaH2PO4·2H2O | Sangon Bitech | A610404-0500 | 0.78 g/L |
| NaHCO3 | Sangon Bitech | A500873-0500 | 3.50 g/L |
| Phenol Red | Sangon Bitech | A100882-0025 | 0.06 g/L |
| Digestion Buffer | |||
| CaCl2 | Sangon Bitech | A501330-0005 | 0.50 M |
| Collagenase IV | Sigma | C1639 | 0.10 mg/mL |
| HEPES | Sangon Bitech | C621110-0010 | 23.80 g/L |
| KCl | Sangon Bitech | A100395-0500 | 4.00 g/L |
| Na2HPO4·12H2O | Sangon Bitech | A501727-0500 | 1.51 g/L |
| NaCl | Sangon Bitech | A501218-0001 | 80.0 g/L |
| NaH2PO4·2H2O | Sangon Bitech | A610404-0500 | 0.78 g/L |
| NaHCO3 | Sangon Bitech | A500873-0500 | 3.50 g/L |
| Tip-wash Buffer | |||
| Fetal Bovine Serum | Gibco | 10091148 | stored at 4 °C |
| DPBS | Gibco | C14190500BT0 | stored at 4 °C |
| Wash Medium | |||
| Advanced DMEM/F12 | Invitrogen | 12634-010 | stored at 4 °C |
| GlutaMAX | Gibco | 35050-061 | 100 X |
| HEPES | Gibco | 15630-080 | 100 X |
| Penicillin/streptomycin | Sangon Bitech | A600135-0025 | 100 X |
| Culture Medium | |||
| A83-01 | Tocris | 2939 | Stock concentration 500 µM, final concentration 2 µM |
| Advanced DMEM/F12 | Invitrogen | 12634-010 | stored at 4 °C |
| B-27 supplement 50x, minus vitamin A | Gibco | 1704-044 | 50 X |
| Chir 99021 | Tocris | 4423 | Stock concentration 30 mM, final concentration 3 µM |
| DMEM/F12 | Gibco | 11330032 | stored at 4 °C |
| Gastrin I | Tocris | 3006 | Stock concentration 100 mM, final concentration 10 nM |
| GlutaMAX | Gibco | 35050-061 | 100 X |
| HEPES | Gibco | 15630-080 | 100 X |
| Matrigel martix | BD | 356231 | stored at -20 °C |
| N-acetylcysteine | Sigma Aldrich | A9165 | Stock concentration 500 mM, final concentration 1 mM |
| Nictinamide | Sigma Aldrich | N0636 | Stock concentration 1 M, final concentration 3 mM |
| Penicillin/streptomycin | Sangon Bitech | A600135-0025 | 100 X |
| Recombinant human EGF | Peprotech | AF-100-15 | Stock concentration 100 µg/ml,final concentration 50 ng/ml |
| Recombinant human FGF10 | Peprotech | 100-26 | Stock concentration 100 µg/ml, final concentration 100 ng/ml |
| Recombinant human HGF | Peprotech | 100-39 | Stock concentration 100 µg/ml, final concentration 25 ng/ml |
| Recombinant Human TGF-α | Peprotech | 100-16A | Stock concentration 100 µg/ml, final concentration 100 ng/ml |
| Recombinant Human TNF-α | Origene | TP750007-1000 | Stock concentration 100 µg/ml, final concentration 100 ng/ml |
| Rho kinase inhibitor Y-27632 | Abmole Bioscience | Y-27632 dihydrochloride | Stock concentration 10 mM, final concentration 10 µM |
| Rspondin-1conditioned medium | Stable cell line generated in the Hu Lab. Final concentration 15%. | ||
| Others | |||
| 0.22 µm filter | Millipore | SCGPT01RE | |
| 16 # silicone tube | LangerPump | ||
| 24 well, suspension | Greiner bio-one | GN662102-100EA | |
| 37 °C Water Bath | |||
| 70 µm filter | BD | 352350 | |
| Accutase | stemcell | AT-104 | stored at 4 °C |
| Anti-CTNNB1 | BD | 610154 | Mouse |
| Anti-GAPDH | CST | 5174S | Rabbit |
| Anti-HDAC7 | Abcam | ab50212 | Mouse |
| Anti-KI67 | Abcam | ab15580 | Rabbit |
| Biological safety cabinet | ESCO | CCL-170B-8 | |
| Cell Recovery Solution | Corning | 354253 | stored at 4 °C |
| Centrifuge 5430 | eppendorf | 542700097 | |
| CO2 incubator | ESCO | AC2-4S1 | |
| DMSO | Sangon Bitech | A100231 | stored at RT |
| EVOS FL Color Imaging Systems | Invitrogen | AME4300 | |
| Hdac3 siRNA | Guangzhou RiboBio Co., Ltd. | siG2003180909192555 | stored at -20 °C |
| Hdac7 lentivirus | ShanghaiGenePharmaCo.,Ltd | LV2020-2364 | stored at -80 °C |
| Hitrans G A | Shanghai Genechem Co.,Ltd. | REVG004 | Increased virus infection efficiency |
| Image Pro Plus software | Media Cybernetics, Bethesda, MD, USA | version 6.0 | |
| Lipofectamin RNAiMAX Transfection Reagent | Invitrogen | 13778150 | Increased virus infection efficiency |
| Live cell dye | Luna | F23001 | stored at 4 °C |
| Low-speed desktop centrifuge | cence | TD5A-WS/TD5AWS | |
| Nucleofactor-α 2b Device | Lonza | ||
| Opti-MEM | Gibco | 31985070 | stored at 4 °C |
| Pasteur pipette | Sangon Bitech | F621006-0001 | |
| Peristaltic pump | LangerPump | BT100-2J | |
| PVDF membrane | Millipore | IPVH00010 | Activate with methanol |
| PX458 | Addgene | 48138 | stored at -80 °C |
| Refrigerated centrifuge | Thermo Scientific Heraeus | Labofuge 400 | |
| RSL3 | MCE | HY-100218A | Stock concentration 10 mM, final concentration 4 µM |
| Trypsin/EDTA | Gibico | 25200072 | stored at 4 °C |
| Wee1 siRNA | Guangzhou RiboBio Co., Ltd. | siG2003180909205971 | stored at -20 °C |
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Cite This Article
Lian, J., Meng, X., Zhang, X., Hu, H. Establishment and Genetic Manipulation of Murine Hepatocyte Organoids. J. Vis. Exp. (180), e62438, doi:10.3791/62438 (2022).