毛利精密切肝片作为肝脏生物学的Ex Vivo模型
1Hepatic Fibrosis Group,QIMR Berghofer Medical Research Institute, 2School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute,Curtin University, 3School of Veterinary and Life Sciences,Murdoch University, 4School of Medicine,The University of Queensland, 5Madrid Institute for Advanced Studies (IMDEA) in Food, CEI UAM+CSIC, 6School of Medicine,Griffith University, 7School of Biological Sciences,Queen’s University Belfast, 8Department of Gastroenterology & Hepatology,Fiona Stanley and Fremantle Hospital Group, 9School of Medical and Health Sciences,Edith Cowan University, 10Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences,University of Western Australia
Summary
该协议为从小鼠中生产可行的精确切肝片提供了一种简单可靠的方法。前活体组织样本可在实验室组织培养条件下维持多日，为检查肝病理生物学提供了灵活的模型。
Abstract
了解肝损伤、肝纤维化和肝硬化的机制，这些机制是慢性肝病（即病毒性肝炎、非酒精性脂肪性肝病、代谢性肝病和肝癌）的基础，需要实验性操作动物模型和体外细胞培养物。这两种技术都有局限性，例如对大量动物进行体内操作的要求。然而，体外细胞培养物不再现多细胞肝环境的结构和功能。使用精密切肝片是一种技术，其中保持一致的可行的小鼠肝脏切片在实验室组织培养进行实验操作。该技术占据了动物研究和体外细胞培养方法之间的实验空白。提出的协议描述了一种从小鼠身上分离和培养精确切肝片的简单而可靠的方法。作为该技术的应用，前活体肝切片用胆汁酸进行治疗，以模拟胆静肝损伤，并最终评估肝纤维成因的机制。
Introduction
大多数慢性肝病（即病毒性肝炎、非酒精性性性性性性肝炎、胆汁性肝病和肝癌）的发病机制涉及多种不同肝细胞类型之间的复杂相互作用，这些细胞类型驱动炎症、纤维生成和癌症发展11、2。2为了了解这些慢性肝基疾病背后的分子机制，必须研究多种肝细胞类型之间的相互作用。虽然多个肝细胞系（以及最近，器官）可以在体外培养，但这些模型不能准确模拟肝微环境3的复杂结构、功能和细胞多样性。此外，培养的肝细胞（特别是转化的细胞系）可能偏离其原始来源生物学。动物模型被实验性地用于研究多种肝细胞类型之间的相互作用。然而，由于肝外器官（例如，在测试潜在治疗药物时），实验操作的范围可能会显著缩小。
在组织培养中使用精密切肝片（PCLS）是首次用于药物代谢和毒性研究的实验技术，它涉及切割可行的超薄（约100~250 μm厚）肝片。这允许直接实验操作肝组织前活体4。该技术填补了体内动物研究与体外细胞培养方法之间的实验差距，克服了两种方法的许多缺点（即，对整个动物可进行的实验范围的实际限制，以及利用体外细胞培养方法丧失结构/功能和细胞多样性）。
此外，与整个动物研究相比，PCLS大大提高了实验能力。由于一只小鼠可以产生超过48个肝切片，这也方便了使用来自同一肝脏的控制和治疗组。此外，该技术将肝脏组织与其他器官系统物理分离;因此，在测试外源性刺激的效果时，它消除了整个动物中可能发生的潜在非目标效应。
在此协议中，PCLS 使用带有横向振动刀片的振动器生成。其他研究已经成功地使用了克鲁姆迪克组织切片机，如奥林加和舒潘5所述。在振动器中，刀片的横向振动可防止剪切应力引起的超薄组织的撕裂，因为刀片被推入组织。振动片和克鲁姆迪克组织切片器都能有效工作，无需对肝脏组织进行结构嵌入，从而简化切片程序。该技术还可用于从患病肝脏创建PCLS，包括那些从小鼠模型纤维化/肝硬化6和肝硬化7。
除了展示PCLS的制备和组织培养所需的技术外，本报告还通过测量腺苷三磷酸（ATP）水平并检查组织组织组织学来评估坏死和纤维化，来检查这些前活体组织的生存能力。作为一项具有代表性的实验程序，PCLS以三种不同的胆汁酸（糖胆酸、黄骨酸和胆酸）的病理生理浓度进行治疗，以模拟胆静肝损伤。在胆汁性肝损伤的情况下，特别是胆碱酸在囊性纤维化相关肝病8的儿童血清和胆汁中都明显增加。
肝祖细胞也接受了在体外用陶罗基酸治疗，以模拟患者观察到的高灰胆酸水平，这种治疗导致肝祖细胞对胆小（胆碱）表型9的增殖和分化增加。随后，PCLS在体内处理，其陶罗乔酸水平升高，并观察到胆碱基标记的增加。这支持体外观察，在小儿囊性纤维化相关肝病9中，陶罗基酸驱动胆汁增殖和/或分化。
Protocol
所有动物实验均根据澳大利亚关于为科学目的护理和使用动物的法典进行，经该研究所动物伦理委员会批准。雄性C57BL/6小鼠（15-20周大）来自澳大利亚西澳大利亚州动物资源中心。 注：所有与样品接触的解决方案、介质、仪器、硬件和管材都必须用70%乙醇溶液消毒或彻底消毒，并使用无菌技术进行处理，以尽量减少培养污染的风险。 1. 振动器的设置 <ol…
Representative Results
为了确定PCLS随时间的细胞可行性，测量了组织ATP水平。ATP 水平通常与可行性成正比。PCLS（面积约15 mm2区域）在正常威廉的E介质中培养，具有10%的FBS，然后在特定的时间点，肝切片从组织培养中去除，并同质化，测量ATP和蛋白质（用于正常化）浓度（材料表）。Figure 1A对于这样的…
Discussion
该议定书演示了鼠PCLS分离和组织培养的应用，这些程序旨在评估生存能力和效用，以及利用生化测定、组织学和qPCR检查肝病理学外源性中介的影响。PCLS组织培养在啮齿动物和人类的实验效用已在各种应用中得到证明，包括微RNA15/RNA9/蛋白质表达16、代谢17、病毒感染动力学10、18、18感染信号<sup class=…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了澳大利亚国家卫生和医学研究委员会（NHMRC）的研究资助（赠款号） 的支持。APP1048740 和 APP1142394 到 G.A.R. ;APP1160323 到 J.E.E.T.， J.K.O.， G.A.R.）.A. Ramm 基金由澳大利亚 NHMRC 的高级研究研究奖学金提供支持（赠款号APP1061332）。曼努埃尔·费尔南德斯-罗霍得到西班牙马德里的TALENTO方案（T1-BIO-1854）的支持。
Materials
| 10 cm Petri Dish | GREINER | 664160 | Sterile Dish |
| 12 Well Tissue Culture Plate Flat Bottom | Greiner Bio-one | 665180 | |
| 70% Ethanol Solution (made with AR Grade) | Chem-Supply Pty Ltd | EA043-20L-P | Disinfection solution |
| Acetone | Chem-Supply Pty Ltd | AA008-2.5L | |
| Cholic acid | Sigma-Aldrich | C1129-100G | |
| Cyanoacrylate Super Glue | Parfix, DuluxGroup (Australia) | Other brands should work | |
| Disposable Single Edge Safety Razor Blades | Mixed | ||
| Dissection Board | Made in-house | Sterile material over polystyrene | |
| Fetal Bovine Serum | GE Healthcare Australia Pty Ltd | SH30084.02 | |
| Forceps sharp point 130 mm long | ThermoFisher Scientific | MET2115-130 | |
| Forma Steri-Cycle CO2 Incubator | ThermoFisher Scientific | 371 | |
| Glutamine | Life Technologies Australia Pty Ltd | 25030081 | |
| Glycocholic acid hydrate | Sigma-Aldrich | G2878-100G | |
| ISOLATE II RNA Mini Kit | Bioline (Aust) Pty Ltd | BIO-52073 | |
| Ketamine 50 ml | Provet | KETAI1 | |
| Krebs-Henseleit Buffer with Added Glucose 2000 mg/L | Sigma-Aldrich | K3753 | Can also be made in house |
| Laminar Flow Hood | Hepa air filtration | ||
| NanoDrop 2000/2000c Spectrophotometers | ThermoFisher Scientific | ||
| Penicillin-Streptomycin, Liq 100 ml | Life Technologies Australia Pty Ltd | 15140-122 | |
| Picro Sirius Red | ABCAM Australia Pty Ltd | ab246832 | |
| Pipette Tips Abt 1000 µl Filter Interpath | Interpath | 24800 | |
| Pipette Tips Abt 10 µl Filter Interpath | Interpath | 24300 | |
| Pipette Tips Abt 200 µl Filter Interpath | Interpath | 24700 | |
| Pipette Tips Abt 20 µl Filter Interpath | Interpath | 24500 | |
| Precellys Homogeniser | Bertin Instruments | P000669-PR240-A | |
| Protractor | Generic | To measure blade angle | |
| Quantstudio 5 QPCR Fixed 384 Block | Applied Biosystems/ ThermoFisher Scientific | ||
| Scalpel Blade | Mixed | ||
| Scalpel Blade Holder | Mixed | ||
| SensiFAST cDNA Synthesis Kit | Bioline (Aust) PTY LTD | ||
| Small Paintbrush with Plastic Handle | Mixed | Plastic handle resists ethanol | |
| Square-Head Foreceps | Mixed | ||
| Sterile 50 ml Plastic Tubes | Corning Falcon | 352098 | |
| Surgical Clamps | Mixed | ||
| Surgical Forceps | Mixed | ||
| Surgical Pins | Mixed | ||
| Surgical Scissors | Mixed | ||
| Taurochoic acid | Sigma-Aldrich | T-4009-5G | |
| Vibratome SYS-NVSLM1 Motorized Vibroslice | World Precision Instruments | SYS-NVSLM1 | With thermoelectric cooling |
| Williams Medium E | Life Technologies Australia Pty Ltd | 12551032 | 2.0 g/l glucose |
| Xylazine 100 mg/mL 50 mL | Provet | XYLAZ4 |
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Cite This Article
Pearen, M. A., Lim, H. K., Gratte, F. D., Fernandez-Rojo, M. A., Nawaratna, S. K., Gobert, G. N., Olynyk, J. K., Tirnitz-Parker, J. E. E., Ramm, G. A. Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology. J. Vis. Exp. (157), e60992, doi:10.3791/60992 (2020).