新生儿肝外胆管的隔离
Summary
一种技术，从新生小鼠的肝外胆管胆管隔离描述。管道正在认真解剖，然后细胞被生长在厚厚的胶原凝胶中分离。该方法为研究肝外胆管发育和病理的有用工具。
Abstract
肝脏内和肝外胆管发育是不同的，并且可能会不同程度地影响某些疾病。然而，区域内和肝外胆管，新生儿和成人细胞之间的差异，都不能很好地理解。
方法从肝内胆管胆管的隔离是源远流长1-4。肝外导管细胞，特别是从新生儿的隔离，还没有被描述，尽管这将是有很大好处的理解不同的胆管群体之间的差异，并在研究的疾病，如胆道闭锁，似乎靶向肝外导管。这里描述的是一个优化的技术来隔离两个新生和成年小鼠肝外胆管细胞。这种技术产生一个纯粹的细胞群与细胞间质成纤维细胞样污染最小。
这METHOD基于切除肝外管和胆囊，其次是细致的解剖和刮去除脂肪和成纤维细胞层。结构被嵌入在胶原和培养的厚层为大约3周，以允许在胆管上皮细胞单层，然后可以用胰蛋白酶消化并重新接种于实验使用的产物。
Introduction
的内部和肝外胆管的起源和发展是明显不同的。肝脏从腹侧前肠内胚层5的憩室发展。憩室的尾部区域形成肝外胆管树，而颅区域产生肝内胆管树5。肝内胆管结石胆管是从祖细胞在围门 地区6得出周围的胆管板。这些细胞具有分化成肝细胞或胆管6的能力。这给了cholangiopathies可能专门针对一类胆管的显著临床意义。例如，胆道闭锁最初影响到新生儿的肝外管道，而Alagille综合征会影响肝内管道。
有肝内胆管和胆管单元从小鼠和大鼠的分离的多描述。在vestigators已经分离出单个细胞通过管道隔离和随后的产物，或者通过肝脏的消化和抗拔胆管表达特定的细胞表面标志物1-4下来。功能和偏光胆管单位已分离肝脏的消化和大小过滤7。胆管单元能够响应与分泌刺激物，并证明分泌液7，而孤立胆管细胞已被证明开发导管结构体外 1,2。这些方法的局限性包括需要专门的技术知识和专用设备肝脏灌注消化酶。此外，存在污染与间充质细胞3的危险。
阿来分离肝外胆管胆管，特别是从新生儿肝外胆管方法，过去一直没有被描述。本文概述了一个简化的技术来隔离一个新生儿结果好，成人肝外胆管细胞在高水平的纯度。这种技术将促进区域内和肝外胆管和研究像胆道闭锁的疾病，涉及肝外管机制之间的差异研究。
Protocol
整个过程是在室温下，除非另有规定。所有的动物工作应经当地机构动物护理和使用委员会（IACUC）的协议下，人道的条件下进行。 设备1。制备及解决方案在靠近所述组织培养罩和孵化器（ 图1A）设置在鼠标手术台。注：需要的设备包括一个解剖显微镜，光源，12.5厘米长的直虹膜剪，6寸非锯齿弯钳细的技巧，以及4英寸的锯齿钳尖端弯曲（ 图1B）。…
Representative Results
在具有优良的纯度新生小鼠肝外胆管，具体表现为K19免疫荧光染色（ 图2A和2B）人口的隔离使用此协议的结果;我们实现了类似的结果从成年小鼠中分离细胞。我们观察到，它需要3周的时间从新鲜分离的胆管细胞形成的厚胶原凝胶单层。在胆管以线性方式增长整个厚胶原，形成细胞从分离的管道片。细胞应该分开并重新镀上很薄的胶原凝胶前单层变得杂草丛生，因为孔出现在细胞的表?…
Discussion
这里描述的是一种技术，从各年龄段，包括新生儿的小鼠的小鼠的肝外胆管分离出纯的胆管。该技术的优点是肝外胆管可分别从肝内胆管细胞进行研究，并可能促进研究，以确定细胞的这些人群之间的主要区别。我们最近发表了一份研究报告，证明在肝外胆管分离这种方法和感染恒河猴轮状病毒8下降纤毛。缺点包括该技术是劳动密集，细致的解剖要求，以防止成纤维细胞污染;另外，生长…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢宾夕法尼亚大学NIDDK中心分子生物学研究中消化的分子病理学和影像核心和肝脏疾病（P30 DK50306）与成像协助。这项工作是由赠款从美国国立卫生研究院（R01 DK-092111）和弗雷德和Suzanne比泽克小儿肝病中心（以RGW），并从儿童肝脏疾病研究和教育网络（向SK）的奖学金支持。
Materials
| Name of Material/Equipment | Company | Catalog Number | Comments/Description |
| DMEM/F12 (1:1) | Gibco/ Life technologies | 11320-033 | 500ml, used in BEC media |
| FBS | Atlanta Biologicals | S11150 | 25 ml, used in BEC media |
| MEM with non-essential amino acids | Gibco/ Life technologies | 11140-019 | 5 ml, used in BEC media |
| Insulin-transferrin-selenium | Gibco/ Life technologies | 51300-044 | 5 ml, used in BEC media |
| Na Pyruvate | Cellgro | 25-000-CL | 5ml, used in BEC media |
| Chemically-defined lipid concentrate | Gibco/ Life technologies | 11905-031 | 5ml, used in BEC media |
| Penicillin-Streptomycin | Cellgro | 30-002-CI | 5ml, used in BEC media and 500 ul in the isolation |
| Gentamicin | Gibco/ Life technologies | 15750-060 | 0.2ml, used in BEC media and 500 ul in the isolation |
| Ethanolamine | Sigma Aldrich | E9508-100ml | 0.13ml, used in BEC media |
| MEM vitamin solution | Gibco/ Life technologies | 11120-052 | 5ml, used in BEC media |
| Soybean trypsin inhibitor | Biowhittaker | 17-605E | 5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration |
| L-glutamine | Cellgro | 25-005-CL | 5ml, used in BEC media |
| Bovine pituitary extract | Gemini | 500-102 | 1.1ml, used in BEC media |
| Dexamethasone | Sigma Aldrich | D4902 | 0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol |
| 3 3',5-triiodo-L-thyronine | Sigma Aldrich | T6397 | 0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol |
| Epidermal growth factor | millipore | 01-101 | 0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA |
| Forskolin | Sigma Aldrich | F6886 | 5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO |
| Fungizone | Gibco/ Life technologies | 15290-018 | 1ml, used in BEC media and 500 ul in the isolation |
| Rat-tail collagen | BD Biosciences | 354236 | variable depending on concentration of collagen |
| PBS 10X | USB Corporation | 75889 | use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration |
| dH2O | N/A | N/A | sterile, used to make collagen, amount used depends on collagen concentration |
| NaOH 10N | Fischer Scientific | ss255-1 | Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration |
| collagenase type XI from Clostridium histolyticum | Sigma Aldrich | C7657 | dilute in DMEM and sterile filter before use |
| trypsin-EDTA (1x) 0.25% | Gibco/ Life technologies | 25200-056 | 3 ml, incubate max 10 minutes |
| trypsin-EDTA (10x) 0.5% | Gibco/ Life technologies | 15400-054 | 3 ml, incubate max 10 minutes |
| Dissecting microscope | Nikon | SMZ645 | Other models acceptable |
| Light source (fiberoptic illuminator) | Schott-Fostec | Ace EKE LR 92240 | Other models acceptable |
| 12.5 cm straight iris scissors | Kent Scientific | Other models acceptable | |
| 6" non-serrated curved forceps with fine tips | Electron Microscopy Sciences | Other models acceptable | |
| 4" serrated stainless forceps with fine tips | Electron Microscopy Sciences | Other models acceptable |
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Cite This Article
Karjoo, S., Wells, R. G. Isolation of Neonatal Extrahepatic Cholangiocytes. J. Vis. Exp. (88), e51621, doi:10.3791/51621 (2014).