人肝细胞通过一个两步胶原酶灌注过程隔离
Summary
对于人肝细胞的分离的修饰的两步胶原酶灌注过程进行说明。此方法也可应用到其它哺乳动物的肝脏。孤立的肝细胞有很高的产量和活力提供，使他们在诸如肝再生，药代动力学和毒理学领域一个合适的模型进行科学研究。
Abstract
肝脏，具有特殊的再生能力的器官，进行了广泛的功能，如解毒，代谢和内环境稳定。因此，肝细胞的大量的各种研究问题的重要模式。特别是，使用人肝细胞是在药代动力学，毒理学，肝脏再生和转化研究的领域尤其重要。因此，这种方法提出了一个两步胶原酶灌注过程所描述的Seglen 1分离的肝细胞的修改版本。
先前，肝细胞已被分离用机械的方法。然而，酶的方法已被证明是优良的肝细胞分离后保留其结构完整性和功能。本文提出的方法相适应前面对大鼠肝脏对人体肝片，结果在大产量的肝细胞为77±10％生存力的设计方法。主在此过程中不同的是血管cannulization的过程。此外，这里描述的方法也可以应用于从其它物种具有可比性的肝脏或血管体积的肝脏。
Introduction
的肝细胞悬液可以从肝脏通过机械或酶的方法来制备。用于制备全肝细胞的机械方法包括强迫肝脏通过干酪2，摇晃了肝片与玻璃珠在卡恩振动筛3，用玻璃匀浆器搭配宽松杵4,5 等。多年来，机械方法已经跌出有利于因损坏细胞膜和分离肝细胞6,7的功能丧失。因此，利用酶法的是目前对肝细胞的分离的主要方法。
采用酶法肝细胞的分离时，Berry和朋友8经由大鼠门静脉肝脏灌注胶原酶和透明质酸酶大幅提高。此灌注过程中使用的脉管系统，以允许所述酶接触到的大部分细胞紧密接触，从而导致一个增加6倍于肝细胞8的产率。进一步，这种方法产生的细胞保持其结构的完整性，几乎没有变换面内质网中分离成囊泡和无线粒体损伤8。
此方法是由Seglen 1，谁开创了一种两步灌注过程肝细胞分离改性。在此过程中，大鼠肝灌注用的Ca 2 +的缓冲液随后灌注含Ca 2 +的1胶原酶缓冲液中。的Ca 2 +的在第一步骤中除去，可以破坏桥粒，而所需的最佳胶原酶活性1,9在第二步骤中添加的Ca 2 +的。
鉴于上述公布的工作已在大鼠进行的，本文的目的是证明可用于肝细胞与来自嗡嗡声高生存能力的隔离的变形过程一个肝脏。利用人体肝细胞仍然是转化研究和验证利用动物模型实验非常重要。在这项研究中使用人类肝片通过人体组织和细胞研究基金会，国家控股的非营利基金会10收购与治理同意。经过病理学家删除什么需要诊断，肝片是从剩余的组织收集。由病理切片关组织形态是肝切除后的切缘获得的健康组织。
Protocol
1。灌注和隔离溶液的制备制备所需的肝片和肝细胞，根据表1中的隔离的灌注的解决方案。溶液可以贮存于4℃直至使用。 无菌过滤器使用的是0.22微米的过滤器的所有解决方案。 所有的解决方案，它们与肝脏接触应该是无菌的。 2。灌注设备和解决方案的研制应设置该设备的肝片的灌注， 如图1。 水浴应设置?…
Representative Results
灌注设置所需的肝灌注的设备应根据图1进行设定。 生存能力和人离体肝细胞的产量人离体肝细胞的平均存活率为77±10％和肝细胞的平均收益率为13±11000000肝细胞/克肝，与表达的价值观为平均值±标准偏差。肝细胞隔离的数量进行了获取这些平均值是648隔离开展了1999年1月至2012年12。 适合灌注参数<p class="…
Discussion
该协议将导致人肝细胞具有较高的可行性和纯度的隔离。为了达到这些效果，它开始与适当的一块肝脏是重要的。这件作品肝应该有完整的Glisson囊在所有表面除1切面。另一个重要的因素是特定批次胶原酶使用，因为不同批次可能导致消化11后在肝细胞的存活率显着差异。因此，胶原酶不同批次应进行测试并产生肝细胞具有最佳活力批次应在大量获得。最后，合适的消化时间具有基于获得?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作成为可能，人类组织和细胞研究基金会，这使得可用于研究人体组织。和Hepacult有限公司从德国联邦教育与研究（：虚拟肝络，授权号码0315759补助金名）收到对这项工作的财政支持。我们还要感谢从Grosshadern医院组织库的技术助理的肝脏样本，并从细胞分离核心设备的技术助理的集合进行肝脏灌注和肝细胞分离。特别是，我们要感谢NataljaLöwen酒店为展示在视频这个程序。最后，我们要感谢NataljaLöwen酒店和Edeltraud同时，Hanesch在视频的示意图创建插图图1和数字。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Bubble trap | Gaßner Glastechnik | ||
| Glass jacketed condenser | Gaßner Glastechnik | ||
| 41 °C Water bath | Julabo | 35723-H24/EG | |
| 37 °C Water bath | GFL | 1083 | |
| Compressed gas cylinder (95 % O2/5 % CO2) | Linde | ||
| Gas permeable tubing | Neolab | 2-4440 | |
| Peristaltic pump | Ismatec | IP65 | |
| Scalpel | Feather | 320010 | |
| Forceps | Omnilab | 5171014 | |
| Conical flasks 1 L | Schott Duran | 2121654 | |
| Conical flasks 5 L | Schott Duran | 2121673 | |
| Beakers | Schott Duran | 2110654 | |
| 200 ml centrifuge tubes | Becton Dickinson | 352075 | |
| Crystallizing dish | Omnilab | 5144063 | |
| Curved irrigation cannulae with ball tips | Ernst Kratz GmbH | 1464LL/ 1465LL A+B/ 1472LL | |
| Micro vascular clamps | Ernst Kratz GmbH | ||
| Büchner funnel | Carl Roth | HT38.1 | |
| Nylon mesh 210 μm | Neolab | 4-1413 | |
| Nylon mesh 70 μm | Neolab | 4-1419 | |
| 0.22 μm sterile filters | Peske | 99505 | |
| 500 ml bottles | Schott Duran | 2180144 | |
| 1 L bottles | Schott Duran | 2180154 | |
| Hemocytometer | Peske | 06-0001 | |
| 1.5 ml tubes | Eppendorf | 0030 120,086 | |
| 50 ml conical tubes | BD Biosciences | 352070 | |
| Ice bucket | Neolab | 1508454 | |
| Sterile Pasteur pipettes | Brand | 747715 | |
| Motorised pipette filler (Pipette boy acu) | Integra | 155017 | |
| Refridgerated centrifuge | Eppendorf | 5810R | |
| Laminar flow | Kendro | Hera safe-KS9 | |
| Aspirator (Low-flow surgical suction pump) | Atmos | C361 | |
| Laboratory Gas Burner | Integra | Fire Boy eco | |
| Disposable laboratory coat | Paperlynen GmbH | MD0202414 | |
| Surgical mask with visor | Kimberly-Clark | 48247 | |
| Surgical hood | Barrier | 42072 | |
| Latex gloves | Semper Care | CE0321 | |
| Collagenase (Batch number NB 4G) | Serva | 17465 | |
| Calcium chloride dihydrate | Merck | 2382 | |
| EGTA | Sigma | E4378 | |
| Sodium chloride | Roth | 9265.2 | |
| Hepes | Roth | 9105.3 | |
| Potassium chloride | Serva | 26868 | |
| Albumin | Biomol | 01400-2 | |
| Glucose | Serva | 22700 | |
| Sodium hydrogen carbonate | Serva | 30180 | |
| 0.4% Trypan blue solution | Lonza | 17-942E | |
| Cold storage solution | Hepacult GmbH |
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Cite This Article
Lee, S. M., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure. J. Vis. Exp. (79), e50615, doi:10.3791/50615 (2013).