Name: isolation of human hepatocytes by a two-step collagenase perfusion procedure
Uploaded: 2013-09-03T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure at JoVE.com

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。

1。灌注和隔离溶液的制备<ol><li>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。 </li><li>无菌过滤器使用的是0.22微米的过滤器的所有解决方案。 </li><li>所有的解决方案，它们与肝脏接触应该是无菌的。 </li></ol> 2。灌注设备和解决方案的研制<ol><li>应设置该设备的肝片的灌注， 如图1。 </li><li>水浴应设置?...

<ol>
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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?...

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。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass jacketed condenser</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>41 &deg;C Water bath</td>
			<td>Julabo</td>
			<td>35723-H24/EG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37 &deg;C Water bath</td>
			<td>GFL</td>
			<td>1083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compressed gas cylinder (95 % O2/5 % CO2)</td>
			<td>Linde</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas permeable tubing</td>
			<td>Neolab</td>
			<td>2-4440</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td>IP65</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>320010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Omnilab</td>
			<td>5171014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 1 L</td>
			<td>Schott Duran</td>
			<td>2121654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 5 L</td>
			<td>Schott Duran</td>
			<td>2121673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beakers</td>
			<td>Schott Duran</td>
			<td>2110654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 ml centrifuge tubes</td>
			<td>Becton Dickinson</td>
			<td>352075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Omnilab</td>
			<td>5144063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Curved irrigation cannulae with ball tips</td>
			<td>Ernst Kratz GmbH</td>
			<td>1464LL/ 1465LL A+B/ 1472LL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro vascular clamps</td>
			<td>Ernst Kratz GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>Carl Roth</td>
			<td>HT38.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 210 &mu;m</td>
			<td>Neolab</td>
			<td>4-1413</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 70 &mu;m</td>
			<td>Neolab</td>
			<td>4-1419</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &mu;m sterile filters</td>
			<td>Peske</td>
			<td>99505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Schott Duran</td>
			<td>2180144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 L bottles</td>
			<td>Schott Duran</td>
			<td>2180154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Peske</td>
			<td>06-0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>0030 120,086</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Neolab</td>
			<td>1508454</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile Pasteur pipettes</td>
			<td>Brand</td>
			<td>747715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Motorised pipette filler (Pipette boy acu)</td>
			<td>Integra</td>
			<td>155017</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Refridgerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow</td>
			<td>Kendro</td>
			<td>Hera safe-KS9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator (Low-flow surgical suction pump)</td>
			<td>Atmos</td>
			<td>C361</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory Gas Burner</td>
			<td>Integra</td>
			<td>Fire Boy eco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable laboratory coat</td>
			<td>Paperlynen GmbH</td>
			<td>MD0202414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical mask with visor</td>
			<td>Kimberly-Clark</td>
			<td>48247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical hood</td>
			<td>Barrier</td>
			<td>42072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Semper Care</td>
			<td>CE0321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase (Batch number NB 4G)</td>
			<td>Serva</td>
			<td>17465</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>2382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Roth</td>
			<td>9105.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Serva</td>
			<td>26868</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Biomol</td>
			<td>01400-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Serva</td>
			<td>22700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Serva</td>
			<td>30180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.4% Trypan blue solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cold storage solution</td>
			<td>Hepacult GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of human hepatocytes by a two-step collagenase perfusion procedure

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。

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="...

Watch this Scientific Journal Video about Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure at JoVE.com

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

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。

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Research

JoVE Journal

Biology

Derivation of Human Embryonic Stem Cells by Immunosurgery

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

科研

生物学

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

GST-他的净化：一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...