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>水浴应设置在一个适当的温度，这是不同的每一个特定的实验装置，使得该解决方案是在37℃的温度时，它们到达肝片。在这种情况下，水浴设定为41℃预热的解决方案1，2和3和夹套玻璃冷凝器。溶液4应温热至37℃时在一个单独的水浴中使用，以减少胶原酶活性的丧失。 </li><li>不久肝脏灌注前，打开含有95％O 2/5％的CO 2气充氧设备（ 图1E）液化气罐的调节器。 </li></ol> 3。肝脏灌注<ol><li>一种肝片与尽可能多的完整Glisson囊越好，最好只用1切面应该从一个病理学家灌注得到。 </li><li>将本肝片上的布氏漏斗，其中包含一个穿孔的过滤盘（ 图1B）。 </li><li>灌注系统应填装与解决方案1。 </li><li>用低流量，弯曲灌溉套管用橄榄油提示应该被插入到较大血管的肝片的切割面。当血液从肝脏冲出来，在组织中具有良好的灌注区域变得更亮。用于各种尺寸的套管的数量肝脏是如图2A所示。选择的计大小应导致紧密贴合，将举行插管到位。较小的血管应由开放的灌注缓冲区排出肝片。 </li><li>增加对蠕动泵以取决于肝（ 图2B）之间的大小110-460毫升/分钟的流速。这将导致在44±16平均流速每插管毫升/分钟（ 图2C）。选择的速度取决于肝片，并应导致肝片轻微丰盈。在某些情况下，可能有必要以夹紧关闭一些打开的容器用微血管钳来实现上面提到的轻微丰盈。一个良好的灌注可以在肝脏部分是较亮的颜色在整个观察。 </li><li>用一块纱布浸泡在盐水覆盖它保持灌注期间肝片湿润。 </li><li>用1升溶液1灌注冲洗掉任意的remaining血液在肝脏件。 </li><li>灌注液变则通2和灌注10分钟。 </li><li>灌注液切换到解决方案3和灌注与0.5 L。 </li><li>改变灌注液解决方案4，其中包含胶原酶（ 表2）0.1-0.15％。 </li><li>对于此步骤，灌注应进行的循环方式进行9-12分钟，或直到肝被充分消化，肝组织应该出现相距稍微打破根据Glisson囊和手感变软时，与一个钝边探测手术刀。 </li></ol> 4。肝细胞的分离<ol><li>关闭蠕动泵，并从肝片取出插管。 </li><li>将肝片含有100〜200毫升的解决方案5的结晶皿。 </li><li>小心地取出Glisson囊，轻轻地摇出的细胞。如果有未灌注良好的区域，手术刀可以用来切穿这些区域以释放包含在细胞中。添加更多的解决方案5的过程中，如需要。 </li><li>添加更多的解决方案5，直至500ml的终体积达到。 </li><li>过滤细胞悬浮液两次，先通过一个210微米的尼龙筛后跟一个70微米尼龙筛。接着，倒入细胞悬液入200ml离心管中。 </li><li>离心细胞悬浮液在72×g离心5分钟，在4℃下吸上清，轻轻重悬细胞沉淀轻轻地在200毫升溶液5。 </li><li>重复该洗涤步骤数4.6三次。在最后一步离心，重悬细胞在寒冷的存储解决方案（见材料清单）。细胞应该是使用血球为基础的台盼蓝染色，每毫升约2-5亿元用于肝细胞的产量和可行性评估。 </li><li>进行锥虫蓝排除测定，加0.1毫升适当稀释细胞（≈2-5百万/毫升）加入含有0.5毫升锥虫蓝SOLUTI的微量离心管（0.4％台盼蓝溶于磷酸盐缓冲盐水（PBS）中）和0.4毫升的PBS。彻底混合细胞悬液后，装入血球与悬挂，并在显微镜放大100倍下检查。在显微镜下，死细胞会被染成蓝色，而活细胞出现不染。计数活和细胞总数中的每个标记在血细胞计数仪的1mm 2中网格的数量。活力（％），活细胞的产量（每毫升冷存储解决方案（CSS）的百万肝细胞或每克肝亿元肝细胞）可以使用下面的公式计算。 </li></ol><img alt="式（1）" fo:src="/files/ftp_upload/50615/50615eq1highres.jpg" src="/files/ftp_upload/50615/50615eq1.jpg" /> 注意:在这种情况下，台盼蓝稀释因子为10，血球因子是10,000。

<ol>
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这个协议的优化部分被Hepacult有限公司拨款资助。沃尔夫冈Thasler博士是Hepacult GmbH的创办人之一，仍然是董事会在这家公司的成员之一。 Maresa DEMMEL的就业是部分被Hepacult。玛丽亚·豪纳是由Hepacult有限公司聘用。 Hepacult是从大学分拆生物技术公司，它提供了人类肝细胞同意和开放获取用于研究目的。

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?...

A correction was made to: <a href="http://www.jove.com/video/50615/isolation-human-hepatocytes-two-step-collagenase-perfusion">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure </a>
The changes listed below have been made to Table 1.
1). In the recipe for Solution 2, the Final Concentration of EGTA has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>0.1mM</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>1mM</td>
		</tr>
	</tbody>
</table>
2). In the recipes for Solution 3 and 4, the Final Concentration of Calcium chloride dihydrate has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>0.5&#181;M</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>5mM</td>
		</tr>
	</tbody>
</table>

A correction was made to: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.
The Final Concentration of EGTA in Solution 2 and the Final Concentration of Calcium chloride dihydrate in solutions 3 and 4 have been updated in Table 1.

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

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. ...

isolation-human-hepatocytes-two-step-collagenase-perfusion

Research

JoVE Journal

Biology

30.9K 次观看.  Grosshadern Hospital, Munich. 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。 

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

Derivation of Human Embryonic Stem Cells by 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 medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


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 medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

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

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

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

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 to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of 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

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

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

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

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

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

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

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

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

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

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 ...