这项工作是由国立卫生研究院以R01 DK05823（RGW），F32 DK083213（JWW），F30 DK081265（阿拉伯劳工组织），拨款和弗雷德“苏珊娜比泽克小儿肝病中心（RGW）从。

整个过程是在室温下进行，除非另有规定。 1。酶液的制备<ol><li>根据表1和无菌过滤器采用0.22微米过滤器，准备酶的解决方案。直到使用时间，保持解决方案1和方案2在室温和解决方案3，4°C间。进入孤立的细胞接触的所有的解决方案和设备必须是无菌的。 </li></ol> 2。灌注设备的制备<ol><li>总理与HBSS中减去的Ca 2 + 2 /镁灌注系统，它已被加热到37°C。注：为了确保灌流37℃时，它在到达肝脏，在设置为37°C水浴加热的HBSS后通过灌注泵时，它会通过一个连接到水循环集到40°C（ 图1）夹套玻璃冷凝器。 </li><li>消毒手术来OLS在70％乙醇的烧杯。 </li></ol> 3。肝脏灌注<ol><li>腹腔注射戊巴比妥（50-100毫克/公斤体重必要获得足够的麻醉），麻醉的成年雄性SD大鼠。 </li><li>放置在一个塑料托盘上的仰卧姿势的动物和修复四肢用胶带托盘。 </li><li>清洁腹部，用70％乙醇。 </li><li>使小切口在皮肤上腹部中线。剖析皮肤上腹部大U形切离筋膜。相同的形状，暴露腹腔后的剪刀剪下腹部肌肉。 </li><li>暴露门静脉，通过使用2个棉签轻轻移动到右侧腹部内脏。 </li><li>通过门静脉下方的两个无菌的2.0丝线，配合松散。 </li><li> Cannulate与16-18压力表静脉导管门静脉和导管到位，确保拧紧松散TI从上一步（ 图2）ED缝合。 </li><li>注入稀释肝素通过静脉导管肝素5000 USP HBSS中减去的Ca 2 + / Mg 2 +的 4毫升稀释的单位/毫升（1毫升）。肝脏应当灼。 </li><li>将静脉导管灌注系统。 HBSS中减去的Ca 2 + /镁2 +在10分钟20毫升/分钟的速度灌注。横切劣势下腔静脉到肝脏允许的血液和灌注液引流。汇集血液从腹腔吐气。 </li><li>改变灌注液，以0.3％的胶原酶溶液（500毫升的HBSS加的Ca 2 + / Mg 2 +的 2型胶原酶150毫克）和25分钟的灌注。保持肝脏的湿润与先前解剖腹部皮瓣覆盖。把培养皿盖在腹部皮瓣和区域位置热灯腹腔内的温度保持在约37°C。 </li></ol> 4。钍的制备Ë胆管树<ol><li>起重用钳子从腹腔取出肝，它放置在无菌组织培养与冷Leibovitz的L-15的媒体充满菜。 </li><li>工作在组织文化的吸油烟机，肝包膜剥离，并用钳子轻轻挑逗除了肝实质。移动肝脏通过充满Liebovitz的L-15家媒体的几个特色菜，同时继续逗了实质，直到最后离开孤立的胆管树。肝实质是黄褐色的，而余下的胆管树是白色的。 </li><li>孤立胆管树放入50毫升猎鹰10毫升青霉素/链霉素（10,000 IU / ml的青霉素和10000μg/ mL链霉素）钢管;冰离开15分钟。 </li></ol> 5。门户的成纤维细胞组分的分离<ol><li>放入无菌的50毫升猎鹰管胆道树，并用无菌剪刀切末。 </li><li>添加少量的酶溶胶的ution到管＃1（约5毫升）和继续直言不讳胆管树浆的一致性，直到它到达。 </li><li>浆料倒入无菌玻璃瓶。洗出余下的解决方案＃1隼管，再倒入玻璃瓶。在37°C孵育30分钟100转晃动。 </li><li>通过烧杯覆盖的单层尼龙网（30微米孔径）过滤泥浆。 </li><li>洗涤用酶液＃2网格网格上的任何组织剩余转移到无菌的玻璃瓶。启动解决方案的一半（25毫升）倒入无菌15厘米培养皿。小心地从烧杯中取出的网状网边缘，没有污染的中心区，然后反相网格和它浸泡在培养皿的解决方案，以消除任何剩余的组织网格。丢弃网。在培养皿中转移到无菌的玻璃瓶的解决方案。冲洗培养皿与REMA伊宁25毫升溶液＃2，并将其转移到相同的玻璃瓶。在37°C孵育30分钟100转晃动。 </li><li>在此期间，滤液在烧杯中倒入50毫升的猎鹰管。在每分钟1600转（460×G）离心5分钟，在室温下。 </li><li>吸媒体和悬浮颗粒在25毫升的解决方案＃3。 </li><li>采取步骤5.5解决方案，并与30微米网覆盖到第二个无菌烧杯过滤。 </li><li>丢弃网。在每分钟1600转离心5分钟，在室温下的滤液。吸液和悬浮沉淀用25毫升的解决方案＃3步骤5.7。 </li><li>结合步骤5.7和5.9的分数，并在每分钟1600转离心5分钟，在室温下再次。 </li><li>吸媒体和门户网站的成纤维细胞培养基在10毫升（10％胎牛血清，1％青霉素/链霉素，0.3％庆大霉素，0.2％fungizone的DMEM/F12补充）悬浮沉淀。 </li><li>组织培养皿中的生存能力和板计数细胞。根据所需的应用程序，0.5至5×10 6细胞每10厘米的组织培养板是恰当的。从成年SD大鼠，我们通常会获得2至10万个活细胞。 </li><li>更换培养基次日除去非贴壁的碎片。此后，每2天更换培养基。 </li></ol> 6。代表结果从成年大鼠肝脏中分离的主要门户网站的成纤维细胞在1，3和7天隔离后（ 图3）所示。请注意，这些细胞与成纤维细胞形态典型，被拉长。可染色细胞与抗弹性蛋白抗体（CL55041AP，Cedarlane实验室，伯灵顿，NC），以确认纯度。门户成纤维细胞进行培养的肌纤维母细胞分化。这可以证明α-平滑肌肌动蛋白（α-SMA的A2547，西格玛，圣路易斯，密苏里州）的免疫。 <table边境=“1”&gt; <tbody><tr><td rowspan="2"> 0.3％的胶原酶溶液 </td><td>类型2胶原酶</td><td> 150毫克</td></tr><tr><td> HBSS中具有的Ca 2 + /镁2 + </td><td> 500毫升</td></tr><tr><td rowspan="7"> 解决方案＃1（链霉蛋白酶） </td><td>牛血清白蛋白</td><td> 50毫克</td></tr><tr><td>型胶原酶2 </td><td> 25毫克</td></tr><tr><td>蛋白酶</td><td> 18毫克</td></tr><tr><td> DNA酶</td><td> 3毫克</td></tr><tr><td>的DMEM/F12 </td><td> 47.5毫升</td></tr><tr><td>笔/链球菌</td><td> 1毫升</td></tr><tr><td>胎牛血清</td><td> 1.5毫升</td></tr><tr><td> 解决方案＃2 </td><td>牛血清白蛋白</td><td> 50毫克</td></tr><tr><td rowspan="6"> （透明质酸） </td><td>型胶原酶2 </td><td> 25毫克</td></tr><tr><td>透明质酸</td><td> 22毫克</td></tr><tr><td> DNA酶</td><td> 3毫克</td></tr><tr><td>的DMEM/F12 </td><td> 47.5毫升</td></tr><tr><td>笔/链球菌</td><td> 1毫升</td></tr><tr><td>胎牛血清</td><td> 1.5毫升</td></tr><tr><td rowspan="2"> 解决方案＃3 </td><td> DNA酶</td><td> 3毫克</td></tr><tr><td>培养基中，1640 </td><td> 50毫升</td></tr></tbody></table> 表1。酶活性的解决方案。 <img alt="图1" src="/files/ftp_upload/3669/3669fig1.jpg" /> 图1。灌注成立。A。循环水变暖列。二柱含有灌注媒体。 C.旋塞阀控制泵流出。 D.泵流入。 E.变速蠕动泵。 F.与生物清洁洗涤剂保温瓶。 G.散热灯。 H.水洗澡变暖灌注媒体。 <img alt="图2" src="/files/ftp_upload/3669/3669fig2.jpg" /> figu重2。 原位灌注。门静脉插入静脉导管，缝合和固定，通过活塞灌注管相连。 B.玻璃吸管连接到保温瓶和墙吸删除灌注液引流下腔静脉横断。一肠。属肝。 <img alt="图3" src="/files/ftp_upload/3669/3669fig3.jpg" /> 图3。相衬和免疫荧光染色培养的大鼠门户成纤维细胞培养后1，3，7天的隔离。门户成纤维细胞进行了固定和弹性蛋白，α-SMA和DAPI染色。

<ol>
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没有利益冲突的声明。

门户网站的成纤维细胞发挥胆道纤维化的重要作用。在这里，我们描述了一个隔离大鼠肝门成纤维细胞，提供了一个简单的方法来研究这种细胞在体外人口克鲁格洛夫和Dranoff 8发表的原始协议的修改。 这种方法使用蛋白酶消化和规模为基础的过滤。该方法的主要优点是没有获得人口相对纯的细胞，可以通过在文化，使基因表达的研究或几天后隔离功能细胞的行为。这种技术还提供了隔离健康和患病的肝脏细胞的潜力。 在该协议中最关键的步骤之一是肝实质的酶消化。为了确保成功消化，重要的是确认在初始灌注肝脏血液已完全耗尽，锡安作出肯定，甚至有肝热烫。为了促进这一点，可以用棉签轻轻按摩肝脏，而灌注。消化肝实质结果在一个活细胞的产量低，而在消化将使其难以分开胆管树的肝实质。由于蛋白酶的筹备工作有可能需要在不同的地段，优化使用量之间的酶活性的变异时，有活菌产量低。 该协议可能会被修改为门户的成纤维细胞从小鼠肝或年轻大鼠肝脏隔离使用一个较小的计静脉导管，cannulate门静脉灌注液量，减少动物体重的比例，并减少肝脏灌注率约10毫升/分钟。 门户成纤维细胞培养，经过10-14天的肌纤维母细胞的分化，由α-SMA的前pression 9。肝星状细胞，包括fibulin-2，IL-6，弹性，以区别于门户网站的成纤维细胞已被用于各种标记核苷三磷酸diphosphohydrolase-2（NTPDase2），cofilin的-1和神经突触2，6，10，11，如蛋白质。我们发现弹性是一个门户网站的成纤维细胞的标记，甚至肌纤维母细胞分化后，，而NTPDase2丢失门户成纤维细胞后，经历了9几天后，文化。因此，我们通常确认为弹性蛋白免疫荧光染色门成纤维细胞的分离。 这项技术的限制后，立即门户成纤维细胞的分离，是有污染的细胞，包括Kupffer细胞和胆管细胞的一小部分。门户长大成纤维细胞会在2-3天内这些污染的细胞，然而，产生一个相对纯净的人口（98％）9。 硅，NCE文化门户的成纤维细胞进行组织培养塑料肌纤维母细胞分化，我们通常在7天的隔离研究这些细胞。细胞维持在含10％胎牛血清的媒体门户的成纤维细胞培养，在方法部分所述。然而，这些细胞就会生存含2％胎牛血清几天媒体的增长。我们通常不使用低血清培养基，直到隔离后至少24小时。一旦门户成纤维细胞进行肌纤维母细胞的分化，他们可以传代数次和保持肌纤维母细胞冷冻库存。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>类型2胶原酶</td><td>沃辛顿生化（雷克伍德，新泽西州） </td><td> LS004177 </td></tr><tr><td>蛋白酶</td><td>罗氏（印第安纳波利斯，IN） </td><td> 70290120 </td></tr><tr><td>透明质酸</td><td>西格玛（圣路易斯，密苏里州） </td><td> H3506 </td></tr><tr><td>脱氧核糖核酸</td><td>西格玛</td><td> D4527 </td></tr><tr><td>的DMEM/F12 </td><td> Invitrogen公司（卡尔斯巴德，加利福尼亚州） </td><td> 11320 </td></tr><tr><td> HBSS中没有的Ca 2 + /镁2 + </td><td>敏达（马纳萨斯，弗吉尼亚州） </td><td> 21-021-CM </td></tr><tr><td> HBSS中具有的Ca 2 + /镁2 + </td><td>敏达</td><td> 21-020-CM </td></tr><tr><td>莱博维茨的L-15 </td><td> Invitrogen公司</td><td> 11415 </td></tr><tr><td>培养基中，1640 </td><td> Invitrogen公司</td><td> 11875 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A2153 </td></tr><tr><td>胎牛血清</td><td>双子座生物（西萨克拉门托，加利福尼亚） </td><td> 900-108 </td></tr><tr><td> nitex尼龙网30微米</td><td> ，杰纳西科技（圣迭戈，CA） </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

肝纤维化是指由活化的肌成纤维细胞外基质过度沉积。有多种肝肌纤维母细胞的前体，包括肝星状细胞，门成纤维细胞和骨髓成纤维细胞1。肝星状细胞已经最好的研究，但门户网站成纤维细胞的肌纤维母细胞池的重要因素，特别是在2胆管纤维化，人们日益认识到。胆管上皮损伤门户成纤维细胞进行增殖，可能发挥在胆道疤痕3-5的早期阶段的关键作用。门户的成纤维细胞的分离方法，将允许在这个细胞群的体外研究，并导致更大的门户成纤维细胞在胆道纤维化中发挥的作用的认识。 门户网站成纤维细胞已被隔离，使用各种技术，包括生长6，7和李VER灌流酶消化大小选择8。相比生长技术的消化和大小选择技术的优点是，可以不通过文化的必要性研究细胞。在这里，我们描述了原有技术分离大鼠肝门成纤维细胞，导致在一个相对纯净的原代细胞的人口由克鲁格洛夫和Dranoff 8所述的修改后的版本。

Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

一个从大鼠肝门成纤维细胞的分离技术。肝脏被灌注和消化<em&gt;原位</em&gt;胶原酶，其次是<em&gt;体外</em&gt;消化肝细胞浆和大小选择。这种方法提供了一个门户网站的成纤维细胞的纯人口，而不需要通过文化。

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 ...

isolation-of-rat-portal-fibroblasts-by-in-situ-liver-perfusion

Research

JoVE Journal

Biology

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K 次观看.  The Children's Hospital of Philadelphia. 一个从大鼠肝门成纤维细胞的分离技术。肝脏被灌注和消化原位胶原酶，其次是体外消化肝细胞浆和大小选择。这种方法提供了一个门户网站的成纤维细胞的纯人口，而不需要通过文化。

视频: Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

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 ...

科研

生物学

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

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. ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

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 ...