Name: isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens
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This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

该协议获得从手术患者丢弃的人体组织，并建立原代培养的是经德克萨斯大学医学科和新墨西哥州机构审查委员会的大学大学。用于人体组织标本采购的一般要求如下所述。注意，由于隶属函数可以从冰冻组织，生物银行和商业供应商分离出来的也是可行的办法。 1.获取人体组织<ol><li>提交协议来获得人结肠组织的机构审查委员会批准。 </li><li>建立合作与普通外科医生和/或结直肠外科为了获得人体组织进行研究。与病理科协作也是强制性的，因为它们将被判定为不需要进行诊断和可被指定为研究的组织。 </li><li>奉劝病理学家从肿瘤提供组织，然后grosslŸ正常粘膜至少5厘米的肿瘤。立即放置在冰冷的洗涤介质的样品，并放置在冰上。病理学家将确定可以用于研究被安全地给定组织的量。需要MF隔离最小的组织为1.5 平方毫米。 </li><li>使用获得的组织或者立即建立原代培养物或冷冻并置于在-80℃下以供将来隔离。 <ol><li>如果冷冻购买隔离，切割组织成约1 - 2毫米2件 ，放入低温管与1ml冷冻介质（见下文），并储存在-80℃。注:利用此协议，隶属函数的自组织冻结了超过4年成功分离已被观察到。 </li></ol></li></ol> 2.准备试剂<ol><li>胶原酶的解决方案:准备100 U / ml的胶原酶I，II和IV在汉克的平衡盐溶液（HBSS）与钙 / 镁离子（ 见材料表 </strong&gt;的信息）。小心:胶原酶活性单位浓度和纯度可能变化从厂商而，这可能会妨碍组织消化在粘膜细胞表面表位的和/或保存的效率。为了防止这些潜在的缺陷，使用胶原酶具有最高纯度（≥95％）的，并准备基于活性单位浓度的储备溶液。 </li><li>制备水性DNA酶原液以10毫克/毫升（3.55 UA /毫克）。 </li><li>准备清洗介质:（MEM 89毫升，抗生素 - 抗真菌剂，100×1毫升，热失活的FCS 10毫升） </li><li>准备成纤维细胞生长培养基:MEM:一瓶500毫升; L-谷氨酰胺（200 mM计）:5毫升， MEM非必需氨基酸，100倍的溶液:5毫升，环丙沙星盐酸（10毫克/毫升）:570微升;抗生素 - 抗真菌剂，100倍溶液10毫升，丙酮酸钠（100毫摩尔）:5毫升，热灭活的FCS 50 ml的</li><li>制备冻结介质:成纤维细胞生长培养基（试剂2.4）用加入了10％的二甲亚砜（DMSO）中，然后过滤，韦伯斯特ilize用0.22微米的过滤器。 </li></ol> 3.酶促文摘人体组织<ol><li>如果组织被冻结在冷冻媒体，地方离心管中的温暖（〜37℃）的水，直到组织和媒体解冻。 </li><li>为4 - 5 2 2毫米组织片，两次用10ml的HBSS洗组织不含Ca 2+ / Mg 2 +的。 </li><li>一旦组织已落户在重力作用，精心弃上清没有旋转样品。 </li><li>加入10 mL胶原酶溶液的样品，并在组织的溶液混合物与转移到无菌的，DNA酶/无RNase管内置转子样品解离。用胶原酶以解离细胞外基质包围的上皮细胞的基础方面，形成在粘膜固有层中的非细胞部分。 </li><li>紧紧贴着管颠倒其安装到离解器（ 例如 ，GentleMACs）的袖子。如果实验室不具有接入到t他上述的设备，增加每个消化步骤的时间，以获得比较的结果。 </li><li>运行程序h_tumor_01，预先设定的信息包括与机器（36秒的总的持续时间，与间歇脉冲范围从1000 - 4000转，以268转每运行）。 </li><li>终止程序结束后，取下管从离解。 </li><li>在37℃下连续旋转孵育样品45分钟在摇床上，在140转。注意:对于不同的组织样本的量，消化的时间可以按比例减少或增加（ 即 ，更大的组织的量可能需要额外的时间）。 </li><li>连接管倒到离解的袖子。 </li><li>选择并运行程序h_tumor_02（37秒的总时间，间歇性脉冲，从1000 - 4000转，235发每运行）。 </li><li>终止程序结束后，取下管从离解。 </li><li>加入50微升DNA酶原液。使用DNA酶以解离/去除死细胞和细胞碎片的DNA的级分导致细胞结块。 </li><li>在37℃下在摇床上连续旋转孵育样品30分钟以60rpm。 </li><li>再次将试管倒置到离解的袖子。 </li><li>选择并运行程序h_tumor_03（37秒的总时间，间歇性脉冲，从1000 - 4000转，168发每运行）。 注意:如果组织未完全消化，离心机在250×g的10分钟，在20℃，弃上清，重悬在2毫升细胞解​​离溶液和在室温下孵育10分钟。 </li><li>通过无菌70微米的细胞过滤的细胞悬浮液。 </li><li>离心细胞悬浮液在250×g的10分钟，在20℃。吸去上清液彻底。 </li><li>洗涤细胞沉淀两次用25ml的HBSS不含Ca 2+ / Mg 2 +的并弃去上清液。 </li><li>前的最后被小时，使用一个自动细胞计数系统，可以在实验室或手动使用血细胞计数器计数细胞数。对于MF原代培养一代继续开行步3.19.1，进行分析或隶属函数的使用流式细胞仪进行步3.19.2排序。 <ol><li>分离和纯的MF培养的生长 （图1）中，丢弃从最后离心，重悬细胞沉淀上清液中的成纤维细胞分离培养基的种子高达4×10 6细胞所需要的适当的量在3ml的培养基，每孔中6孔，细胞培养处理板。继续执行步骤3.20。 </li><li>用于分析或微丝的使用流式细胞术，最多放置2×10 6在2 ml成纤维细胞分离培养基的24孔，低的结合板孵育O / N在37℃，5％CO 2的分选。这是恢复可能受影响的酶的过程中的细胞表面表位。然后收集细胞悬液，计数回收的细胞，并继续用于免疫染色和流式细胞仪分析8。 </li></ol></li><li>生长的细胞在37℃，5％的CO 2。更换介质每2 - 3天，直到形成80％汇合MF单层〜（ 图1D-E）。 </li><li>代细胞在T25烧瓶与来自6孔板中以比1:1和生长〜10 - 在成纤维细胞生长培养基中7-14天实现80 - 100％汇合。 </li><li> 2:从T25传代细胞T75细胞比1扩大文化。一旦细胞达到汇合，利用流式细胞仪如前5描述用一个锥形瓶孤立MF纯度的分析。 3:在比1冻结或通过MF文化的另一种T75烧瓶中。 注意:当通过流式细胞术分析，因此预 ​​计间充质来源的胃肠粘膜基质MF当在培养生长将具有以下表现型:EpCAM-，CD31，CD45，波形蛋白+α-SMA +，CD90 +。 </li></ol>

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The authors have nothing to disclose.

虽然该协议是为研究中的应用开发只，在成长为一个潜在的癌症预后标志物和治疗靶点基质细胞至关重要的光，冻结"功能性"生物标本以备后用的能力提供了一个显著优势。这是一个独特的优势，为创作临床biorepository的，可作为额外的工具服务促进个性化医疗10,11的发展，类似于先前针对脂肪间充质干细胞从冷冻的基质血管级分中分离报告的结果，我们进行了未观察到在标记和增殖/免疫/?...

肌成纤维细胞/成纤维细胞（MFS）表示细胞在胃肠道黏膜丰富的人口。这些基质细胞就位于下方的上皮形成和在肠道内的粘膜固有层的互连网络。微丝不仅负责细胞外基质的沉积，但通过旁分泌调节可影响电解质转运，恢复原状的，屏障功能相邻上皮1,2。此外，隶属函数已被证明在炎症和组织中发挥关键作用重塑3,4。肌成纤维细胞是在肿瘤微环境，在那里他们也被称为癌相关成纤维细胞的一个关键部分，并有助于肿瘤细胞的生长，并作为一个利基癌症干细胞3。新出现的数据表明，微丝也可用作本地抗原呈递细胞。此外，隶属函数功能的固有免疫和适应性免疫反应的重要调节器，机生产线CING多种细胞因子和生长因子5。 在健康个体中，微丝细胞被认为从间充质干细胞分化和表达在其细胞表面上，所述间充质标记物，CD90 3,5，这些细胞也表达vimentin，但阴性上皮细胞和造血细胞标记物的EpCAM和CD45分别。肌成纤维细胞被建议是成纤维细胞的活化形式，并且可以从非活化的成纤维细胞用α-SMA 5的表达来区分。 在过去十年中，多种方法用于从人结肠黏膜肌成纤维细胞的分离已公布-大多是基于最初由Mahida 等人 5-8中记载的方法。虽然个别的研究报道来自各种肠粘膜，对于隶属函数的从胃肠道的多个区域（即，胃，小和升隔离没有通用协议分离程序阿尔赫肠粘膜）已经出版。本文提出的方案已经过测试，并成功地用于所有三个类型的组织上面提到的。 。此外，还没有被报道用于从冷冻胃肠粘膜分离的MF流程。 这里，我们提出一种优化方法，它是基于酶消化，并且同时允许对人体肠道粘膜的MF的培养分离和流式细胞术分析在新鲜消化，单细胞，粘膜制剂。这种技术可靠地得到原代培养与MF的表型。此外，同样的方法可以用来从胃和小肠的组织冷冻样品隔离微丝为好。从新鲜的GI组织成肌纤维细胞的分离先前已描述;然而，冷冻样品的利用带来了许多好处。即，研究人员将能够收集和存储组织免受任何数目在全世界合作者谁具有capabi的lity运送冷冻组织样本。此外，研究人员可能会发现从手术室和/或内窥镜套件和立即丢弃组织的处理的组织为隔离与他们当前的实验时间表冲突的集合。此外，由于手术的不可预测性，可能会出现组织采购非常晚了一天，这将限制左为加工组织的时间。冷冻组织进行后续处理会减轻这些挑战。 最后，这些方法已被成功地用于在结肠，胃和小肠肌纤维母细胞在疾病状态如大肠癌和炎症性肠疾病的隔离。

<table><tbody><tr><td>MEM, 1x</td><td>Corning</td><td>MT-10-010-CV</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100x</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>Corning</td><td>61-277-RF</td><td></td></tr><tr><td>L-Glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DL-Dithiothreitol solution (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Cellgro</td><td>46-034-CI</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002139</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type I</td><td>Sigma-Aldrich</td><td>C1639</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type II</td><td>Sigma-Aldrich</td><td>C1764</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Accumax cell dissociation solution</td><td>Sigma-Aldrich</td><td>A7089</td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Cell Strainer (70 &amp;micro;m)</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>PE Mouse Anti-Human Vimentin</td><td>BD Biosciences</td><td>562337</td><td>Clone RV202</td></tr><tr><td>FITC Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma-Aldrich</td><td>F3777</td><td>Clone 1A4</td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Biosciences</td><td>559869</td><td>Clone 5E10</td></tr></tbody></table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

成纤维细胞/肌成纤维细胞（MFS）已获得越来越多的关注他们在发病中的作用及其对肿瘤微环境的两个伤口愈合和推广贡献。虽然目前有很多技术的MF的胃肠道（GI）组织中分离，该协议引入了一种新的从冰冻组织这些间质细胞的分离的元素。冷冻胃肠组织标本不仅允许研究者获得来自世界各地的合作者，生物库，和商业供应商的样品，它也允许新鲜样品的延迟处理。所描述的协议将始终如一地产生特征梭形细胞表达标记CD90，α-SMA和波形对MF型。因为这些细胞来自患者样品衍生，使用原代细胞也赋予密切模仿隶属函数的受益于疾病状态，即癌和炎症性肠疾病。这种技术具有蜂ñ证实胃，小肠和结肠MF原代培养一代。主培养物的MF可以使用在实验繁多在若干通路和它们的纯度评估由两个免疫细胞化学和流式细胞术分析。

使用此酶消化协议中，我们一直能够隔离并从胃肠手术标本和活检（图1）生长的MF人口肠道CD90 +粘膜基质细胞。 5播种单个核细胞悬液到6孔板（ 图1A-B）之后-可见MF菌落数量激增可能在第2天进行观察。对MF原代培养达到〜50 - 70％汇合由7天- 11（图1C-D）。 <img alt="图1" src...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

isolation-cd-90-fibroblastmyofibroblasts-from-human-frozen

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  University of Texas Medical Branch. Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production. 

视频: Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遗传学

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

医学

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

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 ...

生物学

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

人瘢痕疙瘩组织中原代真皮成纤维细胞的分离、培养和表征

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

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 ...