这项工作是由卫生部资助5KO8DK085136-02 JY国家机构的支持。

1。获得结肠组织鼠标的协议进行动物实验，详细介绍了基本原理和研究的目的，已提交并经我们的机构动物研究监督处。 <ol><li>安乐死鼠标与吸入异氟醚后颈椎脱位。 </li><li>使下腹正中切口，收回小肠路程，确定结肠。收回膀胱和子宫（如果存在的话）横向和识别耻骨，切细剪刀，露出直肠。 </li><li>解剖直肠背侧肠系膜断结肠，动员结肠从肛门至盲肠，并横切到断开小肠结肠。 </li><li>将样品在冰冷的磷酸盐缓冲盐水（PBS）。 </li><li>填有10毫升注射器用冰冷的PBS冲洗和结肠腔数次以清除其内容，再切结肠氧化聚乙烯Ñ​​长度方向用剪刀。 </li><li>将结肠在50ml锥形管中加入20ml冰冷的PBS。结肠并没有被放置在任何特定的方位。通过更换PBS洗结肠直至油液清晰，没有颗粒物。 </li></ol>人的一个协议获得人体组织从外科病人，详细介绍了获得该组织以及潜在的风险和收益进行评估的重要性，已提交并经本机构的人类研究保护计划办公室。 <ol><li>与结直肠外科医生的研究合作是必要的，以便能够获得人体组织对你的研究。一个协议，以获得人类结肠组织应提交并批准您的机构审查委员会。 </li><li>书面知情同意从患者得到的外科手术之前。 </li><li>外科医生将在展台进行结肠切除术ARD时尚。一旦结肠已经从患者体内取出，将试样立即送往病理。中的不需要的病理评价结肠壁全层的截面A 0.5提供，并立即放置在冰冷的PBS。这个标本不应包括结肠肠系膜和网膜附属物应删除为好，因为脂肪的存在会影响消化过程。 </li></ol> 2。剥夺上皮细胞<ol><li>孵育整个小鼠结肠中在50ml锥形管中加入25ml 5mM EDTA的HBSS中（室温）。将锥形管在37℃下振荡空气浴（250 rpm）离心15分钟。 15分钟后，小心地倒出液体并重新填充该管，用25ml的5毫摩尔EDTA，重复，共5次洗涤。对于人类结肠癌，获得的结肠壁一个半英寸带从病理学家后，用剪刀剪结肠分成四个相等大小的块。放置两片到一个50ml锥形管中，并将其余两块结肠组织到一个独立的50ml锥形管中。然后孵化用25ml 5mM EDTA的HBSS中，并执行清洗，如上所述。 </li><li>在20ml的冰冷的PBS漂洗两次结肠。 </li><li>将小鼠结肠组织中一个新的50毫升锥形管含有20ml的RPMI-5，10单位分散酶，及2000ü胶原酶的D（室温）。对于人类结肠组织，用中性蛋白酶和胶原酶的两倍（20 U分散酶，4000ü胶原酶D）。 </li><li>将管在37℃下振荡空气浴垂直取向（如果水平放置，有太多的曝气和细胞的损伤）。摇在250转长达60分钟。暴露在分散酶和胶原酶后D，结肠组织将开始消化和结肠组织将开始寻找粘性。这通常需要大约30分钟。当结肠有这个样子，从酶去除结肠。 </li><li>摇各管上下2-4次，打破了组织。 P埃利特组织在200×g离心在台式离心机（4℃）5分钟。小心倒出上清液并丢弃。 </li></ol> 3。肌成纤维细胞的分离与培养<ol><li>通过加入10ml的ACK裂解缓冲液（47℃）重悬每个粒料。再次离心200×g离心5分钟（4℃），倒出并丢弃上清液。 </li><li>通过加入10ml的RPMI-5用移液管重悬浮每个粒料。 </li><li>通过用10毫升吸管成100毫米TC-处理的培养皿一个70微米的筛网过滤器传递一半的细胞悬浮液（5毫升）。然后添加一个额外的15毫升RPMI-5。用其余的5 ml细胞悬液重复插入不同百毫米TC-处理的培养皿中。因此，人们小鼠结肠将产生两根100 mm TC处理的菜肴。人结肠癌组织中的一个半英寸长条将产生共4 100毫米TC-处理的培养皿。 </li><li>放置在组织培养皿中在10％CO 2培养箱中在37℃下培养条件是相同的f或小鼠和人原代细胞。 </li><li> 3小时后，轻轻洗掉非贴壁细胞用HBSS的两种变化。碎片漂浮应该轻轻洗掉。贴壁细胞组成的上皮细胞，巨噬细胞和成肌纤维细胞。一周后播种，只有隔膜肌成纤维细胞，巨噬细胞和上皮细胞中的第一通道后衰老。加入新鲜RPMI-5和成长的细胞在细胞培养。 </li><li>细胞通过胰蛋白酶消化传代5分钟，被分割为2:1。 </li></ol>缩略语 HBSS：汉克平衡盐溶液，RPMI-5：罗斯韦尔公园纪念研究所媒体，ACK​​：氯化铵，钾，FCS：胎牛血清，TC处理：组织培养处理解决方案 ACK裂解缓冲液- （4.15克氯化铵 0.5克KHCO 3，18.6毫克的Na 2 EDTA，加入400ml H 2 O，调节pH值至7.2〜7.4，用1N HCl）。过滤器sterilize到0.2微米的过滤器，并储存于4℃ 的RPMI-5 - （使500毫升：454.5毫升RPMI，将25ml的FCS，加入5ml 200mM的L-谷氨酰胺，将5ml 1M的HEPES，pH为7.4，加入5ml 100 1mM丙酮酸钠，5毫升100X青霉素 - 链霉素，加入500ml的50mM乙-ME的PBS中）。

<ol>
	<li>Armaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cell+targeting+by+TNF+as+a+common+pathogenic+principle+in+chronic+inflammatory+joint+and+intestinal+diseases.">Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases.</a> J. Exp. Med. 205, 331-337 (2008).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> Am. J. Physiol. 277, C1-C9 (1999).</li><li>Yoo, J., Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+mediates+synergistic+MMP-3+expression+induced+by+TNF-alpha+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-alpha and bradykinin in human colonic myofibroblasts.</a> Biochem. Biophys. Res. Commun. 413, 30-35 (2011).</li><li>Yoo, J., Chung, C., Slice, L., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D+mediates+synergistic+expression+of+COX-2+induced+by+TNF-{alpha}+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts.</a> Am. J. Physiol. Cell Physiol. 297, C1576-C1587 (2009).</li><li>Valentich, J. D., Popov, V., Saada, J. I., Powell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+an+intestinal+subepithelial+myofibroblast+cell+line.">Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.</a> Am. J. Physiol. 272, C1513-C1524 (1997).</li><li>Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+potentiates+lysophosphatidic+acid-induced+COX-2+expression+via+PKD+in+human+colonic+myofibroblasts.">TNF-alpha potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300, G637-G646 (2011).</li><li>Yoo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+induces+upregulation+of+EGFR+expression+and+signaling+in+human+colonic+myofibroblasts.">TNF-alpha induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 302, G805-G814 (2012).</li><li>Mahida, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am. J. Physiol. 273, G1341-G1348 (1997).</li><li>De Wever, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+and+potentiation+of+DNA+damage+response+by+fibronectin+in+human+colon+cancer+cells+and+tumor-derived+myofibroblasts.">Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts.</a> Int. J. Oncol. 39, 393-400 (2011).</li><li>Edwards, R. A., Smock, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+arachidonate+release+and+PGE2+production+in+Gi+alpha2-deficient+intestinal+and+colonic+subepithelial+myofibroblasts.">Defective arachidonate release and PGE2 production in Gi alpha2-deficient intestinal and colonic subepithelial myofibroblasts.</a> Inflamm. Bowel Dis. 12, 153-165 (2006).</li><li>Hoang, B., Trinh, A., Birnbaumer, L., Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+MAPK-+and+PGE2-dependent+IL-11+production+in+Gialpha2-/-+colonic+myofibroblasts.">Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1511-G1519 (2007).</li><li>Li, Z. B., Kollias, H. D., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+directly+regulates+skeletal+muscle+fibrosis.">Myostatin directly regulates skeletal muscle fibrosis.</a> J. Biol. Chem. 283, 19371-19378 (2008).</li><li>Saada, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subepithelial+myofibroblasts+are+novel+nonprofessional+APCs+in+the+human+colonic+mucosa.">Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.</a> J. Immunol. 177, 5968-5979 (2006).</li><li>Lahar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+subepithelial+myofibroblasts+support+in+vitro+and+in+vivo+growth+of+human+small+intestinal+epithelium.">Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.</a> PLoS One. 6, e26898 (2011).</li></ol>

作者什么都没有透露。

无论是使用鼠标或结肠癌时，通用的技术是相似的。这种技术也可以用于从小肠分离成肌纤维细胞。具体细节，肌纤维母细胞从1隔离。小鼠和2。人结肠癌在下面讨论。 1。小鼠结肠癌小鼠结肠灌溉是通过附加一个4，为16G波普尔钝头针头至结肠的一端提供便利。这也有助于纵向切割结肠时。您可以手风琴结肠组织到针，然后将剪一个尖与尖底起来，滑过剪?...

胃肠道（GI）道功能的调节涉及粘膜层与下伏间质之间复杂的，动态的相互作用。这些相互作用通常用来维持内环境稳定，但也可能导致疾病状态的发展病理条件下，1。肌成纤维细胞是位于下方的所在，以旁分泌方式进行通信及邻近细胞亚群调节若干关键胃肠道工序，包括粘膜再生，修复和纤维化2的上皮细胞层基质细胞亚群。 的18 CO细胞株是已被广泛用于研究肌成纤维细胞功能的人结肠肌成纤维细胞系的一个例子，因为它有许多共同的初级原位成肌纤维细胞的特性。这些包括的α-平滑肌肌动蛋白的蛋白表达（α-SMA）和波形蛋白，以及一个数的表达式细胞表面受体如表皮生长因子受体，或受体的溶血磷脂酸3,4。因为这些共享超微结构特征，以及在生物功能5的相似性，则18 CO细胞系已被广泛用于研究肌成纤维细胞的功能在许多疾病状态如炎性肠疾病或结肠直肠癌3,6的范围内。但是，也有使用的细胞系时固有的局限性和关注。这些包括基因型不稳定随着时间的推移区别于原代细胞的细胞系，从而改变细胞的表型和生物学功能，包括生长速率，并与其他细胞群的相互作用。细胞系也缺乏GI微环境（上皮，基质和血管成分），这是一个主要的限制其使用的正常组成部分。因此，从实验细胞株的研究得出的结论需要进一步的验证，以降低RISK误解。 初级肌成纤维细胞可以从人类和小鼠的结肠组织得到证实在细胞系7识别的实验结果。该方法最初是由马希达， 等。8描述的，和我们的协议是可以被用来从小鼠和人结肠9隔离初级肌成纤维细胞的许多良好描述的技术之一。对于结肠疾病的任何小鼠模型，这种技术可以用来隔离初级肌成纤维细胞，研究它们与邻近的细胞是如何相互作用或促使正常或病理性的GI处理10,11。这种技术可以用来研究肌纤维母细胞如何促进黏膜愈合，纤维化和结直肠腺瘤和癌的发展。初级肌成纤维细胞还可以从已经在切除操作的时间为良性（炎性肠病，狭窄，憩室炎）或恶性Ç人结肠组织获得onditions。来自人类和小鼠的结肠组织分离的原代细胞可以生长在细胞培养物和利用多通道的数量有限。

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

的肌成纤维细胞是胃肠（GI）道，已获得相当大的关注在许多胃肠功能的关键作用的基质细胞。虽然一些肌成纤维细胞的细胞系是可商购的，研究这些细胞在体外 ，离暴露实验的细胞培养条件的细胞系的研究成果具有因工作过于简单化的性质固有的局限性。使用初级肌纤维母细胞提供了在确认细胞系鉴定实验结果方面有很大的优势。从动物模型隔离初级肌纤维母细胞可为肌成纤维细胞的研究更紧密地模仿所研究的疾病状态条件下。从人结肠组织主要肌纤维母细胞的分离提供了可以说是最相关的实验数据，由于细胞直接来自患者的基础疾病。我们描述了一个行之有效的技术，可以为utilized从小鼠和人的结肠组织隔离初级肌成纤维细胞。这些分离的细胞已被鉴定为α-平滑肌肌动蛋白和波形蛋白阳性的，和结蛋白阴性的，与皮下肠肌成纤维细胞是一致的。初级肌成纤维细胞的细胞可以生长在细胞培养物和用作实验过通道的数量有限。

一旦分离，原代人成肌纤维细胞可以生长在细胞培养物和使用过的通道的一个有限数量（最多通道4）。这些细胞的特征在α- ​​平滑肌肌动蛋白和波形蛋白阳性，结蛋白阴性的（ 图1A），与肠上皮下成肌纤维细胞5,7一致。他们也有一个特点星状形态（ 图1B）。初级成肌纤维细胞随后可以用来确认在细胞系中发现的实验结果。这种方法被用来证明促炎细胞因子TNF-?...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

肌成纤维细胞是胃肠道，调节重要生理过程的正常和疾病状态的一个有影响力的基质细胞。我们描述了一种技术，它允许主肌纤维母细胞从小鼠和人结肠组织，它可以用于在体外实验的隔离。

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 ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Research

JoVE Journal

Biology

19.4K 次观看.  UCLA. 肌成纤维细胞是胃肠道，调节重要生理过程的正常和疾病状态的一个有影响力的基质细胞。我们描述了一种技术，它允许主肌纤维母细胞从小鼠和人结肠组织，它可以用于在体外实验的隔离。 

视频: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

科研

生物学

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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 and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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 ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...