我们感谢慷慨支持和监督这项工作，并为这篇稿子的关键编辑基兰Mellody先生（英国曼彻斯特大学，曼彻斯特）罗伯特A ·温伯格博士（怀特黑德生物医学研究，剑桥研究所）。这个项目得到了英国研究（CR英国）授予数量C147/A6058（AO）

1。原代培养人正常乳腺成纤维细胞的分离隔离原代培养人正常乳腺成纤维细胞的实验程序概述图。 1A。 <ol><li>准备的细胞分解缓冲区，作为描述以前12：10％胎儿小牛血清（FCS），青霉素，链霉素（200单位/毫升），胶原酶型我（1毫克/毫升）和透明质酸（贝科的修改鹰的中等（DMEM） 125单位/ ml）。 </li><li>多次在磷酸盐缓冲液（PBS）从减少隆乳（〜0.5克）洗净乳房组织解剖。切碎成小的片段，用无菌刀片（&lt;1.5毫米3）组织。 </li><li>转移到15 ml锥形管含有适量的上述细胞解离缓冲液（10毫升每0.5克组织）和最高速度为1分钟的旋涡中的组织碎片。 </li><li>在细胞分解缓冲f消化的组织碎片或12-18 h，在37 ° C的缓慢搅拌。*注仍然会有许多未消化的组织碎片留在管件。 </li><li>没有搅拌，在室温下5分钟孵育管。产生的基质细胞丰富上清转移到一个新的锥形管，使用5毫升的血清学吸管。 </li><li>离心5分钟，在250 X克的基质部分，并在PBS重悬细胞沉淀。再次离心5分钟，在250 X克，并在DMEM培养液重悬细胞沉淀，辅以10％FCS。文化的DMEM含10％FCS的一个15厘米的培养皿中，在37 ° C和5％的二氧化碳的细胞。 </li><li>宣传，直到融合细胞。它通常需要8-10天。商店细胞在-80 ° C使用冻结的介质（10％二甲基亚砜和20％FCS的DMEM）。准备作为原始股票的5个冷冻瓶。解冻一小瓶和扩大二级股票含有成纤维细胞的细胞准备5瓶5人口内传倍增（PDS）的后续实验。这些程序最小化克隆选择和文化压力，可能会发生在延长的组织文化。*注意，P1.1 - 7也可以用于隔离乳腺癌乳房切除术 12获得CAFS 。 </li></ol> 2。 GFP标记，嘌呤霉素抗性，永生化人正常乳腺成纤维细胞的生成<ol><li> immortalise在P1.7的主人体正常乳腺孤立的成纤维细胞），介绍一个12逆转录病毒pMIG（MSCV - IRES - GFP）载体，表达hTERT和GFP。培养细胞为4-5天，然后进行排序GFP阳性细胞用流式细胞仪。 </li><li>介绍了一种逆转录病毒pBabe PURO构建嘌呤霉素抗性基因编码成一个GFP标记的不朽的成纤维细胞。文化嘌呤存在5-7天（终浓度为：1微克/毫升）的细胞分离GFP阳性（GFP +），嘌呤霉素抗性（PURO ř </suP&gt;）永生化人乳腺成纤维细胞。 </li><li>下一步，研究这些成纤维细胞是否会产生积极的病毒，从而导致编码GFP和嘌呤霉素抗性基因的水平转移，增长2.5 × 10 5 GFP + PURO ř永生人乳腺成纤维细胞在6厘米2天的Petri菜。 </li><li>过滤介质的空调这些使用注射器过滤器（孔径：0.45微米），成纤维细胞，并添加到10T1 / 2鼠标为12H的成纤维细胞中存在的硫酸鱼精蛋白（5微克/毫升），增加病毒感染的效率。然后介质是额外的2天改为10％FCS - DMEM。 </li><li>由荧光显微镜检查细胞。 10T1 / 2细胞GFP的病毒感染，将成为GFP阳性。治疗5-7天细胞嘌呤（1微克/毫升），如果任何PURO ř 10T1 / 2细胞嘌呤霉素治疗后目前形成菌落，观察板。 </li><li>请注意，基因转录水平sfer由父母GFP + PURO ř人乳腺成纤维细胞所产生的积极的病毒可能使周围的鼠细胞和/或在肿瘤异种移植癌的细胞绿色荧光蛋白+ PURO ř。这可能会妨碍最初注入的GFP + PURO ř人类肿瘤异种移植乳腺成纤维细胞的分离过程。 </li></ol> 3A。与乳腺癌细胞的人类乳腺成纤维细胞共注射到免疫缺陷小鼠实验程序生成EXP - CAFS图所示。 1BA。 <ol><li>混合1 × 10 6 MCF - 7 - ras基因人类乳腺癌细胞和3x10 6 GFP + PURO ř，永生化人在P2.2产生的乳腺成纤维细胞） 。每次注射基质胶50％（V / V），准备在400μL培养基的细胞。 </li><li>免疫缺陷的裸鼠皮下注入细胞的混合物。当咖啡馆是从不同的独立提取肿瘤，植入到每个几个小鼠左，右两翼混合物。 </li></ol> 3B。人类乳腺成纤维细胞注射到免疫缺陷小鼠<ol><li>要生成控制对EXP - CAFS成纤维细胞，准备3x10 6 GFP + PURO ř人在P2.2与50％（V / V）（图1BB）基质胶每次注射400μL培养基产生的乳腺成纤维细胞。不要混合成纤维细胞与癌细胞。 </li><li>植入到裸鼠皮下网站P3b.1准备的成纤维细胞。 </li></ol> *注意，接种成纤维细胞形成皮肤的成纤维细胞组织，但不是肿瘤。 4A。肿瘤异种移植的剖析<ol><li>人道毁灭鼠标窝藏皮下人乳腺癌异种移植后植入（图1BA）42天。*注意，当其他肿瘤细胞和/或成纤维细胞是coimplanted，成纤维细胞可能需要培养在肿瘤异种移植的时间超过42天，以启动他们的肌纤维母细胞表型。 </li><li>收获从侧翼鼠标使用镊子和剪刀钝清扫肿瘤异种移植。沉浸在PBS的肿瘤组织。 </li></ol> 4B。成纤维细胞异种移植的剖析<ol><li>窝藏的成纤维细胞异种移植物植入后42天（图1BB）人道毁灭的鼠标。 </li><li>使用镊子和剪刀，解剖皮下成纤维细胞异种移植。 *请注意，成纤维细胞异种移植，作为一个微小的透明组织出现，可能是很难找到的皮肤下。放入PBS成纤维细胞组织。 </li></ol> 5。从移植瘤的原代培养细胞的制备<ol><li>使用无菌的生物安全柜，解剖的肿瘤（〜0.5克），或成纤维细胞组织（〜0.1克）。切除的组织转移到15厘米的文化ð顶部ISH与1毫升的PBS。切碎成小组织的片段，用无菌刀片（&lt;1.5毫米3）组织。 *请注意，如果成纤维细胞移植瘤的重量小于0.1克，收集了一些额外的成纤维细胞移植瘤和混合在一起，增加细胞隔离的数量。 </li><li>转移到15 ml锥形细胞解离最高速度为1分钟的缓冲液（10毫升每0.5克组织）和涡管的组织碎片。 </li><li> 3小时的细胞悬液，在37 ° C的持续缓慢搅拌。 </li></ol> 6。文化嘌呤霉素抗性细胞的分离<ol><li>的细胞悬液，离心分离，无论是从5分钟，在250 X克的肿瘤或纤维母细胞异种移植，并在PBS悬浮细胞沉淀。再次离心5分钟，在250 × G.造成细胞沉淀重悬在10％FCS - DMEM。文化中存在的嘌呤（1微克/毫升）在一个15厘米的培养皿中的细胞在37 ° C二氧化碳和5％，以消除任何污染的肿瘤细胞和/或小鼠基质细胞。传播，直到细胞融合（2-4周）。商店细胞在-80 ° C使用冻结的介质。 *请注意，由此产生的PURO R细胞，指定42日龄EXP - CAF1（图1BA）或控制成纤维细胞（图1BB），是不朽的GFP阳性。这是建议，以检查是否这些细胞产生活跃的病毒，使用10T1 / 2细胞，在P2.3 - 6所述。 </li></ol> 7A。文化EXP氟化钙细胞的分离为了进一步提高水产科学研究院激活肌纤维母细胞表型，混合，与MCF - 7 - ras基因的细胞42日龄EXP - CAF1细胞，并再次注入他们到一个额外的周期为200天的裸鼠皮下。解剖和游离肿瘤异种移植成单细胞悬液培养的细胞，并在15厘米的培养皿10％FCS - DMEM菜嘌呤（1微克/毫升）的存在，正如在P60.1。传播，直到细胞融合（8-10天）。商店细胞在-80 ° C使用冻结的介质。由此产生的PURO R细胞被命名为242日龄的EXP -氟化钙细胞（图1BA ）。 7B。控制成纤维细胞培养提取要生成控制对EXP -氟化钙细胞成纤维细胞，仅42日龄控制成纤维细胞注入无癌细胞裸鼠皮下另外，为200天。剖析和分解成单细胞悬液，并在15厘米的培养皿菜嘌呤（1微克/毫升）P6.1表示存在10％FCS - DMEM培养细胞的成纤维细胞组织。传播，直到细胞融合（3-4周）。商店细胞在-80 ° C使用冻结的介质。由此产生的PURO R细胞被命名为控制成纤维细胞（图1BB） 。 8。代表性的成果： 控制成纤维细胞- 2和EXP -氟化钙CELls，它已经从乳腺肿瘤异种移植中提取，染色强阳性，表明人类间质的标志物，包括人类特有的波形，脯氨酰- 4 -羟化酶1A，胶原蛋白，纤维连接蛋白，S100A4基因，和成纤维细胞表面蛋白 （图2A）11这些细胞间质的起源和性质。与此相反，细胞角蛋白，上皮细胞的标志物，不染，在这些绿色荧光蛋白+成纤维细胞（图2B）。因此，这些研究结果表明，提取的EXP - CaF2和控制成纤维细胞- 2细胞起源从最初引入到小鼠移植瘤的父母的人乳腺成纤维细胞。 重要的是，EXP -氟化钙细胞的比例较高染色阳性，α- SMA和细胞外基质糖蛋白腱糖蛋白 - C 5，这两个标记的肌纤维母细胞相比，42日龄EXP CAF1和控制2成纤维细胞（图2C，D）11。这些数据表明，居民的人类乳腺fibr州逐步演变成非洲足联肌纤维母细胞在肿瘤异种移植。 <img alt="图1" src="/files/ftp_upload/3201/3201fig1.jpg" /> 图1人乳腺成纤维细胞的分离示意图。 一）减少隆乳组织肉末用无菌刀片（P1.2），转移到15 ml锥形管（P1.3）。消化在细胞内分解缓冲区（P1.4）的小组织片段，在体外（P1.5 - 7）和文化准备。为了immortalise孤立的主人乳腺成纤维细胞，逆转录病毒pMIG（MSCV - IRES - GFP）载体，表达hTERT和GFP，介绍，导致GFP阳性细胞用流式细胞仪（P2.1）进行排序。 pBabe普罗逆转录病毒载体，嘌呤霉素抗性基因编码，然后引入这些细胞。嘌呤霉素治疗后，GFP标记（GFP +）嘌呤霉素抗性（普罗R），immortaliSED人类乳腺成纤维细胞中分离（P2.2）。 BA）要生成EXP - CAFS，绿色荧光蛋白+ PURO ř永生的人类乳腺成纤维细胞与MCF - 7 - ras基因的乳腺癌肿瘤细胞皮下免疫缺陷的裸鼠（P3A）共注射。切除肿瘤异种移植在植入后42天（P4A），分离成单细胞悬液（P5）的。这些细胞，然后以消除任何污染的肿瘤细胞和鼠标（六）基质细胞在体外培养中存在的嘌呤。由此产生的嘌呤霉素抗性细胞被称为实验产生CAF1（EXP - CAF1）细胞。这些细胞，手术植入后42天，再次与MCF - 7 - ras基因细胞混合前（P7a）皮下植入主机鼠标。导致肿瘤增长为200天的额外时期，然后解剖，分离，培养和嘌呤存在。孤立嘌呤霉素抗性细胞被称为EXP -氟化钙细胞（242日龄）。 BB）要隔离控制对进出口水产科学研究院细胞，GFP + PURO ř永生人乳腺成纤维细胞作为纯文化注入裸鼠皮下没有MCF - 7 - ras基因的细胞（ P3B）。成纤维细胞组织，解剖（P4B）植入后42天，被分离，分离成单细胞悬液（P5）和嘌呤霉素抗性细胞命名为控制成纤维细胞，如前面所述，（六）。这些成纤维细胞再次，植入到裸鼠皮下没有的MCF - 7 - ras基因细胞此外为200天（P7B）。成纤维细胞组织解剖，分离，培养和嘌呤存在。隔离嘌呤霉素抗性细胞被称为控制成纤维细胞（242日龄）。 <img alt="图2" src="/files/ftp_upload/3201/3201fig2.jpg" /> 图2 <st荣&gt; EXP - CAFS和控制成纤维细胞来自父母的人乳腺成纤维细胞。 （一）控制成纤维细胞- 2（控制楼）和氟化钙EXP -细胞的免疫荧光分析。两种类型的细胞间质标记（红色），包括人类的波形，脯氨酰- 4 -羟化酶，胶原蛋白1A，纤维连接蛋白，S100A4蛋白，成纤维细胞表面蛋白染色呈阳性反应。（二）与此相反，泛角蛋白，上皮细胞的标志物，没有检测到EXP -氟化钙细胞表达GFP（绿色）。细胞核染色4&#39; - 6 Diamidino - 2 -苯基吲哚（DAPI）（蓝色）。比例尺，50微米（称为小岛等。） （三）免疫氟化钙EXP -细胞和抗体对α- SMA（红色）或腱糖蛋白C（TN - C），（红色），控制成纤维细胞（控制F.） 。细胞核用DAPI染色（蓝色）。比例尺，50微米（四）48％的氟化钙EXP -细胞为α- SMA染色阳性，而142日龄EXP - CAF1 4％和2.5％的控制成纤维细胞- 2细胞群为α- SMA阳性。 （简称小岛等al.11）

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没有利益冲突的声明。

非洲足联特定的标记和异质性水平缺乏观察，其中包括水产科学研究院提供的这种类型的细胞的特性本身就是一个挑战。 在体外研究水产科学研究院也已阻碍了新的复杂情况，这些细胞衰老停止增殖，长期培养。我们以前尝试直接immortalise使用端粒酶逆转录病毒基因构造的主要水产科学研究院是不成功的。因此，要进一步研究肿瘤促进这些细胞的特性，我们开发了一种方法来生成使用coimpla...

<table border="1"><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td></td><td> Invitrogen公司</td><td> 61965-026 </td><td></td></tr><tr><td>胎牛血清</td><td></td><td> GIBCO公司</td><td> 10270 </td><td></td></tr><tr><td>青霉素链霉素</td><td></td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>胶原酶I型</td><td></td><td>西格玛</td><td> C0130 - 1G </td><td></td></tr><tr><td>透明质酸</td><td></td><td>西格玛</td><td> H4272 </td><td></td></tr><tr><td>波形蛋白（V9）抗体</td><td></td><td> Novocastra实验室</td><td> NCL - L - VIM - V9 </td><td></td></tr><tr><td>腱糖蛋白C（BC - 8）抗体</td><td></td><td></td><td></td><td>从礼物 博士<stro吴&gt;卢西亚诺 Zardi </td></tr><tr><td> α- SMA - Cy3标记抗体（1A4） </td><td></td><td>西格玛</td><td> C6198 </td><td></td></tr><tr><td>脯氨酰- 4 -羟化酶</td><td></td><td> Dako公司</td><td> M0877 </td><td></td></tr><tr><td> （5B5）抗体</td><td></td><td></td><td></td><td></td></tr><tr><td>胶原TYPE1 1A抗体</td><td></td><td>西格玛</td><td> HPA011795 </td><td></td></tr><tr><td>泛角蛋白抗体</td><td></td><td>西格玛</td><td> C5992 </td><td></td></tr><tr><td>纤维连接蛋白抗体</td><td></td><td> BD公司</td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1（成纤维细胞特定的蛋白质- 1）抗体</td><td></td><td> Dako公司</td><td> A5114 </td><td></td></tr><tr><td>成纤维细胞表面蛋白 （克隆1B10）抗体</td><td></td><td> Abcam公司</td><td> ab11333 </td><td></td> &lt;/ TR&gt; <tr><td> MSCV - IRES - GFP的构建</td><td></td><td></td><td></td><td>请求的作者</td></tr><tr><td> pBabe - PURO兴建</td><td></td><td></td><td></td><td>购买从Addgene </td></tr><tr><td>嘌呤霉素</td><td></td><td>西格玛</td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td>西格玛</td><td> D9564 </td><td></td></tr><tr><td> 15毫升锥形管</td><td></td><td>康宁</td><td> 430766 </td><td></td></tr><tr><td>裸鼠</td><td></td><td>泰康利</td><td> NCRNU - F </td><td>女性NCR裸体</td></tr><tr><td> C3H/10T1/2细胞</td><td></td><td> ATCC </td><td></td><td> CCL - 226 </td></tr></table>

experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts

癌肿瘤细胞和非癌变的车厢称为“基质”组成的复杂的组织。基质由细胞外基质（ECM）和各种间质细胞，包括成纤维细胞，肌纤维母细胞，内皮细胞，周细胞和白细胞1-3。 肿瘤相关基质是邻近的肿瘤细胞释放大量的旁分泌信号作出反应。在疾病的过程中，基质往往成为人口，包括大量的肌纤维母细胞癌相关成纤维细胞（CAFS）。这些细胞先前已提取他们在体外培养来自许多不同类型的人类癌的。一个亚群水产科学研究院是通过上调α-平滑肌肌动蛋白（α- SMA）的表达4,5区别。这些细胞是一种“激活成纤维细胞”的标志，与肌纤维母细胞彗星类似物业ommonly观察受伤和纤维化组织6。这种肌纤维母细胞非洲足联子集的存在是高度相关，高品位的恶性肿瘤，并与患者的不良预后相关。 许多实验室，包括我们自己，已经表明，咖啡馆，当肿瘤细胞注射到免疫缺陷小鼠，能够大大促进tumourigenesis 7-10。然而，从癌患者准备的咖啡馆，经常接受各地正在进行的实验使用的广泛性，传播文化的限制期间衰老。为了克服这个困难，我们开发了一种新技术，对实验产生永生非洲足联的人类乳腺细胞株人乳腺成纤维细胞（EXP - CAFS），使​​用coimplantation乳腺肿瘤异种移植模型。 为了生成EXP -水产科学研究院，父母的人乳腺成纤维细胞，减少隆乳组织获得，分别为第一immortaliSED与端粒酶，端粒酶全酶的催化亚基，并表达GFP和嘌呤霉素抗性基因工程。 coimplanted与MCF - 7人乳腺癌表达小鼠异种移植成一个激活的ras癌基因（MCF - 7 - ras基因细胞）的细胞，这些细胞。最初注射的人乳腺成纤维细胞体内孵化期后，分别提取其嘌呤霉素抗性11的基础上，在肿瘤异种移植。 我们观察到，居民人乳腺成纤维细胞分化，采用肌纤维母细胞表型和后天在肿瘤进展过程中促进肿瘤的性质。重要的是，这些细胞，定义EXP - CAFS，密切模仿从乳腺癌患者解剖隔离CAFS的肿瘤促进肌纤维母细胞表型。因此，我们的肿瘤异种移植派生EXP - CAFS提供一个有效的模型来研究人类breas的生物学水产科学研究院吨癌。所描述的协议也可以用于生成和表征各种非洲足联衍生出其他类型的人类癌的人群扩展。

Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

癌相关成纤维细胞（CAFS）在肌纤维母细胞肿瘤间质内的丰富，推动肿瘤进展发挥了重要作用。从人类乳腺成纤维细胞的实验产生CAFS我们开发了一个coimplantation肿瘤xengraft模型。协议描述了如何建立非洲足联的肌纤维母细胞的获取能力，以促进tumourigenesis。

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

experimental-generation-carcinoma-associated-fibroblasts-cafs-from

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

23.3K 次观看.  University of Manchester. 癌相关成纤维细胞（CAFS）在肌纤维母细胞肿瘤间质内的丰富，推动肿瘤进展发挥了重要作用。从人类乳腺成纤维细胞的实验产生CAFS我们开发了一个coimplantation肿瘤xengraft模型。协议描述了如何建立非洲足联的肌纤维母细胞的获取能力，以促进tumourigenesis。

视频: Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

科研

医学

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

人乳内动脉（IMA）移植及内支架置入术：人体模型支架内再狭窄的研究开发

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...