这项工作得到了国家牙医学院和马萨诸塞州和皮肤疾病研究所（张（（R01AR069653）和美国国家卫生研究院（5T32AR062496-03至CA）的支持。

所有动物实验均得到UCSD机构动物保护和使用委员会的批准。 初级小鼠KC从新生儿皮肤中分离和培养<ol><li>使用剪刀将C57B / 6野生型小鼠品系的产后0-2新生儿斩首。切断手腕上方的四肢和踝关节，然后完全切断尾巴，留下一个小洞。 </li><li> 要剥离整个皮肤，首先将尖锐的剪刀穿过尾部的孔，将身体的背部中线的皮肤沿着颈部的开口切开。接下来，使用一个镊子抓住暴露的身体和其他镊子抓住皮肤，并用一个连续的运动轻轻地将整个皮肤从身体和腿部残余物上剥离。 <ol><li>剥离皮肤作为一个完整的部分，并小心不要将皮肤分解成碎片，因为这将导致细胞丢失步骤。 </li></ol></li> &lt;li&gt; 用10 cm培养皿中的15 mL无菌PBS冲洗去皮皮肤，然后将每只小鼠皮肤放入装有冰冷分散酶消化缓冲液的2 mL管中，该缓冲液在KC生长培养基中含有4 mg / mL的分泌酶（KC基础培养基具有0.06mM CaCl 2 ，dGS和抗生素/抗真菌剂）。 注意:可以将多个皮肤分离物合并并在含有12 mL分散液的一个15 mL管（每管最多5个皮肤）中孵育。在旋转器上的4℃冰箱中孵育皮肤管过夜。 <ol><li>确保皮肤没有折叠在管中，因为这将导致折叠区域暴露于分散液的不足。 </li></ol></li><li>在第二天（分泌12-18小时后），将皮肤与分散液一起转移到培养皿中。将每个组织块转移到具有15mL无菌PBS的新培养皿中，以洗涤多余的分散素。 </li><li>使用两对镊子，从PBS抓住皮肤，抬起皮肤，并转移到一个新的培养皿，表皮一面朝下，真皮面朝上。仔细拉伸皮肤褶皱，使其完全延伸到培养皿上。 </li><li> 在新的培养皿中放置500μL每种皮肤的基于胰蛋白酶的消化液。使用镊子，缓慢地将真皮提起并远离表皮，同时保持表皮下降。将真皮处理为生物危害材料。转移分离的表皮，并将基底层向下漂浮在每滴胰蛋白酶溶液上。 <ol><li>如果将表皮折叠在胰蛋白酶溶液上，则使用镊子去除任何褶皱，以确保从上皮细胞中有效消化基底KCs。 </li></ol></li><li>在室温下（盖上盖子）在胰蛋白酶溶液中培养培养皿上的皮肤，轻轻搅拌20分钟。 </li><li> 每个表皮添加2 mL补充的KC生长培养基（每平方英尺约1平方英寸）表皮）到培养皿。使用镊子抓住表皮，大力擦拭表皮，从表皮片上释放单细胞。 <ol><li>随着细胞与表皮分离，观察中等转变越来越浑浊。倾斜培养皿收集和转移细胞悬浮液到一个新的管，留下剩下的表皮片在盘子上。 </li></ol></li><li>在加入2 mL KC生长培养基后再重复两次摩擦表皮片，并将细胞悬液混合在同一根管中。 </li><li>将细胞溶液轻轻吸取数次，以使用适当的血清移液管破碎任何细胞团块，然后将其通过100μm过滤器，直至新的50 mL离心管。 </li><li>将过滤的细胞离心5分钟，180×g。 </li><li>吸出上清液，轻轻将细胞沉淀重悬于1 mL冷的KC生长培养基/表皮中。 </li><li>用血细胞计数器计数细胞。 <br/&gt;注意:KCs可以在悬浮液中自发分化，因此细胞应在分离后尽快铺板，以确保细胞的最佳性能。我们建议在细胞计数时将细胞置于冰上，因为这会降低KC自发分化的速度。 </li><li> 将细胞以5×10 4 / cm 2的密度接种在用促进细胞附着的细胞外基质（ECM）产品预培养的培养皿中的KC生长培养基中。 注意:我们使用商业用途（例如附着因子，它是一种明胶基涂层材料（详见材料表），细胞在37℃的5％CO 2的潮湿培养箱中培养，新鲜培养基每隔一天补充一次<ol><li>对于涂层，在细胞接种之前，将温热至室温的细胞附着涂层材料包被培养皿10分钟至2小时。删除附件因素加入细胞悬液。 </li></ol></li><li>在初次铺板后24小时更换培养基以除去未附着的细胞，每天更换培养基直到细胞在实验前达到所需的汇合度。 </li></ol> 2.成年尾部皮肤的主要小鼠KC隔离<ol><li>根据动物设施法规，牺牲成年人C57BL / 6小鼠（优选在6-15周龄之间;男性或女性）。从尾座上切下身体的尾巴，切断〜2 mm的尾尖，使尖端有一个可见的孔。 </li><li>要剥离尾巴皮肤，首先使用锋利的刀片将尾皮从基部切割到尾端。接下来，使用一个镊子抓住暴露的尾骨和其他镊子抓住皮肤，并用一个连续的运动轻轻地将尾皮从皮肤上剥下。从中线切下去皮的皮肤，使每片不到2-3厘米长。 </li><li> 去皮去皮皮肤在10cm培养皿中加入15mL无菌PBS，然后将每个尾部的皮肤放入装有冰冷分散酶消化缓冲液（KC生长培养基中的4mg / mL分散酶）的2mL管中。 注意:还可以将多个皮肤片合并，并在含有12 mL Dispase溶液的一个15 mL管（每管最多5尾）中孵育。 <ol><li>在旋转器上的4℃冰箱中孵育皮肤管过夜。 </li></ol></li><li>执行步骤1.4至1.13以从尾部表皮分离单细胞悬浮液。 </li><li>在用胶原蛋白预包被的培养皿中的KC生长培养基中以10×10 4 / cm 2的密度（通过血细胞计数器计数）种成虫细胞。 注意:我们使用市售的胶原基涂层套件（详见表格 ）。细胞在37℃的5％CO 2的潮湿培养箱中培养，每隔一天补充新鲜培养基。一般来说，c在细胞播种之前，将超声波培养皿涂上胶原蛋白30分钟至1小时。我们观察到，与作为明胶基涂层材料的附着因子相比，当涂覆胶原时，成人KC附着到培养皿上显着增强。 </li><li>在初次铺板后24小时更换培养基以除去未附着的细胞，每天更换培养基直到细胞在实验前达到所需的汇合度。 </li></ol> 3.高钙对KCs的体外分化<ol><li>为了诱导终末分化，在培养基中加入CaCl 2至0.2mM。 </li><li>或者，在添加高钙之前，将培养的KCs饿死过夜而不加入低钙KC基础培养基中的生长补充剂。 注意:生长因子饥饿将增强细胞对早期分化和早期分化标志物的诱导，如K10 5 </SUP&gt;。 </li></ol> UVB照射介导的细胞死亡和TNF释放从KCs <ol><li>在KC生长培养基（37℃，5％CO 2的加湿培养箱）中培养小鼠KCs，每天培养基更换直到细胞达到融合。在UVB照射之前，取出生长培养基，并用500μLPBS代替。然后使细胞达到25mJ / cm 2的 UVB或模拟对照（无UVB）。 注:UVB照射是使用手持UVB灯与如先前所述10 2个8瓦灯泡（312纳米）上进行。 UVB照射后，抽吸PBS，加入新鲜培养基。 </li><li>测量和在6,8量化由CCK-8细胞生存力试验中的细胞生存力，以及制造商的指令11以下24小时后UVB治疗。 </li><li>从所需时间点用UVB处理的KCs收集条件培养基，并测量释放的TNFα在由小鼠TNF（单/单）ELISA试剂盒制造商的指令7，12以下的条件培养基。 </li></ol>

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皮肤表皮作为分离和保护身体免受外界环境的损害，水分流失，病原体，热量和紫外线照射的关键障碍。 KCs是表皮的主要细胞系，表皮KCs的原代培养是研究和了解屏障形成的生物学过程和KCs对体外环境侮辱的反应的有用工具。 在这里我们描述从新生儿和成年鼠皮肤分离和培养初级表皮KCs的方法。虽然原代小鼠KCs可以方便地从新生小鼠皮肤分离，但成年KCs的分离和成功?...

皮肤是身体最大的器官，表皮最外层。表皮在形成完整的表皮屏障以将身体与环境分离的过程中发挥关键作用，从而防止水分流失，并且防止环境污染，例如过敏原，病原体和UVB暴露。表皮从单层未分化基底角质形成细胞（KCs）发展成由基底层组成的多层分层上皮，其次是棘突层，颗粒层和角质层。由表皮干细胞和转运扩增细胞组成的基础KCs是增殖和未分化的。由于基底KCs离开细胞周期，细胞承诺分化，并逐渐迁移到表皮表面，伴随细胞细胞连接的成熟和表皮渗透屏障（EPB）的形成。棘突中的KCs表达早期差异离子标记如角蛋白10（K10）;随着KCs迁移到颗粒层，细胞表达晚期分化标志物，如丝胶蛋白（FLG），Loricrin（LOR）和Involucrin（INV）。在角质层，KCs成为终末分化的角质细胞，当新细胞取代它们时，它们最终通过脱屑而脱落。 钙被认为是表皮中最生理的药物，并以类似的方式在体外和体内触发分化。在正常皮肤表皮，钙离子形成的特性"浓度梯度"，在浓度朝向皮肤表面1，2，3增大。钙浓度从最低子层（基底和棘突层）的低水平上升到上部颗粒层中的峰值，然后在最表层（角质层）中降至可忽略的水平。<html>钙梯度也与成分渗透性屏障的出现同时发生，其支持钙信号传导起KC分化的关键作用。 体外 ，低钙（0.02-0.1mM）维持基底KCs作为单层的增殖，而高钙（&gt; 0.1mM）诱导细胞对终末分化的快速和不可逆的承诺，如紧密连接形成和诱导LOR和INV的在高钙处理至基础枯否4,5。 除了屏障形成之外，表皮KCs也是皮肤先天免疫系统的重要组成部分。针对在UVB照射或损伤时释放的病原体或损伤相关分子模式（DAMP），KCs可以产生大量的炎性细胞因子，如TNFα，IL6和IFNβ，导致免疫系统激活&gt; 6，7，8，9。虽然从枯否正确炎性信号所需的病原体清除，失控性炎症反应可以触发自动炎性皮肤疾病，如牛皮癣和酒渣鼻6,8的发展。 总体而言，KCs在维持完整的皮肤屏障和启动对病原体入侵或环境侮辱的免疫应答方面起着至关重要的作用。因此，表皮KCs的原代培养是研究上皮生物学，KC分化以及KC刺激的先天免疫应答的有用技术。由于KC对各种外部兴奋剂的敏感性和敏感性，原代小鼠表皮KCs的分离和培养可能是一个具有挑战性的过程。这里我们描述一种从新生小鼠皮肤中分离和培养KCs的方法成年老鼠尾皮。对于成年KC分离，我们不使用小鼠背部皮肤，因为从这个组织中分离出足够量的活KC可能是困难的，原因如下:首先，在头发周期的休眠阶段的成年背部皮肤（休止期）由薄的表皮只有1-2层细胞，导致低细胞产量和表皮与真皮的低效分离，这是成功进行KC分离的关键步骤。第二，存在于成年背皮上的高毛囊密度进一步有助于分离表皮与真皮的困难。相反，我们通常使用尾皮作为成年小鼠KCs的来源，因为上皮更厚，3-5层表皮KCs。它也具有较低的毛囊密度，其不会干扰表皮分离，从而允许KC从任何成年小鼠尾部皮肤分离，而不管小鼠的年龄和毛发循环阶段。孤立的霓虹灯将tal KCs接种到明胶包被的培养皿中，而使用胶原蛋白包被的培养皿来种植分离的成年KCs，这是因为与新生儿相比，成人细胞的粘附能力受损。为了培养小鼠KCs，补充含有表皮生长因子（EGF），牛转运蛋白，胰岛素样生长因子c1（IGF1），前列腺素E2（PGE2），牛血清白蛋白（BSA）和氢化可的松的dGS的低钙基础培养基。在初次铺板2-4天之后，大多数分化的KCs可以在日常培养基的变化过程中被洗去，剩下的贴壁细胞显示典型的鹅卵石形态4 ，正在增殖，并且不表达早期的分化标志物K10。

<table width="633">
	<tbody>
		<tr height="100">
			<td height="100" style="width:171px;height:100px;">C57BL/6 neonates or adult wildtype mice</td>
			<td style="width:177px;">Jackson Laboratory</td>
			<td style="width:119px;">000664</td>
			<td style="width:167px;">Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">KC basal medium (EpiLife)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">MEPICF500</td>
			<td style="width:167px;">basal medium for keratinocyte culture with 0.06 mM CaCl2</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">Defined Growth Supplement (dGS)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S0125</td>
			<td style="width:167px;">defined growth supplements for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Dispase powder</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">17105041</td>
			<td style="width:167px;">enzyme to dissociate the epidermis from dermis</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Attachment Factor</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S006100</td>
			<td style="width:167px;">gelatin-based coating material</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Coating Matrix</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">R011K</td>
			<td style="width:167px;">Collagen-based coating material</td>
		</tr>
		<tr height="80">
			<td height="80" style="width:171px;height:80px;">TrypLE</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">12604-013</td>
			<td style="width:167px;">A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">100 &#956;m Cell Strainer Nylon mesh</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">352360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">CCK-8 cell viability Kit</td>
			<td style="width:177px;">Dojindo Molecular Technologies, Rockville, MD</td>
			<td style="width:119px;">CK04-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Mouse TNF (Mono/Mono) ELISA Set II</td>
			<td style="width:177px;">BD Biosciences, San Jose, CA</td>
			<td style="width:119px;">555268</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Corded Hand-Held UV Lamps</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">EB-280C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">8-watt UV tubes</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">BLE-8T312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Light Inverted Microscope for cell culture</td>
			<td style="width:177px;">ZEISS, Jena, Germany</td>
			<td style="width:119px;">Axio Observer</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">Fluorescent Microscope</td>
			<td style="width:177px;">Olympus</td>
			<td style="width:119px;">BX41</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

角质形成细胞（KC）是表皮中最主要的细胞类型，即皮肤的最外层。表皮KCs通过形成完整的皮肤屏障来抵御环境污染，例如UVB照射或病原体，以及通过对这些侮辱发起炎症反应，在提供皮肤防御方面发挥关键作用。这里我们描述了从新生小鼠皮肤和成年小鼠尾皮分离KCs的方法。我们还描述了使用定义的生长补充剂（dGS）与穿梭胎牛血清（cFBS）相比的培养条件。在功能上，我们显示新生儿和成年KCs对高钙诱导的末端分化，紧密连接形成和分层具有高度反应性。此外，培养的成年KCs对UVB触发的细胞死亡敏感，并且可以在UVB照射时释放大量的TNF。一起，这里描述的方法对于体外模型设置的研究人员将是有用的以研究新生小鼠和/或成年小鼠中的表皮生物学。

高钙诱导的新生儿和成人KCs的终末分化。以0.06mM CaCl 2铺平并保持的主要小鼠表皮KCs作为单层生长，并且单个细胞具有不同细胞间隙的多边形形状，当汇合时显示出鹅卵石的外观（ 图1A和图2A ）。将CaCl 2升高至0.2mM导致细胞的快速形态变化。高钙开关后8 h内，细胞变平，细胞间隙明显不明显，24 h时?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

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

表皮角质形成细胞形成功能性皮肤屏障，位于主体防御的前线，防止外部环境污染。在这里，我们描述了从新生儿和成年小鼠皮肤分离和原代培养表皮角化细胞的方法，以及从角质形成细胞诱导终末分化和UVB引发的炎症反应。

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-culture-primary-mouse-keratinocytes-from-neonatal-adult

Research

JoVE Journal

Biology

30.5K 次观看.  UC San Diego. 表皮角质形成细胞形成功能性皮肤屏障，位于主体防御的前线，防止外部环境污染。在这里，我们描述了从新生儿和成年小鼠皮肤分离和原代培养表皮角化细胞的方法，以及从角质形成细胞诱导终末分化和UVB引发的炎症反应。 

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

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...