这项工作得到了中国国家自然科学基金（NFSC 82101224 to YG）的支持

所有动物实验均获得习交通大学动物使用与护理委员会批准（编号：2021-847）。
1.灭菌和材料准备
<ol><li>使用高温高压蒸汽消毒对解剖工具进行灭菌，并在50°C培养箱中干燥过夜。</li>
	<li>制备100mL含有10%胎牛血清（FBS）和10mg / mL青霉素/链霉素的培养基（将10mL FBS和10μL青霉素储备溶液加入90mL的Dulbecco改良Eagle培养基（EMEM）中）并储存在4°C。</li>
</ol>2.颞骨的解剖和切除以收集听觉上皮
<ol><li>通过在冰上斩首对总共10只新生小鼠（年龄在P3和P5之间）实施安乐死。如果需要，使用其他合乎道德的方法。</li>
	<li>将头部固定到位，使用微操作剪刀沿矢状缝合线打开头皮，并用手指将头皮分开并双侧固定，如图 1A所示。</li>
	<li>使用小型骨膜升降机取出大脑，并将基底颅骨一分为二。用剪刀剪开头盖骨，向前翻转以打开头骨;用剪刀尖刮开大脑，露出头骨底部。</li>
	<li>观察颅骨底部的双侧颞骨（图1C）。用剪刀沿中线切开颅骨底部，刮掉皮肤，去除任何不必要的骨头（图1D，D'）。</li>
	<li>将颞骨保留并转移到含有新鲜Hank平衡盐溶液（HBSS）的35mm无菌培养皿中（图1E）。</li>
	<li>使用两个#5尖镊子从颞骨的岩部去除大疱和周围组织（图1E）。 注意：在这个阶段，小鼠颞骨中的骨迷路不会完全钙化，并且很容易使用镊子解剖。</li>
	<li>一只手握住镊子固定颞骨的半圆形部分，另一只手将镊子的下脚插入圆窗龛，将耳蜗的外侧骨与鳞状前庭分开。小心地去除颞骨的岩部，不要接触OC上皮。随后，小心地分离并移除耳蜗的骨迷宫，从基底端到顶端（图1F）。</li>
	<li>使用#5尖镊子小心地将Corti感觉上皮器官与modiolus进行微分离（图1G）。</li>
	<li>握住螺旋韧带，用微操作镊子小心地将其与血管纹分离，并使用200μL移液管将干净的听觉上皮转移到含有HBSS的3mm无菌培养皿中（图1H）。</li>
	<li>从每只动物中收集20个标本，并将它们快速转移到含有HBSS的100mm无菌培养皿中，用于下一步制备步骤（图1）。</li>
</ol>3. 酶解聚获得听觉毛细胞
<ol><li>将听觉上皮转移到含有0.25%胰蛋白酶的10mL新鲜DMEM中，并在37°C孵育12分钟。</li>
	<li>使用200μL移液器吸头，在手术显微镜下轻轻地将毛细胞与基底层和其他细胞分开。</li>
	<li>再加入 10 mL 培养基以抑制分解。</li>
	<li>通过70μm过滤器过滤培养基中的悬浮细胞，将滤液收集在干净的50mL管中，并以300× g 离心5分钟。</li>
	<li>使用 1,000 μL 移液器吸头轻轻上下移液，将毛细胞重悬于至少 5 mL 培养基中。避免引入气泡。</li>
	<li>提前将盖玻片放在六孔板的底部。计数细胞，并在六孔板中以 106 个细胞/mL 的密度培养它们。</li>
	<li>在37°C和5%CO2下，在2mL DMEM（含有10%FBS，100单位/ mL青霉素和100μg/ mL链霉素）中培养贴壁细胞。每天更换培养基。 注意：应该提到的是，使用此协议不会导致获得纯毛细胞。基于这种原代细胞培养方法，我们建议使用 d2 或 d3 细胞进行进一步研究，因为毛细胞可能约占培养细胞的 70% 并且处于良好状态。</li>
</ol>4.免疫荧光染色
<ol><li>从d1到d6收获培养的细胞（每天一个孔）。吸出培养基并用磷酸盐缓冲盐水 （PBS） 冲洗细胞 2 次。</li>
	<li>在室温（RT）下用4%多聚甲醛固定细胞15分钟。</li>
	<li>取出固定剂并用PBS冲洗细胞3 x 3分钟。</li>
	<li>在室温下用含有0.2%Triton X-100的PBS透化细胞10分钟。</li>
	<li>在室温下，将透化的细胞与含有10%FBS的PBS封闭溶液孵育20分钟。</li>
	<li>用抗肌球蛋白单克隆抗体（在PBS中以1：200稀释）在4°C下染色细胞过夜。</li>
	<li>用无菌 PBS 洗涤细胞 3 次，并用二抗（Alexa Fluor 594 山羊抗兔 MYO7A，在 PBS 中以 1：500 稀释）和荧光素标记的鬼笔环肽（Alexa Fluor 488，以鉴定细胞结构）在室温下孵育 2 小时。</li>
	<li>用 PBS 冲洗细胞 3 次以去除二抗。</li>
	<li>在载玻片上加入1-2滴含有DAPI的封固剂，安装盖玻片，并将它们置于激光扫描共聚焦显微镜下以捕获细胞的照片。</li>
</ol>5. 统计分析
<ol><li>进行双向方差分析 （ANOVA），然后进行 Tukey 事后检验，以分析肌球蛋白-VIIa 和鬼笔环肽灰度值随时间的变化。使用字母标记方法标记统计差异。</li>
	<li>执行额外的单因素方差分析，然后进行 Tukey 的事后检验，以比较第 1 天至第 6 天细胞样本之间的鬼笔环肽阳性细胞比率和同一天的肌球蛋白-VII 阳性细胞比率。</li>
</ol>

小鼠耳蜗毛细胞的高效体外培养

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作者没有要披露的利益冲突。

与HEI-OC1细胞系相比，毛细胞的原代培养更准确地复制了体内细胞的生理状态。因此，通过将活细胞从耳蜗器官中分离出来并立即培养它们而建立的听觉原代培养方法似乎是广泛研究听觉系统的宝贵工具。某些技术对于成功的文化至关重要。首先，最小化Corti器官与颞骨分离的持续时间可增强毛细胞持续活动的可能性。因此，研究人员必须尽量减少器官解剖和浸入PBS之间的时间间隔。根据我们的实?...

耳蜗毛细胞在声音检测和信号传递到听觉神经中起着重要作用。毛细胞是机械细胞，在脊椎动物中充当主要感觉受体并将声音振动转化为电信号。哺乳动物内耳的感觉上皮包括单排内毛细胞和三排外毛细胞。在不同的基本膜区域，毛细胞感知不同频率（20 至 2,000 Hz）的声音1。外毛细胞的功能是一个主动的机械放大过程，有助于微调哺乳动物的内耳，赋予对声音的高灵敏度。内毛细胞负责检测声音。分级去极化后，声学信息通过听觉神经纤维2传递到大脑。
听力损失可能是由遗传缺陷、衰老、噪音创伤或过度使用耳毒性药物引起的，这些都是世界范围内的主要健康问题 3,4。听力损失主要由毛细胞不可逆的损伤引起5.关于噪声引起的听力损失，尽管研究人员已就其病因的几个细节达成共识，但缺乏对众多潜在机制的全面了解。外毛细胞特别容易受到声学过度暴露的影响6.机械敏感的耳蜗毛细胞与年龄相关的听力损失有关;然而，毛细胞变性的分子和细胞机制仍然未知。分子过程的几种变化导致毛细胞老化、氧化应激、DNA 损伤反应、自噬以及与毛细胞特化相关的基因表达和转录失调7.
由于内耳被包裹在颞骨中，深入身体最坚硬的骨骼，因此在实验上无法进入，这对研究毛细胞修复和再生机制提出了挑战。因此，建立用于研究毛细胞功能的体外培养物已成为研究内耳再生和损伤机制的理想方法。制备耳蜗器官型培养物的程序已在早期研究中描述8,9,10。世界各地的研究人员采用了各种人工耳蜗显微切割和表面处理技术。尽管存在持续的挑战，但各种原代毛细胞培养系统已成功在体外建立。耳蜗器官培养物包含多种细胞类型，包括毛细胞、Deiters 细胞、Hensen 细胞、支柱细胞和听觉神经纤维。深入了解损伤后毛细胞在细胞和分子水平上的变化将有助于开发更强大的研究工具。本研究旨在证明从新生小鼠中分离耳蜗器官并酶促分离丰富的毛细胞用于体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm BioLite cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130182</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>35 mm Nunc cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>150318</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>6-well palate</td>
			<td>Thermo Fisher Scientific</td>
			<td>310109005</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainers</td>
			<td>BD Company</td>
			<td>352350</td>
			<td>using for filter</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientifc</td>
			<td>A11008</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>day 3-5 neonatal murine&#160;</td>
			<td></td>
			<td></td>
			<td>provided by Xi&#39;an Jiaotong University</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>5#</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Leica anatomy microscope</td>
			<td>Germany</td>
			<td>S9i</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Rabbit plyclonal to Myosin VIIa</td>
			<td>Abcam company</td>
			<td>ab92996</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Belevor</td>
			<td>10cm/04.0524.10</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich&#160;</td>
			<td>9036-19-5</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td>using for culture</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary cochlear hair cells from neonatal mice

耳蜗毛细胞是听觉系统的感觉受体。这些细胞位于内耳骨迷宫内负责听觉的感觉器官Corti器官中。耳蜗毛细胞由两种解剖学和功能上不同的类型组成：外毛细胞和内毛细胞。其中任何一个的损坏都会导致听力损失。值得注意的是，由于内部毛细胞无法再生，并且对它们的损害是永久性的。因此，原代毛细胞的 体外 培养对于研究耳蜗毛细胞的保护或再生作用是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。 
手动去除耳蜗侧壁后，在显微镜下从耳蜗中仔细解剖听觉上皮，在由0.25%胰蛋白酶-EDTA组成的混合物中在37°C下孵育10分钟，并使用200μL移液器吸头轻轻悬浮在培养基中。将细胞悬液通过细胞过滤器，离心滤液，并在 24 孔板中培养细胞。根据毛细胞表达参与运动张力的机械转导复合物肌球蛋白-VIIa的能力以及通过使用鬼笔环肽选择性标记 F-肌动蛋白来鉴定毛细胞。细胞在培养 4 天后达到 >90% 的汇合度。该方法可以增强我们对体外培养毛细胞生物学特性的理解，并证明耳蜗毛细胞培养的效率，为进一步的听觉研究奠定了坚实的方法学基础。

按照该协议，我们接种了分离的细胞。如果原代耳蜗毛细胞种子没有漂浮在培养基中并在 24 小时内扩散，则认为这些细胞是成功的。我们确定了毛细胞粘附并扩散成培养皿底部的扁平聚集体后的数量。1天后，将活毛细胞紧紧粘附在培养皿底部，并用PBS冲洗除去非贴壁细胞。通常，培养3天后细胞数量增加一倍（图2）。
免疫荧光（IF）显示肌球蛋白-VIIa和...

Watch this Scientific Journal Video about Advancements in Cultivating Mouse Hair Cells for Auditory Research at JoVE.com

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

在此，我们提出了从小鼠中分离和培养原代耳蜗毛细胞的详细方案。最初，在显微镜下从新生儿（3-5天龄）鼠耳蜗中解剖Corti的器官。随后，将细胞酶消化成单细胞悬液，并在培养数天后使用免疫荧光进行鉴定。

新生小鼠原代耳蜗毛细胞的分离和培养

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...

内耳被包裹在颞骨中，深深地扎在身体最坚硬的骨头中，对调查构成了挑战。原代毛细胞的培养对于研究耳蜗毛细胞是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。我们演示了从天然小鼠中分离耳蜗器官和不对称分离丰富的毛细胞以进行体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。从耳蜗器官中分离活细胞，并立即指导它们，成为广泛研究的宝贵工具，主要培养方法有三种，这三种系统都是三种。该方法可以增强我们对体内培养毛细胞生物学特性的理解，并证明耳蜗毛细胞培养的效率，为进一步的听觉研究奠定了坚实的方法学基础。

isolation-culture-primary-cochlear-hair-cells-from-neonatal

Research

JoVE Journal

Biology

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

2.1K 次观看.  The Second Affiliated Hospital of Xi’an Jiaotong University. 内耳被包裹在颞骨中，深深地扎在身体最坚硬的骨头中，对调查构成了挑战。原代毛细胞的培养对于研究耳蜗毛细胞是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。我们演示了从天然小鼠中分离耳蜗器官和不对称分离丰富的毛细胞以进行体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。从耳蜗器官中分离活细胞，并立即指导它们，成为广泛?...

视频: 作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

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 ...

科研

生物学

Isolation and Primary Culture of Rat Hepatic Cells

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

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

大鼠肝细胞的分离和小学文化

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

Isolation of 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 ...

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 ...

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 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 Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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 Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...