This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1.主动脉的小鼠中分离这里所描述的所有程序均通过的韦恩州立大学的机构动物护理和使用委员会批准。 <ol><li>把鼠标放到麻醉机的感应室。设置异氟烷流至4％结合25％的新鲜空气和75％ 的 O 2。 通过感应腔随后由专用的鼻锥异氟醚（诱导4％，±1.0％用于保养）用25％的新鲜空气和75％的O 2一起吸入麻醉鼠标。注:麻醉用异氟烷可以在75％氧气/ 25％空气中或在100％的氧气递送。 </li><li>检查鼠标每隔5分钟，直到达到麻醉的适当水平（缺乏撤退到脚趾捏）。 </li><li>取鼠标移出感应腔，并继续吸入异氟烷虽然连接到麻醉机专用的鼻锥。切换异氟烷流至1％，以保持麻醉。 </li><li>虽然这是麻醉下，把鼠标到手术垫，放置在它的后面。使用磁带的实验室，以确保四肢。 </li><li>使用加热灯，保持鼠标的温暖。保持该灯在适当的距离从鼠标，使得鼠标不会过热。 </li><li>喷用70％乙醇的胸部。 </li><li>用解剖剪来自中线打开腹部，并露出腹主动脉。打开胸腔暴露心脏和肺部。 </li><li>切腹主动脉与在解剖剪刀释放血液中的中间。 </li><li>填补了1毫升注射器（用25g针），用1毫升的PBS含1000 U / mL的肝素。注入含1000单位/毫升肝素到左心室的PBS中，灌注主动脉。推心脏和肺钳在大动脉到小鼠的右侧，露出胸主动脉。 </li><li>快速使用微迪取出胸主动脉ssection镊子，把它放在冰冷的1X PBS（无菌），那么运输容器放入一个层流罩。 </li><li>插入一个1毫升的注射器装配用25g针进入主动脉的一端，并轻轻冲洗以冰冷的PBS主动脉以除去血液。 </li><li>使用显微解剖钳去除尽可能多的附加脂肪组织的和小的横向容器越好。 </li><li>马上转移的主动脉内皮生长培养基。 </li><li>切主动脉成用无菌手术刀刀片1毫米环。收获大约8 - 每10主动脉环。 </li><li>打开使用一对显微解剖剪的每个主动脉环。 </li></ol> 2.种子矩阵的主动脉段<ol><li>在不使用时，保持生长因子减少基质中-20℃，以防止凝固。把生长因子减少基质中4℃至少过夜以允许完全解冻。 </li><li>预冷6孔板枪头到-20℃，至少10分钟。置于冰上并涂覆6孔板一个孔用1mL基质的板，而不会引入任何气泡。放置板在37℃培养箱中20分钟，以使基质固化。 </li><li>植入主动脉片上使用无菌显微解剖镊固化的基质。放置件内腔面朝下上的基质不接触内皮。放置3 - 4彼此接近的矩阵主动脉段。 </li><li>加入足够的血管内皮细胞生长培养基，以保持湿润段（约200微升）。 </li><li>孵育在37℃的板在5％CO 2的4至6小时。在一天结束时，加入足够的媒体覆盖主动脉段。 </li><li>观察主动脉段相差显微镜下周期性地发芽。检查中等水平和细胞生长的每一天。添加媒体如有必要，要盖上主动脉段。 </li><li>在第 4天，轻轻除去培养基并取出主动脉曲线段F使用无菌针而不中断生长的内皮细胞ROM基质。 </li><li>添加2mL的新内皮细胞生长培养基中，并允许内皮细胞继续增殖基质2 - 3天。 </li></ol> 3.小鼠主动脉内皮细胞的初始传代<ol><li>涂层用明胶（0.1％）的新T12.5烧瓶中，孵育在37℃下30分钟。 </li><li>用无菌37℃PBS仔细清洗矩阵板。 </li><li>加入2毫升中性蛋白酶（50U /毫升）到矩阵板中，在与偶尔摇动摇杆平台室温孵育。检查相衬显微镜下的细胞，以确保大部分细胞被分离。 </li><li>加入2毫升D-瓦尔的灭活中性蛋白酶。 </li><li>仔细收集上清在15毫升离心管中。用2mL的D-缬氨酸洗涤板，并收集上清。 </li><li>离心在900 xg离心所述细胞悬浮液在袋鼠5分钟米温度。 </li><li>重悬在涂有明胶的T12.5烧瓶在4mL内皮细胞生长介质和板的细胞沉淀。孵育细胞在37℃，5％CO 2的2小时。 </li><li>更换介质。孵育细胞在37℃，5％的CO 2，直到它们的85％ - 90％汇合。 </li></ol> 4.传代的小鼠主动脉内皮细胞的<ol><li>外套两个新T12.5烧瓶用明胶（0.1％），孵育在37℃下30分钟。预热胰蛋白酶-EDTA（0.25％胰蛋白酶，0.02 EDTA在PBS中）和无菌PBS在37℃下约15分钟。 </li><li>用无菌37℃PBS仔细洗涤细胞。 </li><li>添加0.5毫升胰蛋白酶-EDTA（0.25％胰蛋白酶，0.02 EDTA在PBS中）中，并在37℃孵育约1分钟，或直到大部分细胞转身。 </li><li>添加2mL的内皮细胞生长培养基以停止消化。吸取细胞悬液上下烧瓶几次内。如有必要，可使用电池刮板以收集细胞。 </li><li>收集在15毫升离心管中的细胞悬浮液。离心细胞悬浮液在900×g离心，在室温下5分钟。 </li><li>重悬在涂有明胶2 T12.5烧瓶在4mL内皮细胞生长介质和板的细胞沉淀。孵育细胞在37℃，5％CO 2的2小时。 </li><li>更换介质。孵育细胞在37℃，5％的CO 2，直到它们的85％ - 90％汇合。 </li><li>使用鼠标主动脉内皮细胞后的2 - 3代。 </li></ol> 5.表征小鼠主动脉内皮细胞的<ol><li>重新板上的细胞。 <ol><li>涂层用明胶（0.1％）的6孔板，孵育在37℃下30分钟。用PBS冲洗板。 </li><li>预加热胰蛋白酶-EDTA和无菌PBS中至37℃约15分钟。 </li><li>清洗细胞，用无菌PBS中的3倍。添加0.5胰蛋白酶毫升/ EDTA对细胞，孵育约1分钟，在37℃õ- [R，直到大部分细胞转身。 </li><li>添加2mL的内皮细胞生长培养基以停止消化。吸取细胞悬液上下烧瓶几次内。如有必要，使用细胞刮刀收集细胞。 </li><li>收集在15毫升离心管中的细胞悬浮液。离心细胞悬浮液在900×g离心，在室温下5分钟。 </li><li>弃去上清液，再悬浮在内皮细胞生长培养基中的细胞。种子涂有明胶的6孔板的细胞以3×10 5 /孔的密度。 </li><li>孵育细胞在37℃，5％的CO 2过夜以使细胞附着到培养表面。 </li></ol></li><li> DIL-AC-LDL摄取和荆豆 -lectin约束力。 <ol><li>存储1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindo-羰花青高氯酸盐标记在-20℃乙酰低密度脂蛋白（语-AC-LDL）。染色之前，解冻冰语-AC-LDL。该股语-AC-LDL SOLU灰是1毫克/毫升。储存在4℃的FITC标记的荆豆凝集素（ 荆豆 -Lectin，1微克/毫升）。股票荆豆 -Lectin溶液是1毫克/毫升。商店赫斯特33342在4℃。股票的Hoechst 33342溶液为100微克/毫升。 </li><li>添加的语-AC-LDL原液10微升至1毫升培养基中，以使10微克/毫升的最终浓度。 </li><li>染色前，删除旧的介质，并用新的培养基洗涤细胞每孔。 </li><li>取出新媒体，并添加语-AC-LDL染色网上平台。 </li><li>孵育细胞在37℃，5％CO 2的1小时。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>加入1毫升10％福尔马林固定细胞。放于室温下的板约30分钟。 </li><li>清洗细胞，用无菌1×PBS中的3倍。避免光线。 </li><li>加入1毫升无菌1X PBS包含荆 -Lectin的10微升。 </li><li>孵育在室温temperatu细胞重新在黑暗中保持1小时。 </li><li>通过用无菌1×PBS中3次洗涤细胞除去荆豆凝集素。 </li><li>添加的Hoechst 33342至1微克/毫升的最终浓度，孵育在黑暗中10分钟。 </li><li>查看语-AC-LDL（红色，576纳米的激发波长）， 荆豆 -Lectin（绿色，519 nm的激发波长）和赫斯特的荧光（蓝色，361 nm的激发波长），用倒置荧光显微镜。 </li></ol></li><li>荧光染色CD31，VEGFR2，eNOS的，VE-cadherin和钙调节蛋白。 <ol><li>商店FITC缀合的抗小鼠CD31抗体，eNOS的抗体，VE钙粘蛋白抗体，FITC缀合的抗兔IgG在4℃下在黑暗中。商店VEGFR2抗体在-20℃。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>加入1毫升10％福尔马林固定细胞。放于室温下的板约30分钟。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>染色CD31，加入1毫升无菌1X的PBS包含CD31-FITC抗体10μL的。染色要么VEGFR2，eNOS的，VE钙粘蛋白或调宁蛋白，添加包含4μLVEGFR2，eNOS的无菌1X PBS 1毫升，VE钙粘蛋白或调宁一抗，分别为。 </li><li>在冰上孵育细胞，在黑暗中1小时。 </li><li>通过用无菌PBS 3次洗涤细胞除去抗体。 </li><li>对于VEGFR2的染色，eNOS的，VE-钙粘蛋白或调宁蛋白，添加包含FITC缀合的抗兔IgG的10微升无菌1×PBS中的1毫升。在冰上孵育细胞，在黑暗中1小时。通过用无菌PBS 3次洗涤细胞除去IgG的。 </li><li>添加的Hoechst 33342至1微克/毫升的最终浓度，孵育在黑暗中10分钟。 </li><li>查看CD31，VEGFR2，eNOS的荧光，VE钙粘蛋白，调宁（绿色的518-535纳米的激发波长）和赫斯特（蓝色，361 nm的激发波长），用倒置荧光显微镜。 </li></ol></li></ol>

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The authors have nothing to disclose.

这项研究表明了一个简单的方法来分离，培养内皮细胞从鼠标主动脉细胞无任何特殊设备。免疫荧光染色显示，大部分细胞是第二通道后内皮细胞。有人建议，细胞在第二至第三通道适于在体外 和体内实验来研究内皮生物学。 从本议定书的重要注意事项 有在步骤五个关键点。首先，血管腔与含肝素的PBS冲洗，以尽量减少内皮?...

血管内皮不仅是分隔的血液和组织的屏障层，它被认为是一个巨大的内分泌腺，绵延在整个血管树具有400平方米1的表面积。内皮的福祉是血管稳态的关键。的内皮机能障碍参与各种心血管疾病，包括动脉粥样硬化，血管炎和缺血/再灌注损伤， 等 2-4。迄今为止，涉及这些疾病的设置特定的细胞和分子机制尚未很好地理解，由于内皮的漫射解剖性质。 鼠标是研究的重要模型，因为遗传操作技术在小鼠比较发达的比任何其他哺乳动物物种。然而，由于主动脉的小尺寸使得enzymat原代鼠主动脉内皮细胞的分离被认为是特别困难内皮不切实际的IC消化。一些报道的方法分离和纯化的EC要求5-7。 该协议的目标是用一个简单的方法来从鼠标主动脉隔离和扩大血管内皮细胞，而无需使用任何特殊的设备。在这个协议中，新鲜分离的主动脉切成小片段，并接种到朝下以允许内皮出芽内皮的基质。段被删除后，内皮细胞在血管内皮青睐中等扩大，准备好两三个代后实验。所描述的方法的优点是:1）相当高数量的内皮细胞来自单一主动脉收获的; 2）细胞存活率保存良好; 3）不需要特殊的设备或技术。它提供了鉴定内皮细胞病理生理学特异性细胞和分子机制的一种有效手段。对于那些有兴趣谁在学习公关从任一基因敲除小鼠imary培养的内皮细胞，基因敲除小鼠中，或鼠疾病模型中，该协议是非常有用和易于实践。

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			<td>4- or 6-week-old mice (Jackson Laboratory, #000664).</td>
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			<td>Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023).</td>
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			<td>Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149).</td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium (Dulbeccos&#8217; Modified Eagle&#8217;s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100&#956;g/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]).</td>
		</tr>
		<tr>
			<td>Growth factor reduced matrix (BD Biosciences, #356231).</td>
		</tr>
		<tr>
			<td>Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos&#8217; Modified Eagle&#8217;s Medium, low glucose[Gibco, #12320-032 ]).&#160;</td>
		</tr>
		<tr>
			<td>Gelatin (100&#8209;200 &#956;g/cm2, Sigma, #G1393).</td>
		</tr>
		<tr>
			<td>1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484),&#160; FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006),&#160; Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372).</td>
		</tr>
		<tr>
			<td>Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005)</td>
		</tr>
		<tr>
			<td>One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222).&#160;</td>
		</tr>
		<tr>
			<td>100mm Peri dishes (Fisher Scientific,&#160; #07-202-516).</td>
		</tr>
		<tr>
			<td>Six-well cell culture plates (Fisher Scientific, #08-772-1B).</td>
		</tr>
		<tr>
			<td>T12.5 cultuer flask (Fisher Scientific, #50-202-076)</td>
		</tr>
		<tr>
			<td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc)</td>
		</tr>
		<tr>
			<td>Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp</td>
		</tr>
		<tr>
			<td>Centrifuge machine.</td>
		</tr>
		<tr>
			<td>Inverted phase-contrast microscope.</td>
		</tr>
		<tr>
			<td>inverted fluorescence microscope.</td>
		</tr>
	</tbody>
</table>

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.

内皮细胞发芽 自发的内皮细胞出芽来自小鼠主动脉段开始。鼠标主动脉段被允许在低生长因子基质生长然后在内皮细胞生长培养基中4天。内皮细胞出芽通常出现在2 - 4天。显微照片拍摄第4天（ 图1）。正如图片所示，无数的内皮细胞迁移从段路程。新成立的豆芽继续从段和分支延伸。 内皮细胞表型 <p class="jove_...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.3K 次观看.  Wayne State University. 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.

视频: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

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 of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 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 Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

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 ...

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 ...