The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

伦理声明:在本研究中所用的血管收获是从新鲜尸体犬获得的剩余材料（N = 4）安乐死其他无关的研究（大学3R政策）健康的狗。异常血管（内和肝外门体分流术，N = 1个）收获从提交给大学医院的乌得勒支大学的伴侣动物狗的业主知情同意后验尸。 1.隔离主犬内皮细胞的培养和<ol><li>用2ml /孔的0.5％w / v的明胶和预涂层6孔板，在37℃用5％CO 2的潮湿气氛离开2小时在恒温箱。播种原代细胞之前，除去多余的明胶溶液。 </li><li>无菌删除从新鲜尸体犬（ 图1A）血管（S）的利益（ 例如，主动脉，下腔静脉，肝门静脉）。维持血管系统的解剖结构通过将直镊子感兴趣的容器的两端。避免与镊子的组织的不必要的操作，以防止对血管内皮细胞的损伤。 <ol><li>切断与手术剪两端夹紧容器，并从尸体除去。运输中Hank氏冰上平衡盐溶液（HBSS）中。 注:大约5厘米的容器长度是优选的此过程，但那些测量1厘米还将向培养提供足够的细胞。 </li></ol></li><li>血管转移至培养皿填充用冰冷的HBSS。用手术剪，从容器的外侧除去任何粘附的组织和脂肪，保持容器本身完整（ 图1B）。确保切割时船舶在任何时候都清晰可见:拉拨周围组织用夹子或镊子最佳视图。 <ol><li>关闭船只的任何分支机构，连字（ 例如 ，聚乳糖3-0），随后将其删除与外科剪刀或手术刀。 </li></ol></li><li>小心进入具有弯曲霍尔斯特德蚊子镊子的容器端，在容器的另一端夹住钳子的前端，然后缩回，慢慢倒转容器中，直至其完全内向外（ 图1C-G）。外部现在由内皮细胞层的。如果任何困难是在反相容器中遇到，淹没在HBSS再次以减少摩擦。 </li><li>放置荷包缝合在容器的两端完全关闭它，并防止任何非内皮血管组织的消化过程（ 图1H）的曝光。使用绑带末端操纵倒置容器中。 </li><li>如果消化和文化稍后进行冷冻保存前的消化协议（步骤1.7）倒船。 <ol><li>将倒船只混和，细胞培养冻存液填充。冻结下降到-80℃，用冷冻合作ntainer。储存于-80℃，如果船只要在一周内使用。对于长期储存，在-180℃的冷冻管到位。当执行欧共体隔离，迅速解冻冷冻管在水浴（37℃），并立即放置在冰冷的HBSS。请按照1.7所示。 注意:考虑到该内皮细胞的分离后的总产率将降低，由于在冷冻过程中丧失活力。 </li></ol></li><li>容器转移至50ml管中，并在HBSS冲洗两次以除去红细胞（或剩余冷冻介质在解冻的情况下）（ 图1I）。 </li><li>消化在50ml管中在容器用30 ml胶原酶II型（0.15单位/ ml）和分散酶（0.15单位/毫升）的犬内皮细胞生长培养基（CECGM）1小时，在37℃，间歇温和溶液搅动。 </li><li>从管中取出容器，并在250离心细胞悬浮液5分钟×g下。 </li><li>重悬在CECGM一个细胞沉淀第二，每孔种子2毫升细胞悬液（1 - 3井取决于容器的大小）。培养在5％的CO 2在空气中的潮湿气氛中的细胞在37℃，并改变它的培养基每周两次。 </li><li>通道中的细胞70汇合时 - 在达到80％以上。 <ol><li>清洗细胞与预热HBSS去除死或非贴壁细胞。加入200μl重组细胞解离酶（1倍），以每个孔，并放回培养箱中5分钟或直到所有的细胞分离。 </li><li>转移细胞到15毫升管中并通过加入10ml培养基（CEGM）含10％胎牛血清（FCS）的停止胰蛋白酶消化。离心机在250×g下5分钟。继续培养明胶预涂板或瓶。 </li></ol></li></ol> 2.表征<ol><li>培养犬主动脉内皮细胞（CnAOECs）并比较初级和商业细胞系用显微镜每周两次的形态。 注:t他典型的成长内皮细胞的图案补丁是达到&gt; 70％汇合时，最能观察到。 <ol><li>培养CnAOECs在CECGM上的0.5％重量/预涂-V明胶T75烧瓶中。传代细胞每周一次当汇合为70％ - 80％。 <ol><li>为传代，用预温热的HBSS洗涤细胞一次，并加入1 ml重组细胞解离酶（1×）。放置烧瓶放回培养箱中5分钟或直到所有的细胞分离。通过加入10ml培养基（CEGM）含10％FCS的灭活胰蛋白酶。离心细胞悬浮液5分钟，在250 xg离心，弃去上清，重悬在1ml培养基中的细胞。 </li><li>取细胞悬浮液的10微升等分试样和稀释1:1 0.4％台盼蓝。计数使用自动细胞计数器的细胞，并在新的T75烧瓶积垢4.0×10 5个活细胞。在37℃下在5％的CO 2的潮湿气氛中加入10 mL预热CECGM的烧瓶中并培养。 </li></ol></li></ol></li><li>从培养CaPECs和CnAOECs的通道1分离RNA。 <ol><li>收集至少为1×10 3个细胞作为沉淀，加入20微升样品制备试剂（SPR）的孵育1分钟以裂解细胞。培养后，小心收集含在-70℃下的总RNA和存储的细胞裂解物。 </li></ol></li><li>使用按照制造商的指示的cDNA合成试剂盒（ 例如，的iScript）转换的mRNA合成cDNA。在4℃长达一个星期，或者在-20℃储存的cDNA长期直至准备执行的qPCR。 </li><li>测量血管内皮标记CD31基因的表达，以确认内皮细胞来源。执行实验装置和量化使用MIQE简要记录准则10。对于标准化，测量参考基因GAPDH，RPS19和B2MG 11的表达。 <ol><li>在无核酸酶的水制备得到的cDNA的10倍稀释。 </li><li>准备从合并的cDNA样品的4倍稀释的标准线，并使用不含核酸酶的水作为非模板对照。稀释样本五次以达到定量PCR反应的50倍的总稀释度。一式两份移取10微升的反应中使用4μl的cDNA和6微升反向和正向引物（ 表1）的20皮摩尔混合荧光团的一个384孔格式。 </li><li>程序设定为95℃，在95℃5分钟为Taq聚合酶活化，然后用10秒的39个循环变性，和在Tm 30秒退火和延伸。上述Tm为每个引物组示于表1中 。 </li><li>进行熔解曲线分析下列每一次运行，以确保只有一个产品被放大。正常化使用参考基因​​的平均相对量的样品的表达水平，并计算ΔCT如果反应效率为95％和105％之间。 </li></ol></li><li>与ANGI评估EC功能ogenesis检测。 <ol><li>添加10微升的细胞外基质的一预冷却的血管发生滑动的各孔，并用移液管尖端扩散到覆盖的表面良好。保持在冰上的细胞外基质和避免引入气泡进入凝胶。通过用水浸湿的纸巾在37℃，5％的CO 2将所述滑动在培养皿在培养箱中30分钟固化的凝胶。 </li><li>添加1.0×10 4个主 内皮在50μl内皮生长培养基每孔。孵育6小时，在37℃，5％的CO 2的幻灯片。 </li><li> 6小时后拍照用放大20倍，并确保包括整个很好的形象。 </li></ol></li></ol>

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The authors have no competing financial interests.

在研究中注重对犬内皮细胞的CnAOEC主线路用于狗3，12，13的内皮细胞谱系的模型。在人类的研究中，HUVEC文化仍然被认为是黄金标准。显然，仅仅着眼于从脐带来源的内皮细胞是心血管研究的坚定限制。内皮细胞具有特定的基因表达模式确定动规范。为了占产后血管这些差异，我们提出了一种基于血管内皮细胞的特定解剖位置这种新的隔离法。通常用于主乳油分离方法被冲洗用酶溶液的容?...

狗被用作大型动物模型为心血管疾病的研究和也可以从天生（基因）血管异常1,2受到影响。为了研究这些疾病的商业内皮细胞系通常用于评估内皮细胞（EC）的功能。狗有可用（CnAOEC）一家商业内皮细胞线，从犬主动脉的。该细胞系在研究，作为控制正常内皮细胞3-5个最常用的。在人类心血管疾病研究中最常用的内皮细胞系分别来自人脐静脉和动脉，派生人脐静脉内皮细胞（HUVEC）和人脐动脉内皮细胞（HUAECs）。内皮细胞已被用来作为自1980年代以来6在血管研究的金标准。它们被认为是研究内皮功能和疾病适应的经典模型系统。从不同的血管分离的内皮细胞的变化appearance和由于遗传背景和暴露于微环境7的功能。另外，内皮细胞和HUAECs从脐带衍生的，可能不会完全模拟成人血管相对于该条件下，它们被暴露于并响应于疾病发展的血管结构。因此，在一般翻译内皮细胞中发现的结果和HUAECs心血管疾病是不够的。 当学习适应和成年内皮细胞的行为，从感兴趣的容器主内皮应作为一个更直接的方法。为了分离这些细胞，已报道的几种方法。一种广泛描述的方法，它也用于内皮细胞，用酶消化溶液8冲洗容器。这通常会导致与非内皮细胞污染如平滑肌细胞和成纤维9。为隔离另一种常用的方法是剁碎血管组织的酶消化，随后通过荧光 -激活细胞根据分化（CD）的31 7，8。FACS分选的内皮细胞标记物群集和随后的细胞培养分选（FACS）需要相对大量的细胞的，因此不适合于内皮细胞的小血管的隔离。因此，我们旨在开发用于从各种犬血管具有高纯度的分离出纯的内皮细胞群的新的鲁棒方法。为了测试新的隔离方法的有效性，我们分离并从不同的犬动脉和静脉，大型和小型纯获得主犬内皮细胞（CAPEC）文化。这种方法还可以使从患病和/或异常血管始发如先天内或肝外门体分流，犬2的常见疾病的内皮细胞的培养。该方法允许额外的相关细胞类型相同的容器，如血管平滑肌细胞的分离，因为大多数的船只保持INT在操作过程中起作用。

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SYBR green super mix</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Recovery Cell Freezing Medium</td>
			<td style="width:164px;">Gibco/Life Technologies</td>
			<td style="width:211px;">12648-010</td>
			<td>Keep on ice prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Freezing container, Nalgene Mr. Frosty</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">C1562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Gelatin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Surgical scissors (Mayo or Metzenbaum)</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">BC555R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps&#160;</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB440R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Mosquito forceps curved</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB441R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">polyglactin 3-0</td>
			<td style="width:164px;">Ethicon</td>
			<td style="width:211px;">VCP311H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trypan blue</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated counting chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Counting Slides, Dual Chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Matrigel</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:211px;">BD356231</td>
			<td>Slowly thaw on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#181;-Slide Angiogenesis</td>
			<td style="width:164px;">Ibidi</td>
			<td style="width:211px;">81501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Endothelial Growth Medium</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EGM-2 SingleQuot Kit&#160;</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-4176</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

不同的血管被成功经受所描述的隔离协议（ 图2）。这是可能的剖析和反转主动脉，下腔静脉，肝门静脉和冠状动脉健康的狗（从每只犬的所有船只，N = 4）。用同样的方法进行内皮两个先天性门体分流术（肝和肝内，N = 1个）隔离。虽然主动脉很容易倒，胸主动脉段均高于腹主动脉更具挑战性。在胸段中的主动脉有许多肋间动脉从它的分支，这需要单独连接?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

isolation-culture-primary-endothelial-cells-from-canine-arteries

Research

JoVE Journal

Medicine

11.2K 次观看.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

视频: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...