这项工作得到了曼佩铃木糖尿病基金会（YT）、波士顿儿童医院 OFD/BTREC/CTREC 教师职业发展补助金、波士顿儿童医院眼科基金会、BCH 试点奖、BCH 曼顿中心奖学金和小长颈鹿基金会 （ZF）、德国研究基金会 （DFG; 到 BC [CA1940/1-1]）， NIH R24EY024868， EY017017， R01717-13S1， EY030904-01， BCH IDDRC （1U54HD090255）， 马萨诸塞州狮子眼基金会 （LEHS）.

所述的所有动物实验都得到波士顿儿童医院机构动物护理和使用委员会的批准（ARCH协议号19-04-3913R）。
1. 准备
<ol><li>加入5 mL的青霉素/链霉素（10000 U/mL）和5 mL和10 mL的商用补充剂到500 mL的完整经典介质与血清。最初为 50 mL 的介质的 Aliquot。 注:请勿将任何介质退回库存，以免受到污染。</li>
	<li>在冰上放一完全经典介质。</li>
	<li>使用 70% 乙醇清洁解剖显微镜、钳子和剪刀。</li>
	<li>准备两个细胞培养皿（10厘米），一个在解剖显微镜上，一个在冰上;在每道菜中放入 10 mL 的完整经典介质。</li>
</ol>2. 实验步骤 （图1）
<ol><li>牺牲C57BL/6J小鼠周围的产后（P） 20使用75-100毫克/千克氯胺酮和7.5-10毫克/千克木氨酸注射在陷阱酮。解剖前，在冰上保持眼睛完全的经典介质。</li>
	<li>去除结缔组织（肌肉和脂肪组织）和眼睛上的视神经。</li>
	<li>使用微剪刀圆圆切 0.5 mm 后角膜边缘。去除角膜/虹膜复合物、玻璃和透镜。</li>
	<li>使切削边缘垂直于视神经的切口为 1 mm，并切割 1 mm 宽度的圆周带。将建筑群的中央和外围区域分开。使用钳子从RPE/胆状/四分层复合物中剥离视网膜。</li>
	<li>保持外设胆小球带在冰上完全经典介质;隔离另一只眼睛，并重复这个过程，以削减第二个带。</li>
	<li>将圆形带切成 6 = 相等的方形片（±1 mm x 1 mm）。 注:切勿接触任何边缘。</li>
	<li>根据制造商的指示解冻基底膜提取物 （BME）。将 30 μL/μL/井的 BME 添加到 24 井组织培养板的每个井的中心。确保 BME 的液滴在板底部形成凸形圆顶，而不接触边缘。 注:在冰箱中通宵解冻 BME。解冻后，BME 应随时在冰上。</li>
	<li>将组织放在 BME 的中间。 注:不要压平胆状外植;一般来说，让组织在BME内膨胀。组织的方向（侧向上或向下）不会影响实验结果。</li>
	<li>在37°C下孵育板10分钟，让凝胶凝固。</li>
	<li>加入 500 μL 的完整经典介质/井。</li>
	<li>每隔一天更换一次经典介质（500 μL）。用显微镜观察3天后，可以观察到胆状芽。 注:对于生长因子治疗，使组织挨饿4小时。稀释生长因子降低介质中的试验化合物（1:200 提升而不是 1:50）。</li>
</ol>3.SWIFT-Choroid计算机化定量方法17 （图2）
注:使用计算机化方法测量生长容器覆盖的区域。在量化之前，需要 ImageJ 软件的宏插件（有关更多详细信息 ，请参阅 补充信息）。
<ol><li>使用 ImageJ 打开 choroid 发芽图像并检查"图像|类型|8 位 "与灰度。</li>
	<li>转到"图像|调整|亮度/对比度（Ctrl/shift/C）"并优化对比度。</li>
	<li>使用魔杖功能从图像中轮廓和去除存在于芽的中心的胆状组织（使用快捷键"F1"）（图2A，B）。 注:将魔杖的容差率设置为 20-30%。</li>
	<li>使用自由选择工具移除图像的背景（图 2C）。转到"图像|调整|阈值 （Ctrl/shift/T）"。使用阈值函数在背景和外围定义微血管芽（图 2D）。</li>
	<li>单击"F2"，将显示摘要。单击"保存"来保存所选区域的图像。保存与原始图像相同的文件夹中，供将来参考。</li>
	<li>测量一组样本后，复制记录以进行数据分析。 注:也可以通过"分析"测量面积（μm2）|使用带比例条的图像设置比例"。</li>
</ol>

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提交人没有财务披露。计算机化方法通过作者免费提供给学术机构。

胆瘤发芽分析辅助研究的新血管AMD9，10，18，19，20。胆龙外植可以从老鼠以及大鼠和人类17，21分离。胆道外植包括ECs，巨噬细胞，和心状体17。在此测定胆道DC与相邻细胞（如RPE细胞）之间的相互作用，有助于阐?...

胆血管生成失调与新血管年龄相关的黄斑变性（AMD）1。胆小球是视网膜色素上皮（RPE）下的微血管床。研究表明，胆瘤中血流量的减少与AMD2的进展有关。血管内皮、RPE、巨噬细胞、心肺和其他细胞之间的复杂关系是组织3、4、5的平衡。因此，可重复的测定建模对新血管AMD的研究至关重要。
前活体血管生成测定和体外内皮细胞培养可以补充对体内微血管行为的研究，用于测试新药和机制学研究。内皮细胞，如人类视网膜微血管内皮细胞（HRMEC），人类脐带静脉内皮细胞（HUVEC），分离的原兽脑或视网膜ECs，常用于体外研究，用于眼血管生成研究6，7，8。特别是HRMEC已被广泛用作体外胆状新血管化（CNV）9的模型，通过评估内皮增殖、迁移、管状形成和血管渗漏来评估干预6、10。然而，由于在胆道中与其他细胞类型缺乏相互作用，并且由于这些测定中使用的大多数EC并非源自胆瘤，培养中的EC作为CNV的模型受到限制。在培养中，小鼠巧克力球状电子能难以分离和维护。
大动脉环测定被广泛用作大血管增殖的模型。来自大动脉外植的血管芽包括 ES、周食和巨噬细胞11。大音环测定模型大血管发生井12，13，14。然而，它作为胆囊新血管化的模型有局限性，因为大动脉环是缺乏特征的胆囊微血管环境的大血管组织，而来自大型血管的芽可能与涉及微血管病理学的毛细管网络的芽不同。最近，一个小组发表了一个前维维视网膜测定15，16。虽然，它适用于视网膜新血管疾病，但它不像在AMD中所看到的那样适合胆状新血管化。
利用小鼠RPE、胆球和胆碱外植组织进行胆瘤发芽测定，以更好地模拟CNV。组织可以很容易地从小鼠（或其他物种）的眼睛17分离。这种测定允许重复评估药理化合物的亲血管原位和抗血管原位，并评估特定途径在胆道新血管化中的作用，利用转基因小鼠和对控组18。这种胆瘤发芽测定已在许多后续出版物9，10，18，19，20被引用。在这里，演示了使用这种测定的方法。

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

病理胆瘤血管生成，年龄相关的黄斑变性的显著特征，导致视力障碍和失明。使用人类视网膜微血管内皮细胞（HRMECs）或分离原发性视网膜EC进行内皮细胞增殖检测，在体外模型中广泛使用，用于研究视网膜血管生成。然而，分离纯杂音视网膜内皮细胞在技术上具有挑战性，视网膜ECs可能有不同的增殖反应比胆状内皮细胞和不同的细胞/细胞相互作用。开发了一种高度可重复的外体胆瘤发芽测定，作为胆球微血管增殖的模型。该模型包括胆道血管（EC、巨噬细胞、心状物）和视网膜色素上皮（RPE）之间的相互作用。小鼠RPE/胆状/细胞外植在生长因子减少基膜提取物（BME）（第0天）中分离和孵育。中等每隔一天改变一次，胆瘤发芽在第6天被量化。使用倒置相显微镜拍摄单个胆瘤外植的图像，并使用本实验室开发的 ImageJ 软件半自动宏插件对发芽区域进行量化。这种可重复的外体胆瘤发芽测定可用于评估化合物的潜在治疗和微血管疾病研究，以评估使用野生型和转基因小鼠组织参与胆小血管增殖的途径。

每天胆小球发芽生长的比较
我们解剖了带sclera的胆小球，嵌入到BME中，并培养了6天（图1）。第3天至第6天C57BL/6J小鼠的胆瘤发芽用显微镜检查，用SWIFT-Choroid对图像J中的半自动定量方法进行量化。在一个代表性的案例中，第3天（图3A）的胆状发芽区（从外植延伸的容器，不包括外植本身）为0.38毫米2（图3A），?...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

该协议提供胆瘤发芽测定，微血管增殖的外体模型。这种测定可用于评估增殖胆小血管所涉及的途径，并评估使用野生型和转基因小鼠组织的药物治疗。

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

an-ex-vivo-choroid-sprouting-assay-ocular-microvascular

Research

JoVE Journal

Biology

5.5K 次观看.  Boston Children's Hospital, Harvard Medical School. 该协议提供胆瘤发芽测定，微血管增殖的外体模型。这种测定可用于评估增殖胆小血管所涉及的途径，并评估使用野生型和转基因小鼠组织的药物治疗。

视频: An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

体外机械装载肌腱

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

牛膝外生软骨重塑的外骨组织培养模型

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...