陈竺承认达特茅斯创业基金和 Branco 的科学研究协会的支持, 由瑞士联邦管理。作者感谢 Drs. 泰伯、Benjamen a. Filas、Qiaohang 郭和云飞仕为有益的讨论, 以及匿名评论者的意见。该材料是根据国家科学基金会研究生研究金资助的工作提供的。DGE-1313911。本材料所表达的任何意见、发现和结论或建议都是作者的观点, 并不一定反映国家科学基金会的观点。

1. 组织培养培养基的制备
<ol><li>使用0.5 升瓶 Dulbecco 的改良鹰的培养基 (DMEM) 与4.5 克/升葡萄糖, 碳酸氢钠和 l-谷氨酰胺作为培养基的基础。</li>
	<li>在无菌层流罩中, 在 DMEM 的0.5 升中加入10毫升抗生素。</li>
	<li>使用无菌吸管, 将50毫升的 DMEM 抗生素溶液转移到无菌50毫升圆锥管。</li>
	<li>添加50毫升的小鸡血清到其余的 DMEM 抗生素溶液中的0.5 升瓶在消毒罩。</li>
	<li>存储最终解决方案 (以下简称鸡文化媒体 [CCM]) 在50毫升圆锥管整除数在-20 °c。</li>
</ol>2. 孵化蛋
<ol><li>使用精致的湿巾与70% 乙醇清洁受精特定病原体无白利佛诺鸡卵。在支架上排列蛋的纵向方向。</li>
	<li>打开一个鸡蛋孵化器设置一个目标温度为37.5 摄氏度, 并保持湿度在 48-55%。湿度是通过向孵化器中添加适量的水来控制的。</li>
	<li>孵化卵到 HH11-13, 大约 40-44 小时。</li>
	<li>让鸡蛋在室温下降温约 15-30 分钟之前喷洒/清洁70% 乙醇。</li>
</ol>3. 拉玻璃毛细血管
<ol><li>在微拉拔上安装10厘米长的玻璃毛细管, 外径为1.0 毫米, 内径为0.5 毫米。</li>
	<li>分别将热和拉参数设置为750和400。按下拉按钮, 将毛细管拉入细针。</li>
</ol>4. 过滤纸载体法
<ol><li>切圆直径约3厘米的过滤纸。</li>
	<li>用打孔器将矩形从圆上切约1厘米到2厘米。请确保删除任何突出或尖锐的角落。</li>
</ol>5. 胚胎的收获和准备
<ol><li>从底部裂蛋, 将壳轻轻地拉出, 小心地放入150毫米 x 15 毫米培养皿中。为了确保胚胎的一侧, 使卵在相同的方向, 他们被孵化时, 他们开裂。</li>
	<li>使用一次性的巴斯德吸管去除薄蛋白。</li>
	<li>用钝端钳将蛋黄中的浓白蛋白分离出来。通过用钝端钳轻轻刮掉蛋黄的顶部, 确保粗蛋白被除去。</li>
	<li>用细尖钳将过滤纸环放在胚胎上, 将环长轴与胚长轴相匹配。</li>
	<li>用剪刀把滤纸环周围的蛋黄切开。</li>
	<li>将环和胚从蛋黄中拉出, 朝第一个被切蛋黄的部位倾斜。</li>
	<li>用室温1x 磷酸盐缓冲盐水 (PBS) 将两个连续100毫米的盘子中的胚胎冲洗干净。</li>
	<li>先将滤纸环放入35毫米培养皿中。然后将胚背侧向上放置在35毫米培养皿中的滤纸上。</li>
	<li>将不锈钢环放在过滤纸三明治的顶端。确保不要破坏胚胎。</li>
	<li>添加3毫升以前制备的 CCM 到每个培养皿。</li>
	<li>移除每个胚胎的 VM, 通过轻轻地掠过胚胎顶端的拉毛细针, 然后从前端 (右上方的前脑) 开始剥离 vm, 然后继续脊索 (图 1A)。</li>
	<li>将八35毫米的培养皿放入一个 150 mm 的盘子里, 里面衬着水饱和细腻的任务湿巾 (以保持湿度)。</li>
	<li>将150毫米的培养皿放入一个密封塑料袋中, 然后用由 95% O2和 5% CO2组成的气体混合物填充袋子。</li>
	<li>密封袋, 并把它放在一个37.5 摄氏度孵化器。</li>
	<li>孵化胚胎27小时, 直到 HH15-HH16 (图 1B)。</li>
</ol>6. 诱发表面张力
<ol><li>从孵化器中取出胚胎, 并使用光学相干层析成像 (OCT) 系统对其进行图像处理。使用 OCT 确定神经管 (NT) 的扭转角度 (图 2)。</li>
	<li>将胚胎移植到光显微镜下, 并以10X 倍的放大可视化。使用200微升吸管增量从培养皿中移除0.2 毫升培养基。</li>
	<li>每隔一段时间取 brightfield 图像, 观察培养基-空气界面对胚胎的影响。</li>
	<li>移除介质, 直到整个胚胎表面张力诱发扭转 (图 1C)。</li>
	<li>再次利用 OCT 系统对胚胎进行图像, 建立与控制胚胎的最终扭转角。 注: 使用解剖显微镜获得明亮的图像。采用附有显微镜的光学相干层析成像系统获取活胚的横断面图像栈。图像是在 3 x 10 毫米 x 3 毫米3扫描域中获得的, 然后在成像软件中进行处理。最后, 采用数字式单透镜反射相机对物理模型图像进行了分析。</li>
</ol>7. 表面张力/VM 力的物理模拟
<ol><li>开发一个简化的3D 几何, 它类似于商业建模软件中 HH14-17 之间的胚胎 (图 3A)。</li>
	<li>设计了商用3D 计算机图形学软件中3D 几何的负模。</li>
	<li>使用3D 打印机加载1.75 毫米丙烯腈丁二烯苯乙烯灯丝3D 打印设计的模具, 以 stereolithographic (. stl) 格式。</li>
	<li>铸造模具, 将硅橡胶弹性体元件 A 和 B 混合在同等部位, 并及时倒入模具中;设置铸件模具在室温下固化12小时 (图 3B)。</li>
	<li>将胚胎的物理模型标记在背侧的 NT 上, 以可视化扭转。</li>
	<li>使用盖玻片复制用于模拟 VM 或表面张力的3D 物理模型的力(图 3D)。</li>
	<li>在物理模型的侧面插入一系列相同长度的刚性导线。盖玻片在大脑模型上施加外力后, 导线就会倾斜, 这取决于位置。通过 arctan 每根导线的原始长度来确定旋转角度 (图 3E)。</li>
</ol>8. 改变心脏回路的方向
<ol><li>按照3.1 和3.2 中的步骤, 获得拉毛管。</li>
	<li>按照5.1 到5.10 的步骤准备胚胎。</li>
	<li>使用一对镊子翻转过滤纸, 使胚胎成为腹侧。</li>
	<li>用拉毛管切开 splanchnopleure (SPL) 膜中的狭缝。</li>
	<li>用毛细管施加机械力, 将心脏从右手侧推到左手边。</li>
	<li>按照5.12 到5.15 的步骤观察扭转的变化。</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Peebles, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+proton+spectroscopy+and+diffusion+weighted+imaging+of+chick+embryo+brain+in+ovo.">Magnetic resonance proton spectroscopy and diffusion weighted imaging of chick embryo brain in ovo.</a> Dev. Brain Res. 141, 101-107 (2003).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerberus+regulates+left-right+asymmetry+of+the+embryonic+head+and+heart.">Cerberus regulates left-right asymmetry of the embryonic head and heart.</a> Curr. Biol. 9, 931-938 (1999).</li></ol>

作者声明没有利益冲突。

虽然物理现象在形态发生中扮演一个不可分割的角色26,27,28,29,30, 具体的机械机制, 以及协调机械和分子机制, 仍然主要未探索。据了解, 原脑的腹壁弯曲和向右扭转是导致早期胚胎形态发生的两个中心过程18,31,...

现代发展生物学研究主要集中在从分子遗传学的角度理解发展问题1,2,3,4,5,6,7,8,9,10,11,12,13. 众所周知, 物理现象在形态发生中起着中心作用, 或产生生物形态14,15,16,17;然而, 具体的发展机械机制仍然很大程度上研究忽略。在汉堡包-汉密尔顿阶段11 (HH 11)18中, 原始脑管的腹壁弯曲和向右扭转是导致胚胎形状变化的两个主要过程1920。特别是, 胚胎大脑扭转发育的物理机制仍然不完全理解。
鸡胚的胚胎扭转是左-右 (l-R) 发育不对称的最早形态发生事件之一。当 l-R 不对称的过程受到扰动时, 诸如内脏、异构或heterotaxia等出生缺陷将发生在21中。 在这里, 我们提出一个协议, 其中结合前蛋实验22,23与物理建模, 以表征机械力在早期胚胎脑发育。所提出的方法的目的是确定在早期开发过程中负责大脑扭转的机械力和影响扭转程度的因素12。通过实验观察, 卵黄膜 (vm) 对胚胎的背侧有一定的约束, 我们假设 vm 提供了诱发发育脑扭转的必要力量。因此, 在这个方法中, 我们删除了覆盖大脑区域的 VM 的一部分, 以找出对大脑扭转的影响。此外, 采用流体表面张力的方法, 以确定 VM 的机械作用, 并提供了对大脑扭转所需的力量的估计, 这是以前没有做过的。在胚胎形态发生过程中测量力是一项艰巨的任务。值得注意的是, 在一项开创性的研究中, Campàs 和同事24开发了一种新的方法, 用注射 microdroplets 量化细胞应力。然而, 这种方法仅限于测量细胞水平的力量, 因此不适用于组织或生物体级的探针力。本文提出的协议部分填补了这一空白。

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Fertilized Specific pathogen-free White Leghorn chicken eggs</td>
			<td style="width:177px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:288px;">Optical Coherent Tomography Microscope</td>
			<td style="width:177px;">Thorlabs</td>
			<td style="width:119px;">GAN220C1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:288px;">Silicone elastomer</td>
			<td style="width:177px;">Smooth-On, Inc.</td>
			<td style="width:119px;">EcoFlex 00-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:119px;">MZ8</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:288px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Lonza</td>
			<td style="width:119px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Antibiotics</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">P4083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Chick serum</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">C5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Micropipette puller</td>
			<td style="width:177px;">Sutter Instrument</td>
			<td style="width:119px;">Model P-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Filter paper</td>
			<td style="width:177px;">Whatman</td>
			<td style="width:119px;">5202-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Phosphate buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Comsol MultiPhysics</td>
			<td style="width:177px;">Comsol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">3D computer graphics software</td>
			<td style="width:177px;">Rhino 5</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Microscope attached with OCT</td>
			<td style="width:177px;">Nikon&#160;</td>
			<td style="width:119px;">FN1</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:288px;">Digital single-lens reflex camera</td>
			<td style="width:177px;">EOS&#160;</td>
			<td style="width:119px;">Rebel T3i</td>
			<td></td>
		</tr>
	</tbody>
</table>

probing the roles of physical forces in early chick embryonic morphogenesis

胚胎发育通常是从生物分子遗传学的角度来研究的, 但力学在形态发生中的基本重要性越来越被人们所认识。特别是, 胚胎小鸡心脏和脑管, 经过剧烈的形态学变化, 因为他们的发展, 是主要的候选者, 研究物理力量在形态发生中的作用。小管状胚胎鸡脑的渐进腹侧弯曲和向右扭转发生在鸡胚胎发育的器官水平左-右不对称的最早阶段。卵黄膜 (VM) 约束胚胎的背侧, 并有牵连提供必要的力量, 以诱发发展中的大脑扭转。在这里, 我们提出了新的前蛋实验和物理建模的组合, 以确定大脑扭转的力学。在汉堡-汉密尔顿阶段 11, 胚胎是收获和培养的前蛋 (在媒体)。然后使用拉毛管将 VM 移除。通过控制流体的水平和将胚胎置于流体-空气界面上, 介质的流体表面张力可以用来代替 VM 的机械作用。同时进行显微外科实验, 改变心脏的位置, 以发现脑扭转的手性改变。该协议的结果说明了力学在驱动形态发生中的基本作用。

在本研究中, 胚胎在 HH11 的 VM 从前端移除到胸椎弯曲。胚胎被一个 OCT 系统所成像。在这个阶段, 脑管的扭转尚未启动 (图 1A)。在孵化到 HH15-16 后, 胚胎与其 VM 删除显示减少脑管扭转, 约35度 (图 1B) 相比, 控制胚胎, 显示扭转约90度。当培养基水平降低, 以诱导胚胎的背端表面张力与其 vm 移除, 大脑扭曲的水平可比控制胚胎 (<strong c...

Watch this Scientific Journal Video about Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis at JoVE.com

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

早期雏鸡胚胎形态发生中物理力的作用探讨

在这里, 我们提出了一个协议, 介绍了一套新的前蛋实验和物理建模方法来研究早期鸡胚脑扭转的形态发生机制。

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

probing-roles-physical-forces-early-chick-embryonic

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Dartmouth College. 在这里, 我们提出了一个协议, 介绍了一套新的前蛋实验和物理建模方法来研究早期鸡胚脑扭转的形态发生机制。

视频: Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

测微探针进行远程磁驱动<em&gt;原位</em&gt; 3D细菌生物膜物理性质的映射

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...