作者要感谢过去的库存实验室的成员:铭敏李凯瑟琳Plichta，维多利亚米兰达 - 汤普森和萨姆 - 金圭他们对许多这些协议的改进作出贡献。这项工作是由美国国家科学基金会资助的IOS-0840932和IOS-0724978 SP的股票投资部分

1 在体内的线虫的饲养与他们Symboitic细菌<ol><li>翻转100×15毫米的塑料培养皿中，并将滤纸（90 MM）两张碟的菜的盖子。 </li><li>均匀分布1毫升的IJ（感染性幼虫）的水悬浮液（在1000-2000 IJ / ml浓度）在滤纸上。 注:IJS不需要进行表面灭菌。 </li><li>添加更多的waxmoth 大蜡螟的10末龄幼虫的菜。的目标是提供大约100-200 IJS /幼虫。 </li><li>盖上培养皿（ 图1）的底部的盖子和标签的培养皿中。何况下面的信息:线虫种类名称（如已知）;隔离代码/名称;最新感染的陷阱设置。 </li><li>里面放置一个松散的密封塑料袋的菜（以保存水分），并保持它在黑暗中，在室温或在20-25℃的恒温箱。检查每天的菜 注:黑暗便于线虫感染，因为它模仿自然感染状况，在土壤中。 </li><li> 3-5天后，取出尸体线虫感染的迹象，以修改后的白色陷阱7。 注意:如果尸体腐烂的气味，这可能是一个迹象表明，培养不成功和/或有污染。有一批新的线虫和昆虫树立了新的传染箱。 </li></ol> 2， 体外线虫的饲养与他们共生菌<ol><li>肝肾阴虚型琼脂法9,19 <ol><li>斩肝肾小块（2 毫升或更小），并放入用NaCl，琼脂搅拌机及300ml水并混合，直到肉变成液体浆，厚浆或果浆。然后，转移混合物至1升的锥形烧瓶中。 </li><li>添加最终200毫升水，以搅拌机容器中，并弃去剩余的存储介质插入锥形烧瓶中。高压釜中进行15分钟，在121℃。可高压灭菌后的琼脂冷却浇注前触及。 注:中移液不建议，因为这种媒介往往有大块的肉。 </li><li>倒〜20毫升琼脂到5（或6）厘米的培养皿中，同时用手搅拌或纷飞的锥形瓶中，注入了更均匀的介质。让琼脂在4℃下固化和存储菜肴，直到需要 注意:使用火焰消毒金属铲，打破了任何大块的肉倒入培养皿中。 </li></ol></li><li>脂琼脂法9,19 <ol><li>通过营养肉汤，琼脂和酵母提取物混合在一起制备脂质琼脂培养基倒入混合到2升的锥形烧瓶中。加蒸馏水H 2 O和氯化镁2•6H 2 O和高压灭菌15分钟，在121℃。 </li><li>添加玉米油和玉米糖浆混合无菌混合，搅拌或摇动剧烈，经常均匀地分散油滴。 <br/&gt;注:通过将所需量的紫外线交联剂消毒玉米油和糖浆。中石油将不会溶解到液体中，但要确保它是在微小液滴。 </li><li>倒〜20毫升琼脂到5（或6）厘米的培养皿中，并允许琼脂在4°C，直到需要巩固和存储菜肴。 </li><li>数所需的共生细菌到脂质琼脂平板上，孵育板在28℃，24〜48小时。 注:价差100-200微升过夜（12-16小时）的LB亚文化。 </li><li>一天后，加入约表面消毒线虫悬浮液（或鸡蛋感染幼体）0.4毫升培养板在室温下在黑暗中或在孵化器在22±3℃。 注意:不要添加超过0.4毫升线虫悬浮液，因为它可以创建一个过于潮湿的环境，不利于线虫生长。 </li><li>日常监测的文化。文化应该生产出新一代IJS在12天左右（ 图2）。</li><li>转底菜用琼脂时，线虫看到爬行的菜（ 图3A）的两侧，以修改后的白色陷阱（见奥罗斯科等 12）收获IJS（ 图3B）。执行下在层流罩此步骤，以降低介质的污染。 </li><li>出苗后，收集修改的白色陷阱IJS和冲洗IJ停牌，以消除任何媒体碎片。商店IJ在无菌蒸馏水在10℃下（在冷温度下孵化或冷室中）或立即使用，如果需要的。 注:根据本研究的目的，与种子或者共生，殖民或aposymbiotic线虫板。 </li></ol></li></ol> 3， 体外 Aposymbiotic的饲养（共生免费）昆虫病原线虫<ol><li>感染10-20 G.蜡螟幼虫所需的线虫种类IJS（见第1节）。后3或4天（此时IJS应该已经成熟第一代成虫）收集尸体。 注:到期日，并需要获得卵子足够数量将种不同的女性数量。感染多G。蜡螟幼虫如果有必要，并早在48小时后感染，以评估他们是否在正确的发展阶段开始解剖。 </li><li>单独将每个尸体在培养皿用生理盐水溶液（M9缓冲18）。拉它的头用细尖镊子解剖尸体。 </li><li>收集至少20妊娠（里面的蛋）女性用细针（L型最好），并放置在含有10毫升M9的缓冲区（ 图4A）手表玻璃。 </li><li>通过考虑下列成分制备axenizing解决方案:6.75蒸馏水，1.25毫升5N的NaOH和2.25毫升市售漂白水稀释成3％的次氯酸盐。 注:氢氧化钠是碱性溶液，手套，护目镜和其他适当的保护衣服应穿。 注:准备新鲜axenizing溶液各一次，如漂白剂降解的光。准备axenizing液多批次，并在多个钟表眼镜进行表面灭菌的鸡蛋和收获。 </li><li>冲洗女性至少三次，M9缓冲填充表玻璃用缓冲溶液，吸干缓冲用移液管。确保女性留在表玻璃。重复此步骤两次。 </li><li>立即添加axenizing溶液并让女性保持在该溶液中不超过10-15分钟以上。观察线虫组织开始崩解，而鸡蛋（它们是耐axenizing溶液）保持不变。 </li><li>手动扰乱女性的身体，以加快这一进程，通过削减他们用针或手术刀（ 图4B，C）。吹打成的1.5 ml离心管中，避免将任何不溶解的组织取出鸡蛋。 注:使用为M任何微量离心管中根据需要来收集鸡蛋，并迅速工作，如卵的存活力将减少曝光的长时间来axenizing溶液。 </li><li>使用"高速设置"涡流管，持续15秒，以进一步除去附着在卵线虫组织。另外，如果一个涡流，则无法使用吸管上下溶液数次。 </li><li>沉淀蛋离心大约在18,000 g下2分钟。提高速度，如果鸡蛋没有足够的沉淀。除去axenizing溶液并用无菌蒸馏水和离心机填充离心管中再一次在900×g离心1分钟，冲洗两次。 </li><li>最后一次洗涤后，悬浮鸡蛋300-500微升的无菌蒸馏水，并将其放置在无菌3厘米培养皿或消毒手表玻璃。请记住，鸡蛋是现在表面消毒，所以用无菌吸管和/或pippetter提示和水，以保持蛋的清洁度。 </li><li>检查解剖显微镜（30-50X倍）下的蛋，以确认他们是否完好（ 图4D），并将它们转移到5cm的薄层板上，以肝肾阴虚型琼脂（参见2.1节） 注意:不要在板上增加超过400微升的卵悬浮液，因为它会使琼脂过度潮湿。 </li><li>标板的相关信息和在黑暗中直立存放在28℃。种蛋应该是很明显过夜。 注:上冷凝水盘的盖子，但术后1〜2天就会蒸发。在这个时候，将培养皿在塑料袋中，以保持在琼脂的水分并避免其干燥。 </li><li>定期观察菜肴和丢弃显示真菌或细菌污染的迹象，任何菜肴。一旦IJS被视为迁移了培养皿的壁，取下盖子，并用琼脂转让的底部培养皿，以修改后的白色陷阱12。转碟入modifie时，要考虑无菌条件d白平衡陷阱，以避免暴露介质的污染。 </li><li>继续监测白色陷阱和收获IJ需要。 注:Aposymbiotic线虫会继续入主陷阱的水中迁移了好几天，甚至几个星期。 </li></ol>

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没有利益冲突的声明。

使用合适的主机是成功的 EPN 体内饲养的关键因素。通常情况下，无论steinernematids和heterorhabditids可以复制，成功地完成其生命周期中的大蜡蛾， 大蜡螟 （鳞翅目:螟蛾科）幼虫。然而，其他的昆虫物种，从不同的家庭和/或单可以考虑。一些目前描述的线虫种类已知有特异性特定昆虫宿主。例如，S。 kushidai和S。 scarabaei都不是很致命的，以鳞翅目幼虫，需要鞘翅目...

昆虫病原线虫（EPN）， 斯氏和异小杆菌。 （斯氏，异小）及其共生细菌， 致病杆菌属和Photorhabdus菌（肠杆菌科）被认为是陆生动物，微生物的共生关系2-4,6,10,19。 嗜和Photorhabdus属一个新兴的模式。被包藏的共生体中的线虫的唯一自由生活阶段的肠，也被称为感染性（IJ）或第三阶段感染性8,10,13。细菌线虫对是致病性，适用范围广的昆虫，并已成功地在生物防治和综合虫害管理方案的全球6,8落实。 在这里我们将展示一系列的体内和被频繁运用的EPN的实验室条件下饲养体外技术， 我Ñ ​​体内方法考虑的昆虫宿主线虫的饲养。通常，各种昆虫的订单（ 即鳞翅目 ， 鞘翅目 ， 双翅目 ， 等 ）的未成熟阶段被认为是合适的宿主， 在体内方法通常被认为是对维护线虫培养物在实验室中。考虑到大规模生产的线虫时，这种方法可能不适合。可能需要用于此目的，要求更多的时间和相关的养虫额外成本大量昆虫宿主的。 昆虫致病性线虫，也可以在体外培养的几个媒体。根据不同的研究的目标; 体外方法可以或者考虑的共生细菌在媒体的掺入。在这份报告中，我们将介绍两种常用的方法EPN的传播。媒体的成份提供营养的共生杆菌的来源第二固醇源线虫。 体外方法提供了优势EPN的饲养没有昆虫宿主。 起初，许多体外媒体发展的被用于EPN的乘法时适合昆虫宿主不可用。然而，在过去几年中， 在体外饲养方法已被广泛应用在研究旨在了解EPN及其共生细菌17,19之间的互惠关系。 在此演示文稿详细的技术对应于那些由不同作者的库存实验室，亚利桑那州（亚利桑那州图森，美国）大学描述和细化。这些技术是从的技术，在大规模生产这些生物虫害管理的目的所使用的身体明显。

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		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:485px;">For in vivo Infections</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td style="width:488px;">25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:485px;">Filter Paper, 9 cm Grade 1 Cellulose</td>
			<td style="width:260px;">Whatman/VWR</td>
			<td style="width:488px;">28450-081</td>
			<td style="width:595px;">Grade 1 filter paper recommended</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:485px;">Insect hosts</td>
			<td style="width:260px;">Timberline</td>
			<td style="width:488px;">http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td>
			<td style="width:595px;">Galleria mellonella are recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:485px;">For Liver-Kidney Agar (for 500 ml)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:485px;">60 x 15 mm Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-092</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Beef Liver, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:485px;">Beef Kidney, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Sodium Chloride 2.5g ( 0.5% final concentration )</td>
			<td style="width:260px;">Acros/VWR</td>
			<td>200002-434</td>
			<td style="width:595px;">any research grade NaCl can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Agar, 7.5 g (1.5% agar, final concentration)</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">500 ml distilled H20&#160;</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">For Lipid Agar (for 1 L)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Nutrient Broth, 8 g</td>
			<td style="width:260px;">BD/VWR</td>
			<td>90002-660</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Yeast Extract, 5g</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM1.03753.0500</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Magnesium Chloride Hexahydrate,&#160; 10 ml (0.2g/ml)</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM-MX0045-1</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">Corn Oil, 4 ml</td>
			<td style="width:260px;">Any brand</td>
			<td style="width:488px;">Locally source; supermarket</td>
			<td style="width:595px;">Any brand of corn oil can be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:485px;">Corn Syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H20 and swirl for homogeneity</td>
			<td style="width:260px;">Karo</td>
			<td style="width:488px;">Locally sourced; supermarket</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar, 15 g</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Distilled H20,890 ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

昆虫病原线虫（EPN）（ 斯氏和异小 ）与革兰氏阴性伽马变形菌的肠杆菌科细菌， 嗜菌与steinernematids线虫相关的家人互惠的伙伴关系，而Photorhabdus是heterorhabditids的共生体。一起线虫和细菌形成一个强有力的杀虫复杂，杀死了广泛的昆虫物种中的亲密和特殊的伙伴关系。在此，我们证明在体内和这些线虫在实验室条件下饲养常用的体外技术。此外，这些技术代表了成功建立EPN文化的关键步骤，并且还形成了基础，利用这些微生物为研究其它生物检测。的aposymbiotic（共生免费）线虫的生产往往是一个深入和多方面的方法来共生的研究是至关重要的。这协议不要求另外的抗生素，并且可以在时间与标准实验室设备很短的量来完成。以这种方式生产的线虫是比较稳健的，虽然根据所使用的种类及其在存储存活率可能会发生变化。在此演示文稿详细的技术对应于那些由不同作者的P.股票的实验室，亚利桑那大学（图森，亚利桑那州，美国）描述和细化。这些技术是从的技术，在大规模生产这些生物虫害管理的目的所使用的身体明显。

体内饲养方法使用活虫为线虫生长和繁殖的主机。感染室是用于曝光的昆虫IJS的有效方法。这是线虫的生命周期载体的细菌共生的一种昆虫主机之间的唯一舞台。 图1显示了设立感染室，以及建立该室所需要的材料， 在体外饲养方式让EPN成长没有昆虫宿主，但也实施了aposymbiotic（共生免费）线虫饲养。 图2显示了一个脂质琼脂平板上用不同的线虫阶段。脂质...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

本介绍的目的是在体内和体外技术用于昆虫致病性线虫的饲养证明， 在体内方法考虑这些线虫的饲养与昆虫宿主，而在体外方法利用丰富的琼脂培养基。

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.4K 次观看.  University of Arizona. 本介绍的目的是在体内和体外技术用于昆虫致病性线虫的饲养证明， 在体内方法考虑这些线虫的饲养与昆虫宿主，而在体外方法利用丰富的琼脂培养基。 

视频: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

整个肝脏的脱细胞和Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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. ...

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 ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

在体内和昆虫病原线虫体外培育（斯氏和异小）