这项工作由奥地利科学院 (星期三) 的一个分开的津贴支持, 捷克共和国项目的技术代办处没有。TA04010877 (星期三、VH 和 JGS) 和捷克科学基金会 (GACR) 项目没有 GA16-01246S (JGS)。我们感谢 w 泰康的建议, 他使用假牙固定霜, 和 Ing。p. Liska 从捷克陆军森林服务处获得帮助, 获得了 larynges 的切除鹿。

本文分析的动物标本是按照捷克共和国奥洛穆茨 Palacky 大学的标准伦理要求进行处理的。它们来源于在森林中生活的红鹿, 在一个正常狩猎季节, 它们被捷克陆军森林局猎杀。
1. 半喉标本的制备
注意: 如4所示, 只应使用正确准备的标本。快速冷冻喉部25在切除和储存-80 ° c 后立即将组织退化和生物力学特性改变的可能性降到最低, 并允许在任何方便的时间进行实验。
<ol><li>
除霜喉
	<ol>
<li>将冷冻喉插入两个高压釜袋或任何其他带有防水密封的塑料袋中。密封袋, 并把它们放入水浴加热到30° c, 直到喉完全解冻。所需的时间范围从几个小时到超过一天, 取决于喉的大小和冻结温度。</li>
	</ol>
</li>
	<li>
清洁喉部
	<ol>
<li>喉解冻后, 将其从袋中取出, 用生理盐水 (0.9% 氯化钠) 彻底清洁。</li>
		<li>小心地去除多余的组织 (如外颈部肌肉, 舌骨等) 而不损害主喉结构, 并将气管缩短至足够的长度, 以将喉安装在供气管上 (通常约4-5 厘米)。</li>
		<li>检查喉组织的潜在组织异常, 如伤口, 有机变形, 或裂缝可能发生的冻结过程, 这可能使喉不适合的实验。</li>
	</ol>
</li>
	<li>
甲状腺和环状软骨软骨的暴露
	<ol>
<li>用手术刀除去甲状腺和环状软骨软骨周围的喉部肌肉组织的部分, 从而使软骨在准备 mid-sagittal 切口时产生半喉。此准备阶段在图 1A和1B中描述。</li>
	</ol>
</li>
	<li>
甲状腺软骨中矢状切口
	<ol>
<li>通过甲状腺软骨的前部进行初始垂直切口。</li>
		<li>小心地将切口稍微放在将要被移除的一侧, 以免破坏需要保持的声音褶皱。如果可能, 使用手术刀切割。如果软骨是僵化的, 使用一个小锯。</li>
	</ol>
</li>
	<li>
切割环状软骨软骨
	<ol>
<li>引导切口垂直 (下) 从在杓软骨之间, 然后通过环状软骨软骨到一个近似水平水平的下等甲状腺切口。</li>
	</ol>
</li>
	<li>
切除一声带, 在喉部形成 L 形切口
	<ol>
<li>从先前做的环状软骨软骨的垂直切口的下端开始进行水平切口, 并将新的切口引向下甲状腺切口。前折叠喉的一侧将被删除。</li>
		<li>通过在甲状腺软骨内侧的软组织上进行垂直切开--要小心, 同时将声带前部附着之间的切口引导到甲状腺软骨, 从而避免声带损伤。</li>
	</ol>
</li>
	<li>
切开通过甲状腺软骨的提炼
	<ol>
<li>使用手术刀、锯子或锉, 以便在甲状腺软骨中应用精确的直切口, 并尽可能接近先前检查过的声带的前部。</li>
		<li>也去除后甲状腺软骨的一个小部分, 为了创造空间为插入的刺为 adducting 杓软骨和因而声音折叠 (如下所示)。此准备阶段在图 1C和1D中描述。 注: 根据研究问题, 可能需要对整个声带进行充分的阐述, 以使其可见性。在这种情况下, 应该去掉 (真实的) 声带上方的结构 (即心室或前庭的褶皱, 如适用于标本的解剖)。在一些标本中, 声带上方的喉部软组织可能会失去与甲状腺软骨的连接, 并在振动时干扰声带, 有可能造成假 (大多不规则) 振荡模式。在这种情况下, 仔细清除该组织是不可避免的。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg"> 图 1: 半喉准备和安装.(A) 和 (B) 清除喉标本、内侧和后视, 然后去除左声带;(C) 和 (D) 准备了 L 形切口 (左声带移除)、内侧和后视的半喉。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

2. 半喉实验
<ol><li>
半喉安装
	<ol>
<li>使用一个供气管, 提供温暖和湿润的空气进入喉。</li>
		<li>构造两个垂直排列的透明板作为替代切除喉部。</li>
		<li>使用尖头4提高喉部的稳定性, 并通过将剩余的声音折叠 adducting 到垂直玻璃板 (参见图 2A), 从而创建适当的 pre-phonatory 喉配置。 注: 理论上, 在滑轮-杠杆系统26中, 声带和重量也可能内。然而, 对这些作者最好的了解, 这种方法尚未被尝试进行 hemilarynx 的准备。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg">
图 2: 半喉设置.(A) 支承结构: 供气管、L 形玻璃板排列、收叉。(B) 装入的半喉准备。(C) 和 (D) 半喉准备的特写镜头, 分别从侧面和顶部观看。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>
安装半喉
	<ol>
<li>用假牙固定膏盖住供气管, 用气管的剩余部分安装喉部。定影霜的作用是粘合剂, 并关闭潜在的间隙, 从而产生密封。</li>
		<li>用塑料收紧带或软管钳固定气管。</li>
		<li>还要用固定膏覆盖切口通过甲状腺软骨的边缘, 同时避免在声带或喉内软组织上涂上固定乳膏。</li>
		<li>附上透明的盘子。</li>
	</ol>
</li>
	<li>
甲状腺软骨的稳定, 声带的内收, 使用尖头
	<ol>
<li>用尖头将声带加到板上, 稳定甲状腺软骨。</li>
		<li>固定乳膏设置后, 应用气流以建立声带振荡, 并检查在半喉和玻璃板之间可能出现的渗漏。</li>
		<li>通过添加更多的固定乳膏来密封最终出现的间隙。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

半喉预备具有 "常规" (完全) 切除喉设置的优点: 在这种实验方法中, 物理和生理边界条件和参数 (如门下压力或声带伸长) 可以控制得相当好。hemilarynx 的行为与一个完全的横向对称的全喉是同源的, 除了某些参数的大小 (e. g, 空气流速, 声压) 减少了大约 50%, 但仍然是在实际范围为16。完全切除喉的方法的主要缺点, i. e., 由于在半喉设置中克服了声带表面的不可见性 (caudo-颅) 尺寸, 通过提供振动声乐的侧面视图倍.因此, 半喉设置可以评估声带运动在多个维度, 这是至关重要的, 当试图了解更精细的细节, 生物物理发声机制在人类和非人的哺乳动物。
在这里, 几个示范应用的半喉设置已被证明。从两个摄像机角度的声带振动文献可以进一步定性和定量数据分析。本文新介绍的垂直方向 kymographic 声门运动分析, 允许沿前后 (背腹) 声门轴沿选定位置重建声门的颞几何变化。当重复这一分析的几个点等距间隔沿声门轴, 整个声门运动可以重建。请注意, 这一方法提供了可比的结果, 但与声带运动的评估相比, 通过标记和追踪声带组织中的个别 "fleshpoints" (也在不构成声门的点上),例如,微缝合20或碳化硅粒子5,40。关于三维的时变声门几何的精确知识对于进一步研究声门气流的细节及其与振动喉组织的相互作用至关重要。例如, 在气流射流分离点 41、42、43、44的经验数据中, 可自我维持的声音褶皱振动的计算模型可以得到改进. 45,46,47,48变为可用。
如图 5所示, 半喉准备能够在自我维持的声带振动中评估声带接触面积 (VFCA)。第一, 知识的时变相对大小的 VFCA 是有用的验证结果从声门测量27, 因为鸡蛋是一种广泛使用的方法, 非侵入性评估声带振动在体内。此外, 精确的 VFCA 几何的测量和它的变化随着时间的推移可能被证明是至关重要的, 以更好地理解声带接触深度的概念49及其与 so-called 粘膜波速度的潜在关系50,51,52,53. 在那里, 一个气流驱动的行波发生在声音褶皱组织的表面覆盖层内。这一波的移动最初伴随着从下到上的声音褶皱边缘的跨喉声门气流, 然后它在每一个振荡周期54上横向传播到上面的声音褶皱表面。
考虑到所有的事情, 半喉的方法是一个强大的, 但没有广泛使用的组成部分目前可用的经验方法的基础语音科学的武库。在这里, 介绍了创建半喉准备的教程, 并讨论了未来的一些潜在的应用。给定的指令可能有助于提高不同实验室的实验重复性, 从而为语音科学家提供了更好地理解语音生产生物力学的潜力。

声音通常是由振动喉组织 (主要是声带) 产生的, 它将由肺部提供的稳定气流转换成一系列气流脉冲。声波压力波形 (即, 主要声音) 出现从这个序列的流动脉冲声学兴奋的声带过滤他们, 并由此产生的声音从口和 (在一定程度上) 从鼻子辐射1.所产生的声音的光谱组成很大程度上受声带振动质量的影响, 受喉生物力学和与气管气流的相互作用2。无论是在临床和研究的背景下, 文献和评价声带振动因此是最重要的兴趣时, 研究语音生产。
在人类中, 对喉的直接内窥镜调查在酣然的生产在体内是富有挑战性的, 并且它几乎不可能在非人的哺乳动物, 鉴于当前技术手段。因此, 为了保证仔细控制物理和/或生理实验的边界条件, 使用切除 larynges3,4在许多情况下是充分替代研究的在体内语音生成机制。
声音褶皱振动是一个复杂的三维现象5。传统的调查方法, 如喉内窥镜 (在体内) 或切除喉的准备工作通常只提供一个优越的观点振动声乐褶皱6, 他们不允许完整的三维分析声音折叠运动。特别是在高级视图中, 在振动周期的主要部分中, 声带的下部 (尾缘) 边缘是不可见的。这是由于下 (尾) 与声带的上级 (颅) 边缘之间的相位延迟, 这一现象通常在声带振荡5时出现。直接经验主义的证据为备份发现从数学和物理模型是缺乏的, 知识的几何和运动的更低的声带边缘7, 并且因而几何门下渠道8,9,10对于更好地理解喉气流、声带组织和产生的力和压力之间的相互作用至关重要,1112。声带振动的另一个方面是隐藏在传统的优越观点是垂直 (caudo 颅) 的接触之间的两个声音褶皱的深度。垂直接触深度与声带的垂向厚度有关, 这是歌唱中用于演唱 ("胸部" 与 "假声" 寄存器)1314的潜在指示器。
为了克服常规 (充分) 切除喉准备的缺点, 可以使用 so-called 半喉设置, 其中一半喉被删除, 从而促进评估的振动特性的剩余声带在三维度。令人惊讶的是, 自从在二十世纪六十年代15中引入此设置并在 1993年16中对概念进行了初步验证之后, 没有多少实验室用这种有希望的实验方法进行了实验17,18 ,19,20,21,22,23。对此的解释可能存在于建立一个可行的半喉准备的困难。虽然常规切除 (完整) 喉准备是很好的文件4, 没有这样的 in-depth 指令是目前尚未可用于创建一个半喉设置。因此, 本文的目的是提供一个教程, 建立一个可靠的可再生的半喉设置, 并辅以红鹿标本的实验结果。
一个半喉安装与 "常规" 切除喉设置共享许多功能, 如测量设备, 高速或其他成像技术, 以充分记录在声音产生的喉结构的振动, 或适当供应加热, 湿润的空气。这些一般的设置注意事项将在书的4和国家语音和语音中心的技术报告24中详细介绍。重申这些指示将超出本手稿的范围。在这里, 只有专门的指令, 生成一个半喉设置提出。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td >Surgical blades</td>
			<td >Surgeon</td>
			<td >Jai Surgical Ltd., New Delhi, India</td>
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			<td >Saw</td>
			<td >Hand saw (Lux, 150 mm length)</td>
			<td >Lux, Wermelskirchen, Germany</td>
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			<td >Thermometer</td>
			<td >Testo 922</td>
			<td >Testo Ltd., Hampshire, UK</td>
			<td >K-type Probe, Operating temperature -20 to +50 &#176;C</td>
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			<td >Autoclave bags</td>
			<td >Autoclave bags</td>
			<td >vwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic</td>
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			<td >Conductive glass plates</td>
			<td >Custom made</td>
			<td >UPOL - Joint laboratory of Optics 
			Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td>
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			<td >Denture fixative cream</td>
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			<td >Customized Kanya Al eloxed profiles</td>
			<td >Distributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.;</td>
			<td >&#160;Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs</td>
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			<td >Mounting tube</td>
			<td >Custom made</td>
			<td >UPOL - Joint laboratory of Optics, 
			Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td>
			<td ></td>
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			<td >LED Light</td>
			<td >Verbatim 52204 LED Lamp</td>
			<td >Mitsubishi Chemical Holdings Corporation, Tokyo, Japan</td>
			<td ></td>
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			<td >Camera</td>
			<td >Canon EOS1100D</td>
			<td >Canon Inc.</td>
			<td >18-55 mm lens</td>
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			<td >Humidifier</td>
			<td >Custom made</td>
			<td >Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td>
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			<td >Subglottic tract</td>
			<td >Custom made adjustable subglottic tract</td>
			<td >Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td>
			<td >Hampala, V., Svec, Jan, Schovanek, P., and Mandat, D. Uzitny vzor c. 25585: Model subglotickeho traktu. [Utility model no. 25585: Model of subglottal tract] (In Czech) Soukup, P. 2013-27834(CZ 25505 U1), 1-7. 24-6-2013. Praha, Urad prumysloveho vlastnictvi</td>
		</tr>
	</tbody>
</table>

hemi-laryngeal setup for studying vocal fold vibration in three dimensions

人类和大多数非人类哺乳动物的声音是通过声带的自我维持振荡在喉部产生的。直接视觉文献的声带振动是挑战, 特别是在非人类的哺乳动物。作为替代, 切除喉实验提供了机会, 研究声带振动在受控生理和物理条件。然而, 使用一个完整的喉只是提供了一个顶部视图的声音褶皱, 不包括关键部分的摆动结构的观察期间, 他们与空气动力学的力量的互动。这一限制可以克服, 利用半喉设置, 其中一半喉是 mid-sagittally 移除, 提供了一个优越的和横向的意见, 其余的声带在自我维持振荡。
在此, 给出了半喉结构的解剖学准备和在实验室工作台上安装的 step-by 步骤指南。发声的半喉准备的模范性的记录与高速视频数据捕获的两个同步相机 (优越和横向的意见), 显示三维声带运动和相应的时变接触面积。该出版物的半喉设置文件将有助于在实验研究中的应用和可靠的重复性, 为语音科学家提供了更好地理解声音生产的生物力学的潜力。

如前一节所提到的, 半喉准备的插图及其在供气管上的安装, 分别在图 1和图 2中提供。
从两个摄像机角度来记录声带振动
气流诱导的半喉声带自我维持振荡从顶部和侧面记录了两个同步高速视频 (HSV) 摄像头在6000帧/秒运行, 辅以 time-synchronous 录音的声学和声门 (见下文) 采样44.1 赫的数据。此组作者27,28, 在以前的出版物中可以找到有关数据获取设置 (包括所用设备列表) 的更多信息。从这些 HSV 录音录像显示在伴随的视频。静止图像在振动周期内的代表时刻提取, 如图 3所示。顶部视图 (每个面板的上半部分) 显示译侧声音折叠运动, 在图 3A中表示打开的声门, 允许声门气流, 而在图 3B-d声门关闭 (声带完全接触垂直玻璃板), 从而逮捕声门气流。图 3 b-D中的侧面视图 (每个面板的下半部分) 建议不同程度的声音折叠与玻璃板的接触, 以及该触点的不同几何形状和垂直位置。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55303/55303fig3.jpg"> 图 3: 半喉声带振动.(A-D)静止图像从高速视频素材的顶部 (每个面板的上半部分) 和侧面视图摄像机 (每个面板的下半部分), 提取在代表点在振动周期。注意在 (a) 中没有声带的接触, 以及在 (B-D) 中的声音褶皱接触 (在区域、形状和位置上) 的变化。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
Kymographic 声门运动分析
定量声门运动分析在图 4中进行了说明。声门是在 (振动) 声音折叠29之间的可变开口, 由它们在自我维持振荡过程中的挠度产生。顶部视图的艺术分析的状态 HSV 素材允许追踪声带的侧向挠度30,31。这里描述的半喉准备增加了评估声带振动的垂直 (caudo 颅) 方面的设施。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55303/55303fig4.jpg"> 图 4: Kymographic 声门运动分析. (A) 和 (B) 视频剧照显示半喉的顶部和侧面视图, 取自6000帧/秒的高速视频 (HSV) 录制。黄色垂直线指示在顶部视图的面板 C 和 E 中显示的 kymograms 的 kymographic 扫描线位置, 以及侧面视图的面板 D 和 F。(C) 和 (D) 数字 kymograms 分别从顶部和侧面视图的 HSV 片段中提取。(E) 从 kymogram 中提取的声音褶皱的时变侧移, 并用直线 (短划线) 跟踪。(F) 声音折叠的下边缘和上级边的时变挠度, 由 kymogram 进行评估, 并分别以虚线和虚线进行跟踪。(G) 时变声门结构的天气描述: 侧向声音褶皱偏转 ("顶部"、淡紫色) 和上级 ("侧向"、暗红色) 和下位 ("侧面 inf"、深绿色) 声带边缘提取该 kymograms 显示在面板 E 和 F. (H) 的运动速度从声门结构位移数据所示的面板 G. (I) 和 (J) 声门运动重建从上级的位移数据导出低的声带边缘显示在面板 G。箭头指示旋转运动的方向。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
两个数字 kymograms 是从顶部和侧面视图 HSV 数据 (图4C 和 4D) 生成的。在数字 kymogram (DKG)32,33,3435中, 从一条直线的像素数据 (通常是在最大声音褶皱振动振幅点), 取自数个连续的高速视频帧, 串联在横坐标上形成一个时间轴。DKG 扫描线所覆盖结构的时变位移在纵坐标上是可见的。在图 4C-F中显示的示例中, 使用 Hampala et al所描述的方法, 在声音折叠的前后 (腹背) 维度上选择了顶部和侧面视图的 DKG 扫描线位置。Eq 127。
声门的侧面和 caudo-颅偏转, 由下位和优越的声带边缘划定, 在 DKG 数据 (图4E 和 4F) 内被追踪, 并以公制单位为基础, 以视频帧速率和校准信息为嵌入视频 (图 4G和H)。two-dimensional (横向和垂直) 声门运动在声带中部 (即最大振动振幅的地方) 超过三完整的声门循环的重建显示在图 4E和F。在声门循环的大多数, 声带与玻璃板 (代表声门关闭), 但接触深度不同。在开放阶段 (i. e., 当声带与玻璃板不接触时), 劣质和高级声带边缘保险丝的痕迹, 并且他们陈列一个复杂的循环运动样式, 与其他研究结果的部分协议5,20,36,37 (在人体中发现的运动模式比在这里调查的红鹿标本更容易省略)。有趣的是, 垂直位移达到了一个振动振幅约10毫米,即, 几乎是一个数量级比在人类发现的更大。
声带接触面积的评定
图 (鸡蛋)38是一种广泛使用的非侵入性方法, 用于测量发声期间相对声带接触面积 (VFCA) 的变化。一个低强度, 高频电流是通过两个电极放置在声带水平上的每一侧喉。喉发声过程中声带 (de) 接触引起的导纳变化与时变的相对声音褶皱接触面积39成正比。该卵信号被认为是一个可靠的生理关联的声带振动, 反映了基本频率和振荡制度 (不规则或周期性, 包括分岔)。尽管它的广泛应用, VFCA 和卵波形之间可能的直接关系, 直到最近, 只在一项研究17中进行了测试, 这表明了 VFCA 和卵信号的大小之间的近似线性关系。然而, 在该研究中并没有研究流动诱发的声带振动。因此, 在适当的生理条件下, 对卵子进行严格的经验评估, 以此作为衡量相对 VFCA 的方法仍然是必要的。
在解决这个问题时, 这组作者最近调查了三红鹿 larynges 在切除半喉准备利用导电玻璃板27。在 6000 fps 的矢状面上进行高速视频录制, 对声带与玻璃板之间的时间变化进行了监测, 并与鸡蛋信号同步, 准确度为± 0.167 ms. 代表的结果显示在图 5, 表示卵子信号和 VFCA 之间的平均良好协议–请参阅参考27以了解详细信息)。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55303/55303fig5.jpg"> 图 5: 比较声带接触面积 (VFCA) 和声门 (卵) 波形.(A-D)视频剧照从高速视频数据显示的侧面看, 红鹿半喉在四瞬时在一个声门循环。人工评估的声带接触面积 (即, 在半喉设置中, 声带与垂直玻璃板接触的区域) 以青色叠加。(E) 对一个声门循环的声带接触阶段的规范化卵和 VFCA 数据进行比较。VFCA 数据来源于对声门循环的声带接触面积 (以像素计) 的评估。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions at JoVE.com

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

本文介绍了一种用于制备用于促进声音褶皱振动多维观的半喉标本的协议, 目的是研究人类和非人类哺乳动物发声的各种生物物理方面。

三维声带振动的半喉设置研究

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

hemi-laryngeal-setup-for-studying-vocal-fold-vibration-three

Nanyang Technological University

Research

JoVE Journal

Biology

10.9K 次观看.  Palacky University Olomouc. 本文介绍了一种用于制备用于促进声音褶皱振动多维观的半喉标本的协议, 目的是研究人类和非人类哺乳动物发声的各种生物物理方面。

视频: Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

四重检查板:研究药物组合的三维棋盘的修改

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

三维细胞外囊泡的直接随机光学重建显微镜

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Studying Habituation in Stentor coeruleus

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

Studying Habituation in Stentor coeruleus

We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

研究腔规则支架的习惯化

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display ...