Name: silicon nanowires and optical stimulation for investigations of intra- and intercellular electrical coupling
Uploaded: 2021-01-28T14:00:00
Description: Watch this Scientific Journal Video about Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling at JoVE.com

这项工作得到空军科学研究办公室（AFOSR FA9550-18-1-0503）的支持。

为确保符合道德标准，芝加哥大学机构动物护理和使用委员会（IACUC）首先批准了与啮齿动物心脏隔离心肌细胞有关的所有动物程序。此外，所有动物实验都是按照芝加哥大学亚组会的指导进行的。
1. 制备细胞-SiNW杂交
<ol><li>按照制造商的指南使用商业套件隔离原发性心肌细胞 （CMs）。</li>
	<li>为原发细胞分离准备完整的DMEM，辅以10%的热灭活胎儿血清、1%青霉素链霉素和1%L-谷?...

<ol>
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我们在这里演示了一种执行细胞内电刺激的简单方法。在此演示中，我们使用与 SiGW 预混合的 MF，然后与 CMs 共同培养。一般来说，大多数增殖细胞有内化SiNW的倾向，这允许此方法与许多其他细胞类型使用。此外，虽然我们展示了细胞的细胞内刺激，但同样的原理可以用来对细胞进行细胞外刺激。这可以通过阻止内染色体级联15个细胞自发内化它们， 或使用不内化 SiNW 的细胞来?...

所有生物有机体都使用离子形式的电来调节细胞行为。细胞膜包含各种类型的特定离子通道，允许离子的被动和主动传输。这些离子控制兴奋细胞的功能，如神经元活动、骨骼和心肌收缩。然而，生物电在非兴奋细胞中也起着重要的作用，它控制着许多细胞功能，如细胞增殖1、神经免疫2、3、4和干细胞分化5。
近几十年来，生物电领域引起了越来越多的关注，这促进了生物电子接口众多技术的发展。微电极贴片移液器是细胞内记录和刺激的黄金标准6。在这种方法中，玻璃移液器在特定条件下被拉扯，形成孔径为几微米的锋利边缘。此移液器充满缓冲器，移液器允许缓冲器与细胞内体积直接接触。这会产生一个生物电接口，产生极高的信噪比、对蜂窝电活动的精确控制以及极高的时间分辨率。虽然这种方法是一个极其强大的工具，最近被缩小为纳米移液器配置7，它与几个重要的技术限制有关。细胞胶稀释效应8，以及机械振动，将其效用限制在短期的审讯，它需要昂贵的专业设备和高水平的技术技能。此外，它的体积限制可以同时记录或刺激的细胞数量，并且由于其侵入性，它不能在整个实验中重新配置。为了克服这些限制，开发了微电极阵列，但电极的大小限制了空间分辨率以及细胞内访问。Nanoelectrode阵列允许细胞内记录和刺激，但需要磨蚀性电穿孔才能访问细胞胶9，10。此外，所有这些方法都是基质束缚的，因此仅限于体外细胞培养，或外部表面细胞，无法接触到三维（3D）组织内的细胞。
光遗传学11 被广泛用于解决这些3D和体内的限制。然而，光遗传学方法基于在等离子膜上分布的光激活等离子离子通道的扰动，限制了3D空间分辨率12 和细胞内功能。
我们最近发现，硅纳米线（SiNWs）可用于用亚微米空间分辨率进行细胞内生物电检测，具有不同的非兴奋细胞，即心肌细胞和寡头细胞13。此外，我们使用这些SiNW在3D心脏组织内进行前活细胞特异性询问，调查心脏细胞在体内如何电对14。这种方法的一个主要优点是它简单;它不需要任何基因改造或笨重的仪器。许多细胞会自发内化光反应的SiNW，无需声波或电穿孔15。此外，他们将自发地逃离内体封装，与细胞胶和细胞内细胞器13，15形成无缝整合。这些细胞-SiNW复合材料，称为细胞-硅混合体，具有原始细胞的动态、柔软和多功能特性，以及SiNW的光电功能。杂交后，利用标准组织培养技术采集细胞-SiNW杂交，用于细胞内生物电刺激等各种应用;体外研究细胞间生物电耦合;和体内细胞特定的审讯。由于有效的刺激需要高光功率密度和SiNW的共同定位，因此可以实现2D和3D的高空间分辨率。在该协议中，我们详细描述了方法，以及如何分析结果。重点是体外细胞内和细胞间研究，但这种方法的体内实施可直接用于许多其他生物情景。

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			<td height="21" style="height:21px;width:216px;">35 mm Glass bottom dishes</td>
			<td style="width:121px;">Cellvis</td>
			<td style="width:161px;">D35-10-0-N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3i Marianas Spinning Disk Confocal</td>
			<td style="width:121px;">3i</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein-AM</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">C1430</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellMask Orange Plasma membrane Stain</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">C10045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen I, rat tail</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">A1048301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deluxe Diamond Scribing Pen</td>
			<td style="width:121px;">Ted Pella</td>
			<td style="width:161px;">54468</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, high glucose, pyruvate, no glutamine</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10313039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Anhydrous</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">D12345</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Falcon Standard Tissue Culture Dishes</td>
			<td style="width:121px;">Falcon</td>
			<td style="width:161px;">08-772E</td>
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			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum, certified, heat inactivated,</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10082147</td>
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			<td height="42" style="height:42px;width:216px;">Fibronectin Human Protein, Plasma</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">33016015</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand 112xx Series Advanced Ultrasonic Cleaner</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td style="width:161px;">FB11201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4, AM, cell permeant</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">F14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FluoroBrite DMEM Media</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">A1896701</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">L-Glutamine (200 mM)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">25030081</td>
			<td></td>
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">OKO full environmental control chamber (constant temperature, humidity and CO2)</td>
			<td style="width:121px;">OKO</td>
			<td style="width:161px;"></td>
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			<td height="21" style="height:21px;width:216px;">PBS, pH 7.4</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10010023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:121px;">Thermo Scientific</td>
			<td style="width:161px;">88281</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">25200056</td>
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</table>

silicon nanowires and optical stimulation for investigations of intra- and intercellular electrical coupling

肌纤维细胞可以自发地内化硅纳米线（SiNWs），成为生物电子应用的有吸引力的目标。这些细胞硅混合器件提供无铅光调制功能，对正常细胞行为的扰动最小。光功能由SiNW的光热和光电特性获得。这些杂交种可以使用标准组织培养技术进行收获，然后应用于不同的生物情景。我们在这里演示如何使用这些杂交体来研究心脏细胞的电耦合，并比较肌纤维细胞如何耦合彼此或心肌细胞。除了荧光显微镜和耦合激光线之外，无需特殊设备即可完成此过程。还显示使用定制的MATLAB例程，允许钙在培养中不同细胞内和不同细胞之间进行定量传播。显示肌纤维细胞的电反应比心肌细胞的电反应慢。此外，肌纤维细胞间传播显示稍慢，尽管其细胞内速度相当，表明通过间隙结或纳米管进行被动传播。这种技术具有很强的适应性，可以很容易地应用于其他细胞竞技场，用于体外以及体内或体外调查。

这种方法允许直接访问细胞内细胞体糖的能力取决于SiNW对细胞的自发内化。虽然SiNW将经历自发内化到许多细胞类型15，一些细胞，如心肌细胞和神经元，将需要西努瓦治疗，以允许其内化19。在该协议中，我们描述了直径为200-300纳米和±1-3μm的p-i-n SiNW的内化过程。 图1A 演示了SiNW在透光显微镜下是如何出现的。使用标准相位对比度光学器?...

Watch this Scientific Journal Video about Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling at JoVE.com

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

该协议描述了使用硅纳米线在细胞内光学生物调制细胞在简单和易于执行的方法。该技术高度适应不同的细胞类型，可用于体外和体内应用。

硅纳米线与光学刺激研究细胞内和细胞间电耦合

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

该技术仅使用标准光学显微镜，允许使用极高的空间分辨率和细胞特异性进行细胞内生物电检测。此方法有助于刺激和电质检测细胞和细胞内的特定位置，可以在体外以及 3D 体外组织制剂中进行。该技术能够对心脏或脑细胞等许多细胞领域进行生物电子研究，以及我们所有非兴奋细胞之间的电通信。要将肌纤维细胞与肌纤维细胞心肌细胞悬浮液分离，请将分离的细胞预镀在100毫米组织培养皿上一小时，因为心肌细胞需要一个纤维颈部和治疗表面来粘附。在孵育结束时，收集含有上经应物的富血性心肌细胞，用于下游共培养。用DMEM冲洗肌纤维细胞，消除任何剩余的心肌细胞，并在另外两到四天的孵育前用新鲜培养基喂养细胞。当细胞达到80%的汇合时，使用金刚石划片从晶圆上切割3个3毫米的芯片，该晶圆中用化学气相沉积生长的硅纳米线。并使用锋利的钳子在70%乙醇中冲洗芯片。冲洗后，在生物安全层流罩的紫外线照射下将芯片风干30分钟，然后将芯片转移到无菌微型离心管中。使用完整的细胞培养介质冲洗去除任何剩余的乙醇，然后将芯片在一毫升新鲜培养培养器中声波化1至10分钟。当纳米线释放时，上一层应变成多云。将硅纳米线悬浮液混合到五毫升新鲜培养基中，将纳米线溶液播种到肌纤维细胞的培养皿上。在细胞培养培养箱中四个小时后，用新鲜培养培养剂冲洗盘子五次，去除任何未内部化的纳米线，并继续孵育细胞一小时，使任何部分内化的纳米线完全整合。接下来，在 35 毫米玻璃底盘中加入 500 微升新鲜准备的胶原蛋白涂层溶液。在37摄氏度下孵育一小时后，用无菌PBS冲洗盘子，用3毫升的三毫升三毫升的三辛，在37摄氏度下收获硅纳米线肌纤维细胞杂交剂，两分钟。当细胞分离时，停止与10毫升培养培养剂的反应，通过移液进行大力冲洗，不要离心超过200G，以避免损害细胞。然后将杂交细胞颗粒重新悬浮在一毫升新鲜介质中，并在胶原蛋白涂层玻璃底盘上播种细胞。为了验证纳米线内化，根据标准协议，用荧光细胞胶和膜染料标记细胞，并使用共物显微镜对细胞进行成像。由于硅纳米线具有高反射性，因此可以使用反射光代替荧光进行可视化。为了建立肌纤维细胞硅纳米线混合心肌细胞联合培养，将每种细胞类型的适当数量播种到胶原蛋白涂层玻璃底盘上。并在37摄氏度下培养细胞48小时，使细胞形成细胞间间隙结。在实验当天，用一毫升培养基基代替培养剂，辅以新鲜准备好的钙敏感染料，在37摄氏度下孵育20至30分钟。在孵育结束时，用无菌 PBS 清洗板两次，然后用一毫升预热酚红色无 DMEM 处理细胞。然后让细胞内钙染料在37摄氏度下进行30分钟的脱酯化，然后获取基线图像。在光学成像和刺激之前，在显微镜上预热加湿的微培养箱，用准直激光线耦合到光路径中，用于钙成像和光学刺激至37摄氏度和5%气泡时代的二氧化碳混合物。当显微镜准备就绪时，将肌纤维细胞混合心肌细胞共培养物放入显微培养箱中，通过明亮的场显微镜将纳米线可视化，以找到适当的刺激位点。确定站点后，重新配置荧光模式的光路径，同时保持纳米导线预定义位置的刺激点，并验证每种硅纳米线尺寸和细胞类型的最佳刺激功率和脉冲长度。获得2至10秒的基线细胞内钙活性记录，然后用1至10兆瓦的单个激光脉冲和1至10毫秒的持续时间刺激纳米线，再记录产生的钙波2至10秒。在实验结束时，将光学刺激的录制影片转移到相应的软件程序中，以进行进一步分析。使用标准相位对比度光学器件，光显微镜可轻松查看汇合细胞。然而，硅纳米线几乎看不见，使得它们的位置无法用这种方法来定义。然而，超亮和暗场图像的叠加允许光反射硅纳米线的佩里核排列可视化。共体显微镜可用于可视化荧光标记染色细胞胶和等离子膜，使硅纳米线的细胞内位置更加明显。使用标准显微镜，可以获得明亮的场图像，以确定要刺激的硅纳米线的位置。然后，可以获取自发跳动心肌细胞和休息肌纤维细胞的简短基线视频。刺激后，可以记录共培养中的钙传播。不同细胞兴奋的时间可以通过平均光流变化达到最大值的点来决定。然后可以计算光流，以帮助识别每个细胞区域内的激活时间。可以使用 Khaimah 图形确定每个细胞的细胞内速度，线的斜率表示细胞内传播速度的反比。例如，在这里，可以观察到单个共培养体内细胞相互作用所测量的不同细胞间和细胞内速度的摘要。该方法可用于体内研究，将细胞硅杂交直接注入组织，并利用3D外体体制剂研究体内电气公司。

Research

JoVE Journal

Bioengineering

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

科研

生物工程

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

在体外合成的原住民，纤维长间距“和”分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

硅纳米线场效应晶体管的研制化学生物传感与应用

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

通过光纤使用波前整形多个信号的传输

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...