Name: initial 3d cell cluster control in a hybrid gel cube device for repeatable pattern formations
Uploaded: 2019-03-21T14:30:00
Description: Watch this Scientific Journal Video about Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations at JoVE.com

这项工作得到了日本 JPS KAKENHI (18H04765) 和日本 MEXT 的传播保有权跟踪系统方案的财政支持。

1. 混合凝胶立方体装置的制作
<ol><li>通过使用加工工艺或3D 打印机准备聚碳酸酯 (PC) 立方帧。PC 机架的大小取决于样本大小。在这项研究中, 我们在每一侧使用了一个5毫米的框架, 以便树枝在培养过程中有足够的空间拉长。</li>
	<li>将 PC 机架放置在预冷玻璃滑块或另一个光滑表面的冰盒 (和 lt;0 °c) 中 (图 1a)。</li>
	<li>用移液器将预热的1.5% 琼脂糖 (w v) 从立方框架的顶面添加...

<ol>
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本文提出的方法简单, 无需高科技设备即可实现。同时, 在水凝胶的三维空间中, 可以得到精确的细胞簇形状控制结果。在最初的控制之后, 细胞可以在 HGC 中生长, 就像它们在盘子上培养一样多。多向成像是通过使用任何显微镜系统将样品与 HGC 旋转来完成的, 它显著提高了成像质量。HGC 框架和微工具的材料选择是灵活的, 只要它们是生物相容性的。如果3D 打印机的精度足以满足其应用要求, 则可使?...

3d 培养比2d 培养更能模仿生物环境, 在细胞组织培养1,2,3中得到了很大的强调。细胞与细胞外基质 (ecm) 之间的相互作用为形态发生 4、5 提供了重要线索。许多组织形成只能在三维环境下出现, 如折叠过程6,7, 侵入 8, 和管状形成 9,10。然而, 许多困难使研究人员无法从盘子上的2D 实验转向三维实验。3D 实验的主要困难之一是3D 样品的成像问题。与平面实验相比, 在许多情况下, 获取合适的3D 图像仍然具有挑战性。特别是, 当样品尺寸达到毫米范围时, 由于低放大倍率镜头的焦距较大, 获得合适的3D 图像是一项艰巨的任务。例如, 当使用10倍放大镜时, 焦距超过 50μm, 而单个单元的大小通常小于10μm。为了提高成像质量, 正在开发高技术显微镜系统 (例如双光子显微镜11和光片显微镜系统12), 但由于价格昂贵, 这些系统的供应有限。作为替代方案, 我们以前开发过混合凝胶立方体 (HGC) 设备13。该装置由两种类型的水凝胶组成: 琼脂糖作为支撑凝胶和 ECM, 如胶原蛋白或 Matrigel 作为培养凝胶。HGC 允许我们在培养和旋转立方体的过程中收集样本, 以实现多向成像, 从而解决焦距问题14。
3D 实验的另一个困难是由于3D 环境的可控性较差, 因此其可重复性较低。与塑料盘上的平面培养不同, 初始培养条件的变化很容易发生在被软材料包围的3D 空间中。实验结果的一个显著变化会恶化以下分析, 并掩盖潜在的机制。许多工程技术已经开发出来, 空间对齐单个细胞, 如生物印迹15,16,纤维编织17, 脚手架 18, 但它们需要复杂的预处理或专门设计的设备。相反, 我们开发了一种方法来实现 HGC19中的3d 细胞对齐。
在该协议中, 我们用常用的设备说明了一个简单的过程, 用于控制 HGC 中的三维初始细胞簇形状。首先, 对 HGC 的制造工艺进行了论证。然后, 在 HGC 中放置用光刻或加工工艺制作的微型光片, 以生产 ECM 中任意形状的口袋。随后, 离心后将高密度细胞注入口袋, 以控制 HGC 中的初始细胞簇形状。由于 HGC 的存在, 精确控制的细胞群可以从多个方向进行成像。正常的人支气管上皮细胞 (NHBE) 被用来证明控制最初的细胞簇形状和从多个方向的分支成像, 以提高成像质量。

<table border="0" cellpadding="0" cellspacing="0" style="width:673px;" width="673">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">12-well-plate</td>
			<td style="width:124px;">Corning&#160; Inc.</td>
			<td align="right" style="width:132px;">3513</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">2-Methoxy-1-methylethyl Acetate</td>
			<td style="width:124px;">FUJIFILM Wako Pure Chemical Co.</td>
			<td style="width:132px;">130-10505</td>
			<td>PGMEA, CAS: 108-65-6&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">4% paraformaldehyde</td>
			<td style="width:124px;">FUJIFILM Wako Pure Chemical Co.</td>
			<td style="width:132px;">161-20141</td>
			<td>CAS: 30525-89-4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:251px;">Agarose, low gelling temperature&#160; BioReagent</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">A9414</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Alexa fluor 488 phalloidin</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AZ1512</td>
			<td>Merck</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">BEGM bullet kit</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-3170</td>
			<td>Specialized medium for NHBE cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Bovine Serum Albumin solution (10 %)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">A1595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">EGM-2 bullet kit</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-3162</td>
			<td>Specialized medium for endothelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Lipidure</td>
			<td style="width:124px;">NOF co.</td>
			<td style="width:132px;"></td>
			<td>MPC polymer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Matrigel growth factor reduced basement membrane matrix</td>
			<td style="width:124px;">Corning&#160; Inc.</td>
			<td style="width:132px;">354230</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Normal Goat Serum (10%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">50197Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Normal human bronchial epithelial cells</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-2541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SILPOT 184 W/C</td>
			<td style="width:124px;">Dow Corning Co.</td>
			<td style="width:132px;">3255981</td>
			<td>Base resin and catalyst for PDMS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">SUEX D300</td>
			<td style="width:124px;"></td>
			<td style="width:132px;">DJ MicroLaminates, Inc</td>
			<td>Thick negative photoresist (thichness: 300 mm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Triton X-100 (1%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">HFH10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">TWEEN 20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">P9416</td>
			<td></td>
		</tr>
	</tbody>
</table>

initial 3d cell cluster control in a hybrid gel cube device for repeatable pattern formations

体外3D 培养在细胞组织培养中的重要性得到了很大的强调。然而, 缺乏实验重复性是其限制之一。产生少量可重复的模式形成结果会破坏对自组织机制的分析。减少初始培养条件的变化, 如细胞密度和细胞外基质 (ECM) 中的分布, 对于提高3D 培养的可重复性至关重要。在本文中, 我们演示了一个简单而可靠的过程, 用于控制三维细胞外基质中的初始细胞簇形状, 以获得高度可重复的模式形成。利用光刻技术或加工工艺制备了一种具有所需形状的微极本, 并在混合凝胶立方体 (HGC) 中的 Ecm 中形成了三维口袋。然后将高浓度的细胞注入口袋, 使细胞簇形状与制造的模具形状相匹配。所使用的 HGC 通过旋转实现了多方向扫描, 即使使用了低放大透镜, 也实现了高分辨率成像和整个组织结构的捕获。采用正常人支气管上皮细胞对该方法进行了验证。

利用正常的人支气管上皮细胞, 分别在 ECM 环境中, 演示了所说明的方法, 并控制了初始集体细胞的几何形状, 以实现圆柱形和棱镜形状。给出了圆筒形状 (图 4 a, b) 和棱镜形状 (图 4A, d) 的相位对比和 phalloidin 染色所获得的多方向成像结果。在 ECM 环境中, 这些单元格经过适当的对齐, 以生成所需的3D 形状?...

Watch this Scientific Journal Video about Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations at JoVE.com

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

我们提出了一个控制三维细胞外基质中初始细胞簇形状的过程, 以获得可重复的模式形成。采用含有两种不同水凝胶的立方装置, 实现组织形态形成的多向成像。

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

该协议使用一种简单的方法来提高3D细胞培养模型的可靠性，而无需使用任何特殊设备。该技术的主要优点是，通过控制立方设备内的初始培养条件，可以进行可重复的三D培养实验。准备五分之五毫米聚碳酸酯立方框架后，将框架放在预冷却的幻灯片和冰盒上，并使用移液器将 12 微升预热 1.5% 的 agarose 从立方框架的顶部添加到底部表面。摊开加糖以获得平坦的表面，并允许聚合物固化。使用钳子将框架滑到玻璃的边缘并旋转框架，使打开的一侧朝下，然后再将框架放回幻灯片上。如刚刚演示的，在框架的两个表面填充更多的加糖后，将气糖从开放面放入容器中，在第四面和第五面形成一个加糖墙。设置初始细胞簇状，将感兴趣的细胞培养基体注入适当的细胞外基质，并设置在立方体上制造微模。然后在37摄氏度下将立方体放在二氧化碳培养箱中25分钟。当细胞外基质固化时，小心地提起微模，使基质不会变质。在细胞外矩阵中，将制作所需模具形状的口袋。将细胞在离心后浓缩到适当的实验细胞培养基中，将细胞注入细胞外基质的口袋。将立方体放入二氧化碳培养箱20分钟，温度为37摄氏度，使细胞落入细胞外基质口袋，填充微模产生的空间。在孵化结束时，将立方体放入24井板的一个井中，并在井中加入100微升合适的细胞培养培养。注入额外的细胞外基质以关闭口袋，将立方体返回到培养箱 25 分钟。当细胞外基质固化时，在37摄氏度下将大约10微升20摄氏度的阿加罗斯滴到立方体的顶面上，以关闭表面并固化下降10至20分钟。然后用新鲜的介质覆盖整个立方体，促进渗透压力，促进营养转移到立方体内的细胞。对于通过多向观察进行非侵入性 3D 形状识别，请将板放在显微镜舞台上，通过明亮的场或相位对比度显微镜获取立方体各侧的图像，使用钳子旋转要在捕获之间拍摄的立方体服务。通过多向观察进行免疫荧光成像，首先从含有立方体的井中吸出上流液，并在室温下将立方体固定为4%的甲状甲醛20分钟。在固定结束时，用 PBS 清洗立方体两次，每次洗涤 10 分钟。用0.5%三吨x-100和PBS在4摄氏度下用10分钟对立方体进行渗透。接下来，每次洗涤用PBS清洗立方体三次，10分钟，然后阻止任何非特异性与山羊血清和免疫荧光缓冲液结合60分钟室温。然后根据标准抗体染色方案，用适当的原抗体染色细胞。然后在激光或荧光显微镜上成像立方体的所有六面，正如刚刚演示的。在这个具有代表性的实验中，用正常人支气管上皮细胞培养的混合凝胶立方体的多向成像，通过相位对比度和培养物的免疫荧光成像，展示了初始圆柱形或棱镜状细胞培养的生成。在这里，正常人类上皮细胞的免疫染色最初控制在混合凝胶立方体的圆柱形形状，后代支气管树显示。这些分支演示了垂直于圆柱轴，多维成像显示。将高密度细胞注入细胞外基质口袋非常重要，因为低密度的细胞可能导致孵化后细胞质量恶化。该方法模式是三维细胞模式的可重复形成，通过多向成像进行定量测量，研究菜的发育机理。

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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 ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...