这项工作得到了英国皇家学会的统一报告格式授予UF051616，英国和欧洲研究理事会英镑赠款243261到BS。也支持加州理工学院的再生医学批RB1-01417在锰锌实验室工作。

1。神经祖细胞的分离<ol><li> E14-16小鼠在寒冷的DMEM/F12培养基进行剖析，从全脑。删除所有脑膜转移到35毫米的培养皿下解剖显微镜和大脑。 </li><li>用细镊子机械分解成组织碎片的大脑和他们转移到15 ml管，然后离心3分钟的样本，以消除碎片在800转。 </li><li>加入DMEM/F12培养液含有碱性成纤维细胞生长因子和表皮生长因子和磨碎用1毫升吸管。 </li><li>通过一个细胞过滤细胞悬液，以获得单细胞悬液。 </li><li>板成瓶细胞在2-5×10 4细胞/毫升，然后执行一个完整的介质改变，每3天，并通过细胞每6天。 </li><li>至少有5代后，神经球消化为单细胞用胰蛋白酶和EDTA和增长的poly-D-lysine/laminin-coated electrotactic商会（准备在2以下）。使用日益增长中等含氮补充，碱性成纤维细胞生长因子和表皮生长因子在任何时候都保持的NPC的属性。 </li></ol> 2。编写的electrotactic室<ol><li>准备灭菌22×22毫米一号一半的厚度盖玻片，用金刚石笔除以22×11毫米的玻璃条。 </li><li>创建良好的内部尺寸22×10毫米的独立玻璃胶合在一起四个垂直站立22×11毫米高真空硅脂条。允许井完全干燥变硬过夜。 </li><li>翌日，将两个22×11毫米的玻璃条，相互平行留下一个10毫米的差距，以100毫米文化使用硅脂菜基地。封锁这些带之间的区域，通过放置一个22×22毫米，在每年年底盖玻片，附有油脂三面环盘底部，但不是最接近中心。 </li><li>将玻璃盖2.2跨上准备滑倒，使内墙创建一个播种的密闭空间到板底部的细胞。防水硅脂所有关节。这个密闭的区域与聚-D-赖氨酸然后粘连顺序：大衣添加到室1毫升聚-D-赖氨酸和离开5分钟，让聚-D-赖氨酸绑定到该板块的底部，洗室用无菌PBS两次，然后在无菌PBS稀释产生20微克/毫升和使用覆盖板底部的粘连。在室温下在夜间离开。 </li><li>翌日，收获细胞，并准备1毫升含有1×104个细胞的悬浮。以及从玻璃粘连，使空气完全干燥，并更换1毫升细胞悬液。在37°C，至少4小时，以便附件放置在孵化盘。 </li><li>一旦细胞有足够的融合从室中取出所有介质。小心取出玻璃。细胞在屋顶形式，认真重视，硅脂，蒸压22 x 22毫米盖玻片两个22 x 11毫米带之间的桥接。涵盖中等几滴细胞，以避免干燥。 </li><li>在每个腔年底通过创建两个水密硅润滑脂的障碍，从一个盘子的边缘运行的其他在室的屋顶，形成一个独立的中型水库。新鲜培养基填充室，确保从一个中型水库，通过流动到其他。返回菜孵化器12个小时，使细胞恢复。 </li></ol> 3。电场应用的electrotactic室的<ol><li>准备盖覆盖钻两个孔，定位在每个水库的移民室的菜。 </li><li>在含25毫米的HEPES缓冲介质与文化厅取代所有的介质和传输盘的温度控制的成像系统。成立时间推移和多方位记录实验参数。对齐，使阴极和阳极室上的左，右分别，EF的载体，以确保运行水平看显微镜和成像系统记录。 </li><li>填补斯坦伯格的解决方案（58毫米氯化钠，氯化钾0.67毫米，0.44毫米的Ca（NO 3）2 .4 H 2 0，1.3毫米硫酸镁4 .7 H 2 0和4.6的毫米Trizma基地，pH值7.4）的两个烧杯。连接到每一个中型水库单独的烧杯中，用事先准备好的玻璃桥梁（玻璃管〜13厘米长，3毫米，直径在本生灯火焰加热弯曲成U形），充满了2％（W ​​/ V）Steinberg&#39;s琼脂解决方案，通过孔盖的。完成电路放置Ag / AgCl电极连接到直流电源到每个烧杯Steinberg的解决方案。 </li><li>设置在电源电压为0，打开拨号。电压而转向电压拨号，用电压表测量整个electrotactic室，并调整，以适应实验的要求。 </li><li>开始时间推移录音。执行中的变化和调整电压，根据需要，每隔一小时。新鲜培养基，药物或化学剂可能会增加水库的要求​​。当执行中的变化，两种选择，可以考虑如下： <ol><li>选项​​1 - 暂停录制时间推移，暂时，小心取出从室的玻璃桥避免扰动盖盖，用消毒的巴斯德吸管轻轻地更换新鲜培养基介质和玻璃桥，然后继续录制。 </li><li>选项​​2号 - 或者，自4孔（两个定位在每个水库迁移室），盖上盖子，也可用于使用中改变用途。两个玻璃桥连接，另外两个用于改变介质。选项​​2允许无干扰连续记录介质的变化。 </li></ol></li></ol> 4。器官型脊髓片的制备<ol><li>解剖lumbAR 2周龄C57BL / 6小鼠的脊髓。 </li><li>与McIlwain组织菜刀切成500微米厚的部分脊髓。 </li><li>单独的切片和解剖显微镜下选择片完整的矢状/轴向脊髓结构。 </li><li>板片为35毫米的Petri菜含有30μl的基底膜，并把它们尽可能接近中心，并保持在37℃至少30分钟，直到他们在5％CO 2培养箱基底膜蛋白质自组装生产脊髓片表面的薄膜覆盖。这是非常重要的是要确保基底膜已组装完全，孵化另一个30分钟，如有必要，在37°C的培养皿。 </li><li>加入4-6毫升DMEM/F-12中含有25毫米的HEPES缓冲液和15-20％胎牛血清非常轻柔，避免到介质的流量直接切片。确保片不完全淹没在中期，离开外植体表面以及暴露在空气中。更改介质每周两次。 </li></ol> 5。 Hoechst 33342荧光标记的NPC注入器官型脊髓片<ol><li>准备全国人大暂停1×10 6细胞/μL。 </li><li>预孵育培养基中30分钟与5μMHoechst 33342荧光细胞悬液。 </li><li>用毛细玻璃管到脊髓切片microinject 2μL悬浮在显微镜下缓慢。确保毛细玻璃管穿过基底膜（显微镜下的粉红色部分），并达到脊髓切片（灰色显微镜下的组织）的内部，保持至少30秒内的脊髓切片，以避免细胞悬液辗过。放在培养皿中含有脊髓切片（37°C 5％的CO 2）进入孵化器，并留下过夜。 </li><li>第二天，适用于500毫伏/毫米EF脊髓切片含在electrotactic室赫斯特33342标记的NPC（使用中所述的方法3）。 </li></ol> 6。代表结果当筹备暴露了他们表现出高度定向对细胞迁移的阴极（图1）生理的EF范围。在单细胞水平上的器官型脊髓片体外模型，模仿（图2） 在体内条件下的三维环境也取得了同样的看法。 <img alt="图1" src="/files/ftp_upload/3453/3453fig1.jpg" /> 图1。筹备表明定向迁移的EFS。电脑显示对阴极当暴露在EFS的高度定向迁移，红线和蓝色箭头代表细胞的运动轨迹和方向（一）。 B显示的NPC的迁移路径。酒吧：50微米。 <img alt="图2" src="/files/ftp_upload/3453/3453fig2.jpg" /> 图2。移植的NPC显示在器官脊髓切片直接向阴极迁移 </st。荣毅仁&gt;。 （一）用Hoechst 33342标记的NPC被移植器官的脊髓切片的EF治疗的起点。筹备定向迁移对阴极为2.5小时，而此时的EF极性颠倒（乙）。改变引发对新的阴极（三）外汇极性的电性急剧逆转。 （四）内移植在脊髓片的录制时间推移年底筹备中的形象。 （E）移植的NPC三维重建，脊髓内的切片。三维扫描部分厚度为300微米，从中间开始，并在片底部的结束。虚线表示开始逆转，EF的治疗终点（A类 - C），分别在移植细胞的相同人口的相对位置。箭头表明Hoechst 33342荧光标记的NPC相同的人口。酒吧：50微米。

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

我们什么都没有透露。

5,11以前的研究基础上，我们所使用的协议。使用这些方法，稳定的文化和电目前的条件，可同时申请通过琼脂桥梁，Steinberg的解决方案，Ag / AgCl电极，标准化和精确的尺寸定制设计electrotactic的商会，培养细胞或片的EF维护。钱伯斯的深度可以调整，以适应不同的样品厚度11，在细胞的情况下，室的大小可以修改，以适应但是许多细胞（如Western blot分析需要很多的细胞）。我们已?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>基本的FGF-重组人</td><td> Invitrogen公司</td><td> PHG0026 </td><td> 20毫微克/毫升</td></tr><tr><td>表皮生长因子重组人</td><td> Invitrogen公司</td><td> PHG0311 </td><td> 20毫微克/毫升</td></tr><tr><td> N2 - 补充液体（100X） </td><td> Invitrogen公司</td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12培养基（高糖） </td><td> Invitrogen公司</td><td> 31330-095 </td><td></td></tr><tr><td>聚-D-赖氨酸</td><td> Millipore公司</td><td> -003-E的</td><td></td></tr><tr><td>自然科学小鼠层粘连蛋白</td><td> Invitrogen公司</td><td> 23017-015 </td><td></td></tr><tr><td>生长因子减少基底膜基质（Matr​​igel的） </td><td> BD公司</TD&gt; <td> 354230 </td><td></td></tr><tr><td> HEPES缓冲</td><td> Gibco公司</td><td> 15630 </td><td></td></tr><tr><td> McIlwain组织菜刀</td><td>小数怕长计实验室工程有限公司</td><td> TC752-PD </td><td></td></tr><tr><td>道康宁高真空硅脂</td><td> Sigma-Aldrich公司</td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

内源性电场（EFS） 在体内自然发生和组织/器官的发育和再生过程中发挥关键作用，包括中枢神经系统1,2。这些内源性的EF所产生的细胞与细胞和组织的电阻相结合的离子运输调控。据报道，应用EF治疗可以促进脊髓损伤，动物和人类的3,4功能修复。 EF导向细胞迁移，特别是已被证实在多种类型的细胞5,6，包括神经祖细胞（筹备）7,8。直流电（DC）EF的应用是不是在大多数实验室常用的技术。我们已经介绍了详细的协议，为直流EF的应用细胞和组织培养，以前5,11。在这里，我们提出了一个标准方法的视频示范的基础上计算磁场强度成立2D 1ð3D环境，为筹备和调查外汇基金的刺激，在单细胞的生长条件，在2D，3D的器官在脊髓切片的细胞​​反应。在的脊髓cordslice是学习全国人大前体内的行为，移植后，因为在cytoarchitectonic的组织机构以及保存这些文化在9,10理想收件人组织。此外，这种体外模型还允许程序在技术上并不可行追踪细胞在体内使用在单细胞水平上的时间推移录影。这是极为必要的评估不仅是一个二维的环境中的细胞行为，而且在一个三维器官的条件， 在体内环境mimicks。该系统将允许使用玻璃盖的菜肴，在组织或器官培养， 在体外和体内的单个细胞迁移的3D跟踪，可以移动到前在vi中的中间步骤的高分辨率成像VO典范。

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

此协议表明用于建立定制设计electrotactic的商会的二维和三维环境的方法，它可以追踪细胞<em&gt;在体内/体外</em使用时间推移录制在单细胞水平，以探讨趋电/电性和其他细胞的反应，直接电流（DC）电场（EFS）。

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

electric-field-controlled-directed-migration-neural-progenitor-cells

Research

JoVE Journal

Bioengineering

11.6K 次观看.  Cardiff University. 此协议表明用于建立定制设计electrotactic的商会的二维和三维环境的方法，它可以追踪细胞在体内/体外

视频: Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

触觉机器人适应在3T功能磁共振成像

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

科研

生物工程

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

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

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

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 ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

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 ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

使用机器人微注射操作脑片中单神经干细胞和神经元

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...