作者想感谢他出色的技术支持，克鲁格诺曼。

1。第1部分:PuraMatrix hNPCs文化<ol><li>在棚架与PuraMatrix无层粘连蛋白之一的0.25％浓度的代需要提前准备以下的解决方案</li><li>准备的解决方案，其中包含20％的蔗糖和解决方案，含10％蔗糖溶于无菌蒸馏水。 </li><li>对于解决方案1 ​​120μL与120μL蒸馏水1.5毫升的锥形管20％的蔗糖溶液混合。 </li><li>解决方案2混合60μL60μL20％蔗糖溶液1.5毫升的锥形管PuraMatrix解决方案。 </li></ol>对于棚架制备的补充与层粘连蛋白（8微克每100μL矩阵）需要准备以下的解决方案的0.25％PuraMatrix浓度。 <ol class="lroman"><li>对于解决方案1混合72μl与20％的蔗糖溶液和120μL48μL层粘连蛋白在1.5 ml锥形管蒸馏水的。 </li><li>对于解决方案2混合60μL60μL在20％的蔗糖溶液1.5毫升的锥形管PuraMatrix解决方案。 </li></ol><ol start="5"><li>对于细胞埋设在PuraMatrix准备4孔细胞培养板，放在一个消毒的玻璃盖玻片在两口井（直径13毫米）。存放在以后使用洁净工作台板。玻璃盖玻片仅用于免疫细胞化学研究。如果没有免疫细胞化学染色法的目的是，这一步可以跳过。 </li></ol>要准备封装在一个三维支架hNPCs已经准备了以下解决方案。 Benzonase胰蛋白酶/ EDTA溶液稀释，使用稀释倍数的1:10.000。对于Trypsin-Inhibitor/Benzonase解决方案组合:DMEM/F12 + Benzonase25U/μl+ 1％的人血清白蛋白胰蛋白酶抑制剂（0.5mg/ml的）。 <ol start="6"><li>分离细胞，加入500μL胰蛋白酶/ Benzonase的解决方案，并培育孵化器5分钟（37 ° C，5％ CO 2）。停止Trypsin-Inhibitor/Benzonase解决方案的反应。随后5分钟3000 × G.离心分离细胞5毫升10％的蔗糖溶液冲洗细胞，再次离心。弃上清。 </li><li>重悬细胞，10％的蔗糖溶液中，最后一个单元格的解决方案应包含1 × 10 6细胞/ ml 。 </li></ol>接下来的步骤，1.8至1.11，应尽可能快地完成，因为PuraMatrix解决方案，这可能是对细胞有害的低pH值。 <ol start="8"><li>混合60微升的细胞悬浮液1。 </li><li>新增120溶液1μL，含有细胞，锥形管与溶液2，仔细混合。 </li><li>居住在4孔板，将100μL的混合物在每个盖单。 </li><li>慢慢200μL细胞培养液中添加每孔2-3分钟后，另一200μL。这将启动自组装矩阵。 </li><li>孵育彗星英语学习者在37 ° C，对于在一个加热板或受热面积您的洁净工作台洁净工作台1H。尽可能不动的板块，因为这可能会损害组装矩阵。 </li><li>除去大部分中等，但不是所有的，以避免落入矩阵干1000μL移液器，。加入500μL介质洗为10分钟的矩阵。最后取出介质，并加入500μl新鲜培养基和地方培养板在培养箱（37℃，5％的CO 2） 。由于矩阵敏感休克板块应该是不太尽可能移动和存储在一个单独的孵化器，如果可能的话，。 </li><li>这里使用hNPCs的激增为7天，在三维支架和撤出生长因子13开始分化。换液，每2-3天。 </li></ol> 2。第2部分:整个矩阵的免疫细胞化学染色<ol><li>提前染色过程需要准备:AB锁定缓冲溶液组成的5％正常山羊血清和0.3％TRITON X - 100的PBS，4％多聚甲醛溶液，0.1 M的PBS溶解，如果需要的话，解决方案与4"Diamidin - 2 ,6 -&#39;- phenylindoldihydrochlorid（ DAPI）为原子核含有100毫微克的DAPI /毫升溶解在PBS染色。样品安装Mowiol和DABCO的混合物。 </li><li>洗矩阵删除1000μL移液器培养基，用PBS洗5分钟的棚架一次。 </li><li>要修复支架取出PBS和添加多聚甲醛400μL（4％）在0.1 M PBS，每孔孵育30分钟，在室温下的矩阵。固定支架，可存储在PBS数周长达数月的0.02％NaN的3在4 ° C的补充。 </li><li>在染色之前，用PBS洗5分钟支架。 </li><li>阻断缓冲液孵育的支架。封闭液改变了3次，每次2-3小时，subsequently支架封闭液孵育过夜4 ° C。 </li><li>取出阻塞的缓冲溶液和添加PBS溶解，辅以1％正常山羊血清抗体孵育过夜的矩阵在4 ° C。 </li><li>删除主抗体溶液和洗支架在夜间4次，2小时，随后在4 ° C与PBS。 </li><li>取出PBS和添加与二级抗体溶液，溶解在PBS辅以1％正常山羊血清，室温在黑暗中为4 h。 </li><li> 4-6次为1小时，随后在夜间在4 ° C黑暗环境中，用PBS清洗样品。 </li><li>对于核染色，可以加入400μL为30分钟的DAPI解决方案。 </li><li>要安装一个需要显微镜玻片样品。在显微镜幻灯片，添加50μLMowiol / DABCO。从4孔板取出盖玻片，并把他们精心安装介质。 </li><li>在这里，我们使用了Biozero 8000显微镜（KEYENCE，德国，卡尔斯鲁厄）获得单显微照片和Z -栈。每个堆栈包含30 1-2微米的距离的单一图片。模糊固有荧光，使用相应的分析仪软件被删除之前的Z -栈生产的完整的预测。 </li></ol> 3。第3部分:流式细胞仪分析棚架推出hNPCs <ol><li>提前从支架的细胞释放洗用500μl的HBSS的支架。 </li><li>转让1000μL移液器15毫升含有细胞培养液的锥形管的支架。 </li><li>破坏支架，机械悬浮细胞溶液1000μL移液器多次，随后离心5分钟在3000 XG的解决方案。 </li><li>用吸管取出上清液，加入500μL胰蛋白酶/ Benzonase解决方案和悬浮细胞沉淀数次。 </li><li>细胞孵育5分钟在37 ° C在胰蛋白酶/ Benzonase解决方案。停止反应，添加1毫升Trypsin-inhibitor/Benzonase溶液和吸管上下几次。 </li><li> 3000 XG的细胞悬液，离心5分钟，去除上清，用2毫升的HBSS缓冲细胞。重复此步骤两次。此过程将删除矩阵碎片。 </li><li>通过细胞悬浮液通过一个删除聚合，收集细胞，在细胞培养液中中的细胞过滤器（孔径70微米）。如膜片钳或fluometric测量功能研究的盖玻片上再次获得的细胞可以接种。要处理流式细胞仪检测细胞，立即修复细胞（见4.1）。 </li></ol> 4。第4部分:βIII-微管蛋白的阳性细胞，流式细胞仪定量<ol><li>修正释放的步骤3.7在室温下15分钟获得1％的煤灰的细胞。 </li><li>细胞悬液，离心5分钟在3000 XG和去除电子商务的上清液。如果需要的话，细胞可以准备在4℃保存以供日​​后分析。因此在洗涤缓冲液（PBS 0.5％BSA和0.02％钠叠氮化补充）悬浮细胞。 </li><li> 350 XG，如果要进行流式细胞仪准备，细胞悬液，离心10分钟，弃上清，并在25μL抗体溶液重悬细胞。因此，第一抗体混合皂甙的缓冲区，在浓度为1:100，例如βIII-微管蛋白。皂苷的缓冲区，由皂甙浓度为0.5％，一个BSA的浓度为0.5％和0.02％的叠氮化钠浓度的PBS。 </li><li>细胞悬液，孵育一抗室温在摇床2小时。准备无第一抗体作为阴性对照样本。 </li><li>对于第一次洗涤步骤加入300μl皂素缓冲直接的细胞。的细胞悬液，离心5分钟，在350 × G.对于第二个清洗步骤，弃上清，加入300μl缓冲和5分钟的细胞悬液离心350 × G. </li><li>去除上清，加入25μL溶液中含有二级抗体（例如，1:1000的Alexa Fluor 647，溶解在皂素缓冲）。在黑暗中，在室温下的二级抗体1H孵育细胞。 </li><li>对于第一次洗涤步骤加入300μl皂素缓冲直接的细胞。的细胞悬液，离心5分钟，在350 × G.对于第二个清洗步骤，弃上清，加入300μl缓冲和细胞悬液，离心5分钟，在350 × G. </li><li>弃上清，重悬细胞，流式细胞仪分析500μL洗涤缓冲。 </li></ol> 5。代表性的成果在自组装水凝胶支架PuraMatrix培养hNPCs的一个例子是如图1所示。 hNPCs成长圈状结构，在未修改下午。在这种文化条件S，细胞难以在透射光的认可，虽然捆领域之间的过程是可见的（图1A）。根据所用的细胞类型的培养条件下，可修改矩阵，如添加层粘连蛋白。对于HNPC细胞株ReNcell VM（Millipore公司）层粘连蛋白是必要的，以诱使不太密集的总量的增长模式，但更均匀分布的细胞（图1B）。独立的修改，该矩阵可以用来研究如神经元分化。图1C提出了染色的神经元标记βIII-微管蛋白的hNPCs。在这个例子中的细胞生长矩阵中的7天，其后分化为4天，其中被撤出的生长因子EGF和碱性成纤维细胞生长因子13的胞体和密集网络的进程，建立由细胞诱导分化，可公认的容易。然而，很明显，细胞数量的量化大量耗时，矩阵的不同区域的照片进行分析，以获得可靠的数据的基础上进行了统计分析。在图1D，人们可以看到细胞从基质释放，其后于盖玻片制地图的一个例子。这些细胞可能用于功能研究。 三维支架的细胞释放提供的可能性来分析不同的参数，如标志蛋白的表达或AnnexinV / PI染色或TUNEL法检测细胞存活率。图2显示了一个βIII-微管蛋白+细胞的百分比的流式细胞仪分析的一个例子。阴性对照组（不βIII-微管蛋白标记的细胞），如图2a所示。这些控件用来确定βIII-微管蛋白+细胞后检测门（图2b）。图2c所示的细胞人工计数和流式细胞仪分析比较。 βIII-微管蛋白+细胞的百分比是determ独立非执行董事通过计算细胞总数（核DAPI染色）βIII-微管蛋白+细胞中的荧光图片。 <img alt="图1" src="/files/ftp_upload/3830/3830fig1.jpg" /> 图1。在自组装肽水凝胶PuraMatrix hNPCs培养。）hNPCs PuraMatrix封装（下午）增长球形，密集的包装结构，球的直径可高达几百微米。在这两者之间的领域之一，可以识别细胞（箭头）建立起来的进程捆绑。二）封装在PuraMatrix细胞与层粘连蛋白（PML）生长在较致密的结构，更均匀分布的补充。在补充基质可以识别单个细胞密度较低的地区（箭头）层粘连蛋白，但它几乎是不可能量化的细胞数量。三）hNPCs区别像βIII-微管蛋白（绿色）在PML的表达神经元标记为4天。为了evaluaTE的一个类似的DAPI核染色（蓝色），以确定细胞总数的阳性细胞百分比。显微呈现的Biozero - 8000显微镜拍摄的照片的Z - Stack的投影。 D）从矩阵释放的细胞可以接种如盖玻片，为进一步功能研究。显微图像显示三维培养细胞，随后释放过程。 βIII-微管蛋白染色（红色），揭示了细胞在三维支架主持可比形态。 <img alt="图2" src="/files/ftp_upload/3830/3830fig2.jpg" /> 图2。流式细胞仪分析细胞释放PuraMatrix。为了克服时间消耗我们使用的一个协议释放PuraMatrix访问快流式细胞仪分析培养细胞的显微定量。一）未染色细胞作为阴性对照，为随后的分析门（黑框）例如βIII-微管蛋白的细胞。二）为了量化阴性对照组，同门，βIII-微管蛋白+细胞的百分比。阳性细胞出现在正确的X轴，哪里还观察中间人口（点帧），最有可能代表细胞​​碎片。三）手工计数细胞和细胞计数流式细胞仪比较，发现阳性细胞的比例要高得多，其中βIII-微管蛋白的数量的时间依赖性+相媲美，表明了流式细胞仪协议的可靠性。

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Hydrogel.">Peptide Hydrogel.</a> Guidelines for Use. Catalog No 354250, (2006).</li></ol>

没有利益冲突的声明。

使用三维支架提供学习的机会，发展不同类型的细胞在细胞培养的情况下更接近体内的情况。然而，就如神经元分化或功能的研究，人们必须克服一些障碍，以获得可靠的数据类型的细胞，例如量化分析。 在这里，我们描述了基于脚手架PuraMatrix，并随后在2D情况，流式细胞仪或功能分析提供一个易于访问工具的研究使用的细胞释放的多肽水凝胶的hNPCs文化。最近，我们...

<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> PuraMatrix肽凝胶</td><td> BD生物科学</td><td> 354250 </td><td></td></tr><tr><td>鼠标层粘连蛋白的我</td><td> Cultrex </td><td> 400-2009090 </td><td></td></tr><tr><td>蔗糖</td><td>西格玛</td><td> S9378 - 1KG </td><td></td></tr><tr><td>正常山羊血清</td><td> Dako公司</td><td> X0907 </td><td></td></tr><tr><td> TRITON X 100 </td><td>罗斯</td><td> 3051.3 </td><td></td></tr><tr><td> PBS贝科</td><td> Biochrom公司</td><td>大号1825 </td><td></td></tr><tr><td> HBSS </td><td> Gibco公司</td><td> 14170-088 </td><td>汉克斯"平衡盐溶液1X </td></tr><tr><td&gt;βIII-微管蛋白抗体</td><td>圣克鲁斯</td><td> SC - 51670 </td><td>鼠标，单克隆，1:500 </td></tr><tr><td>的Alexa Fluor 488 </td><td> Invitrogen公司</td><td>一个11029 </td><td>山羊α鼠标，1:1000 </td></tr><tr><td>的Alexa Fluor 568 </td><td> Invitrogen公司</td><td>一个11031 </td><td>山羊α鼠标，1:1000 </td></tr><tr><td>的Alexa Fluor 647 </td><td> Invitrogen公司</td><td>一个21235 </td><td>山羊α鼠标，1:1000 </td></tr><tr><td> Mowiol 4-88试剂</td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> DABCO </td><td>爱秩序</td><td> D2 ,780 - 2 </td><td> 1,4 - 二氮杂双环[2.2.2]辛烷98％ </td></tr><tr><td>细胞过滤器</td><td> BD公司</td><td> 352350 </td><td> 70微米的孔径</td></tr><tr><td>皂素</td><td>默克公司</td><td> 7695 </td><td></td></tr><tr><td>胰蛋白酶/ EDTA </td><td> GIBCO公司</td><td> 25300-054 </td><td></td></tr><tr><td> Benzonase 250 U /μL </td><td>默克公司</td><td> 1.01654.0001 </td><td></td></tr><tr><td>胰蛋白酶抑制剂</td><td>西格玛</td><td> T6522（500毫克） </td><td></td></tr><tr><td> 20％的人血清白蛋白</td><td> Octapharma公司</td><td>人白蛋白Kabi公司20％ </td><td></td></tr></tbody></table>

cultivation of human neural progenitor cells in a 3-dimensional self-assembling peptide hydrogel

3维（3D）棚架上生长，增殖和最后的神经元分化的影响是极大的兴趣，以便找到细胞为基础的，规范化的治疗神经系统疾病或神经退行性疾病的新方法。预计三维结构提供了一个环境更接近体内的情况比2D文化。在再生医学的背景下，生物材料支架与神经干细胞和祖细胞的组合持有作为一种治疗工具的巨大潜力。1-5文化系统模拟三维环境的影响在不同类型的干细胞增殖和分化，并已被证明祖细胞。在这里，3D -微环境的形成和functionalisation重要的是要确定嵌入细胞的生存和命运。6-8在这里，我们用PuraMatrix 9,10（RADA16下午），基于一种多肽水凝胶支架，这是很好的描述和用于研究不同类型的细胞上的3D环境的影响。7,11-14 PuraMatrix可定制容易制造的纳米纤维的合成提供了一个高可靠性的三维培养体系，这此外，异种自由。 最近，我们研究形成的脚手架上的PM浓度的影响，在这项研究中13 PM的使用浓度上形成的三维结构，这是由原子力显微镜表明了直接影响。一个随后的生存和分化的hNPCs分析显示嵌入式细胞的命运时所使用的浓度的影响。然而，生存或免疫荧光技术具有一些障碍，神经元分化的分析。为了获得可靠的数据，来判断一个矩阵内的细胞总数获得的相对数，如。 βIII-微管蛋白标记的神经细胞。这是先决条件的技术来分析，共聚焦显微镜或荧光显微镜能够采取标本Z -栈一样可比技术在所有3个维度的支架。此外，这种分析是极其耗费时间。 在这里，我们展示了一个方法，为以后的分析，如流式细胞仪从三维棚架细胞释放。在这个协议中培养人类神经祖细胞ReNcell VM细胞株（hNPCs）（美国Millipore公司），并在三维支架组成PuraMatrix（下午）或与层粘连蛋白（PML）的补充PuraMatrix有区别的。在我们的手上了0.25％的PM浓度是最适合培养细胞 13，但浓度可能会调整到其他类型的细胞。12可用于释放的细胞如免疫细胞化学研究，并随后通过流式细胞仪分析。这加快了分析和Mo重新结束后，获得的数据的其余部分更广泛的基础，提高数据的可靠性。

Watch this Scientific Journal Video about Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel at JoVE.com

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

在这里，我们描述了自组装的三维支架文化人类神经祖细胞的使用。我们提出了一个协议，以流式细胞仪分析后如释放支架的细胞。该协议可能会调整到其他类型的细胞，进行详细的机械研究。

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 ...

cultivation-human-neural-progenitor-cells-3-dimensional-self

Research

JoVE Journal

Bioengineering

16.5K 次观看.  University of Rostock. 在这里，我们描述了自组装的三维支架文化人类神经祖细胞的使用。我们提出了一个协议，以流式细胞仪分析后如释放支架的细胞。该协议可能会调整到其他类型的细胞，进行详细的机械研究。

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

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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 ...

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

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

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 ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

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

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

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 ...