项目受到Zentrales Innovationsprogramm的Mittelstand（ZIM）DES Bundesministeriums献给Wirtschaft UND科特布斯-KF3010902AJ4资助。出版费用已涵盖的BG创伤医院德国蒂宾根。

注:永生人类间充质基质细胞前体细胞（SCP-1细胞）用于实验。 SCP-1细胞通过的Matthias Schieker 12教授提供。 1. SCP-1细胞的扩展<ol><li>之前与SCP-1细胞的工作，正确清洁用70％乙醇（V / V）戴着手套工作区域（指定的生物安全柜Ⅰ）。 </li><li>在清洗过的生物安全柜中通过如表1所示混合所需组分制备细胞培养基的适当体积。为了维持培养基的无菌性，通过无菌过滤器传递具有0.22微米的孔尺寸添加补充剂。 <ol><li>为防止污染，使用前准备中至少24小时。为了测试其无菌性，孵育在细胞培养板1毫升培养基没有细胞中的标准细胞培养孵化器:37℃，5％CO 2，20％O 2和90％的湿度。后24小时，检查中使用显微镜的至少200倍的放大倍数。 </li></ol></li><li>保持SCP-1细胞与支持条件的标准的细胞培养孵化器:37℃，5％CO 2，20％O 2和90％的湿度。 </li><li>为了维护和扩展，成长SCP-1细胞，直到他们达到80-90％汇合。在这段时间内培养，培养基改变每2-3天。在达到80-90％汇合，拆分SCP-1的细胞（一般为1:2的比例）到下一个通道扩张或板用于实验（指示）。 </li><li>用于分离在SCP-1细胞，温培养基在37℃，使用水浴在37℃下解冻胰蛋白酶/ EDTA。 </li><li>完全吸从80-90％汇合细胞培养液，并弃置于垃圾容器。 </li><li>用DPBS洗涤细胞至少两次（Dulbecco氏磷酸盐缓冲盐水无镁和钙，pH值7.2）。 <ol><li>吸取适当体积的DPBS到细胞上（为一个T75培养瓶中5毫升DPBS和12ml的T175培养瓶）。 </li><li>吸仔细DPBS并丢弃到废物容器中。 </li></ol></li><li>吸取0.25％胰蛋白酶/ EDTA的适当体积到细胞上（1毫升一个T75培养瓶中的DPBS和2ml的T175培养瓶），并在具有5的标准细胞培养培养箱中于37℃孵育5-10分钟％的CO 2，20％O 2和90％的湿度。 </li><li>通过敲击容器逐出细胞并确保所有细胞从培养塑料（胰蛋白酶）通过在显微镜下观察漂浮细胞分离。 </li><li>灭活通过加入10ml培养基的胰蛋白酶反应。混合胰蛋白酶和细胞反复吹打仔细网上平台。 </li><li>细胞悬液转移入反应管中，离心分离，在600×g离心在室温下10分钟。 </li><li>吸出上清液，重悬细胞沉淀在10ml培养中。 </li><li>通过计数台盼蓝排除法的细胞在协议2描述。 </li><li>种子取决于实验设计的细胞。 </li></ol> 2. SCP-1细胞计数<ol><li>执行使用血球的再悬浮的细胞的活细胞计数（锥虫蓝排除法）。 </li><li>前的细胞数，用自来水清洗血细胞计数。干用无绒组织血球成分。濡玻璃盖成反比按下它到两个玻璃选手在计数区域的每一侧组装。确保牛顿环看到在玻璃板上跑步（ 图1）。 </li><li>采取10微升的再悬浮细胞，并用10微升的0.1％台盼蓝溶液混合，以获得2的稀释因子。 </li><li>负载10微升的预清洗和装配血细胞计数器室的总样本。 </li><li>计数活（白/透明细胞）和死亡人数（蓝色核）上的4×4平方细胞（参见图1B）。 </li><li>计算细胞的定式下列的总数: <img alt="图1" src="/files/ftp_upload/54537/54537eq1.jpg" /></li></ol> 3. SCP-1细胞的转染GFP 注意:为了观察并进入针织钛支架上我们标有绿色荧光蛋白（GFP）的细胞在一定培养期间SCP-1细胞的生长。 GFP的过表达是通过感染编码GFP的腺病毒颗粒来实现的。复制无能（-E1 / -E3）编码绿色荧光蛋白（GFP）的腺病毒颗粒用于感染SCP-1的细胞。通过收集重组腺病毒（Ad5的-GFP）转染HEK293T细胞（生物安全实验室II）的培养上清从史蒂芬·杜利教授13获得了病毒颗粒。三反复冷冻（-80℃）和解冻（37℃水浴）循环保证了没有，他K293T细胞保持存活，以产生新的病毒颗粒。使用这种腺病毒种子库可以有效地感染SCP-1细胞，而不会产生新的病毒颗粒。因此，感染的细胞可以在生物安全实验室一处理<ol><li>悬浮用的50,000个细胞/ ml的培养基中接种密度靶细胞（SCP-1细胞）。加入2毫升，每孔到6孔组织培养板。 </li><li>孵育在37℃下在标准细胞培养培养箱5％CO 2，20％O 2和90％湿度，直到SCP-1细胞达到70-80％铺满。 注:会合取决于细胞的接种密度。对于上述的条件时，SCP-1细胞达到70-80％在1.5-2天汇合。 </li><li>在70-80％铺满，吸不培养基每毫升培养液中加入100μl腺病毒种子库。 <ol><li>个别地取决于病毒颗粒在各病毒本身的量确定的浓度范围编辑备料。在情况下，感染效率太低，纯化和浓缩使用各种市售试剂盒的病毒颗粒。 </li></ol></li><li>在37℃下（5％CO 2，20％O 2和90％湿度）孵育在标准细胞培养孵化一小时。 </li><li>删除含有病毒种子储备培养基并将其收集处置。加入新鲜的培养液2毫升，每井的6孔培养板来补充的。 <ol><li>确保在处置之前，含中等病毒颗粒高压灭菌。 </li></ol></li><li>评估细胞内GFP表达（感染效率）使用带有GFP LED立方体/过滤器设置的荧光显微镜在感染后24小时。 <ol><li>观察细胞的形态（ 图2）。细胞从培养塑料分离可以给假阳性结果。 </li></ol></li></ol> 4.针织钛支架的清洁<ol><li> P花边高达5支架到50ml反应管。 </li><li>用30ml蒸馏水，去离子水洗净该支架（6-7毫米厚）三次，在室温下20分钟，使用转子条件（8 XG）。 </li><li>用30ml 1％的取代的蒸馏去离子水（重量/体积）的Triton-X-100溶液（溶解在蒸馏水去离子水），然后洗支架一次，在室温下20分钟，使用转子条件（8 XG） 。 </li><li>丢弃的Triton-X-100的溶液中，接着用30毫升蒸馏水 - 去离子水洗涤两次（每次5分钟，在室温下）保持转子条件（8 XG）。 </li><li>更换蒸馏水，去离子水。冲洗支架依次用试剂级99％丙酮，99％异丙醇和99％的乙醇（30ml每次）为2×5分钟的每个在超声浴（〜50赫兹，50瓦，220-240伏）。 </li><li>用30ml蒸馏水，去离子水再次洗涤三次5分钟，保持超声浴处理常数。 </li><li>放置scaffo为了在无绒布组织LDS至室温空气干燥过夜。 </li><li>用15磅高压灭菌该支架15分钟在121℃。 </li><li>作为协议的5所描述确认通过间接荧光清洗协议。 </li></ol> 5.成像支架结构由间接荧光备注:本协议通过使用荧光磺若丹明B上在586分之565纳米EX / EM波长给出了一个明亮的红色荧光间接免疫荧光支架描述结构的成像。但是，荧光团是可以改变的，以更好地适合于给定的显微镜设置或支架的可能自发荧光。 <ol><li>制备在1％乙酸和存储的磺酰罗丹明B染色溶液（0.04％），在室温下避光。 </li><li>取24孔组织培养板中，并通过将它INS沉浸于500μl的磺酰罗丹明B染色溶液的清洗支架IDE井使用镊子。 </li><li>捕获使用荧光显微镜支架的负像。 <ol><li>拍照与RFP LED立方体/滤波器40分之531纳米的激发波长和四十零分之五百九十三纳米的发射波长设置的。可替代地选择具有荧光光谱观察者20的帮助下适当激发和发射波长。磺酰罗丹明B在578纳米的高峰期激发和593纳米的峰值发射。 </li><li>为了形象化的支架结构，拍照在较低的放大倍率， 例如，4X或10X（ 图3）。使用这些图片，确定的特性， 例如，孔大小和与ImageJ的的帮助下成形。 </li><li>为了检测支架的杂质，拍照时的倍率， 例如，20X或40X，考虑到这会限制分析深度。确保灰尘颗粒/物质没有看到（见代表性的结果; 图URE 3）。 </li></ol></li></ol> 6. 在体外试验生物相容性<ol><li>在37℃的水浴中预加温培养基中。 </li><li>取24孔组织培养板和使用镊子，将清洗和消毒的支架中每个测试孔无菌。在预清洁生物安全柜我下工作！ </li><li>浸泡/孵化用培养基支架为约15分钟（每24孔组织培养板的孔500微升）中，为了从该支架的内部除去空气。 </li><li>与此同时，重悬500000的Ad-GFP感染的SCP1细胞于1ml培养基中。 </li><li>支架浸泡15分钟后，完全吸出介质关闭支架。 </li><li>用于接种细胞在支架上，免除小心在支架上（其被放置在24孔板）表面100微升细胞悬浮液。保持细胞悬浮液的最小体积，而接种的细胞，所以，以确保没有介质流ö支架的UT。 </li><li>孵育30分钟的细胞中，在37℃在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）。 </li><li>加入500μl更培养基并孵育在标准细胞培养孵化24小时（37℃，5％CO 2，20％O 2和90％湿度）。 </li><li>评估细胞附着图案和细胞使用荧光显微镜在支架表面上扩散。 <ol><li>拍照用GFP LED立方体/滤波器二十二分之四百七十零纳米的激发波长和四十二分之五百十纳米的发射波长设置的。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。 GFP具有488nm的高峰期激发和507纳米的峰值发射。 </li><li>为了在较低的放大倍数， 例如，4倍或10倍（ 图4）可视化细胞粘附模式拍摄的照片。为了观察细胞扩散更高的MAGnification（至少100X）需要 - 限制了聚焦深度。 </li></ol></li><li>为进一步评估细胞在支架表面上铺展，固定用4％福尔马林的细胞并用细胞结构的传统的荧光染色， 例如，肌动蛋白丝（鬼笔环肽）进行。然而，适应的孵育时间和抗体的荧光标记，以适合个别脚手架特性， 例如，扩散时间或自体荧光14。 </li><li>次日，更换培养基以除去非粘附细胞。 </li><li>通过如在协议7所述刃天青转换测量计算在支架上的贴壁细胞的百分比。 </li></ol><img alt="图5" src="/files/ftp_upload/54537/54537fig5.jpg" /> 图5: 在体外试验时间安排 （ 一 ）电镀细胞的实验装置。 （B </st程序的荣&gt;）插图，强调直到第7天开始协议和细胞的功能验证的重要性， <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54537/54537fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 7.刃天青转换测量注:刃天青转化测定法用于测量线粒体活性，从而间接细胞增殖。刃天青还原成试卤灵产生荧光信号，这是基于与存活细胞数（ 图7A）相关的线粒体活性。 <ol><li>完全吸从SCP-1细胞的培养液中，并丢弃到废物容器中。 </li><li>用DPBS除去脱落的细胞冲洗SCP-1细胞一次。添加每个含在支架上的SCP-1细胞在24孔组织培养板的孔500微升DPBS。 </li><li>覆盖所需上午细胞无菌刃天青工作溶液（在培养基0.25％刃天青）和&#39;mount孵育在37℃下在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）中30分钟。 <ol><li>记下;孵育时间取决于细胞类型和细胞密度。它可以在10分钟和6小时之间变化。优化的温育时间为:SCP-1细胞是30分钟。 </li></ol></li><li>作为背景对照，包括至少一个孔与刃天青工作溶液，但没有细胞，即在37℃下在标准细胞培养培养箱中培养（5％CO 2，20％O 2和90％湿度）为同一多少时间。 </li><li>转移100μl的空调上清液从24孔组织培养板的各孔于96微孔板。 </li><li>用1毫升DPBS在室温下5分钟洗涤剩余的细胞三次，以去除残留的刃天青工作溶液。第三次洗涤后添加培养基Ť直径:与SCP-1细胞的植入物（每24孔组织培养板的孔500微升），并继续在37℃下温育在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）进一步的时间过程的测量。 <ol><li>确保在该步骤（2-4），以尽量减少移液误差来设置重复。 </li></ol></li><li>在此期间，将96微孔板入酶标仪并测量形成的试卤灵的荧光。 <ol><li>为了降低背景信号，在545纳米的激发波长和585nm的发射波长测量荧光。 </li><li>或者选择适当的激发和发射波长的荧光光谱查看器的帮助。试卤灵具有572纳米的高峰期激发和585纳米的峰值发射。给定的信号强度是25个别读数（每孔25闪烁）的平均值。 </li></ol></li><li>测量氟在条件培养基形成试卤灵的escence，使用底部光学器件。 <ol><li>作出这样的，增益取决于平均量刃天青转换并可以在10和4000之间变化的说明。调整吹打一个试卤灵的标准曲线，以便该荧光信号是低于最大信号强度可检测的80％（在此情况下，20,000）所使用的增益到各个酶标仪。对于SCP-1细胞的优化增益为800。 </li></ol></li><li>减去背景信号（刃天青工作液没有细胞）从所述测试样品的信号。 </li><li>根据实验装置/宗旨，继续进一步的步骤: </li><li>计算上使用的标准曲线的支架的细胞的生存力的百分比。分别使个体的标准曲线对所使用的每个细胞系。 </li><li>通过估算刃天青转换整个培养时间相对增加分析细胞生长/增殖。对于这个计算，设置作为基准1天刃天青转换。刚准备对这种分析的刃天青工作溶液。此外，孵育时间必须是不同的测量之间相等。 </li><li>重复刃天青转换测量的整个协议至少三次，以得到一致的结果。 </li></ol> 8.活死细胞染色<ol><li>板与50,000个细胞/支架的接种密度在支架的SCP1细胞，并允许它在支架上保持的标准细胞培养条件下生长（参见方案1和2）。 </li><li> 24-48小时后完全吸从SCP-1细胞的培养液中，并丢弃到废物容器中。 </li><li>用DPBS除去脱落的细胞冲洗SCP-1细胞一次。加入500微升DPBS每24孔组织培养板的孔，并孵育在室温下5分钟。 </li><li>染色SCP-1用荧光团（同时所有三个污渍）: <ol><li>从现在开始保持在黑暗中的支架，以防止荧光漂白从白天！ </li><li>设定的温育时间为30分钟，以允许在整个支架相等分布。如果转移到其他的支架，以确保最优化的温育时间，以适应个别脚手架特性（孔径，脚手架深度等 ）。 </li><li>为了检测在支架上的活细胞，染色用钙黄绿素AM的细胞以2μM的终浓度（在培养基中）。 </li><li>为了检测在支架上的所有细胞，染色用Hoechst 33342细胞在0.002微克/微升（在培养基中）的终浓度。 </li><li>为了检测死细胞，孵育乙锭同二聚体，支架在4微米（在培养基中）的最终浓度。 </li></ol></li><li>温育时间之后，洗涤细胞用DPBS 3次（1毫升，每孔）在室温下，每次5分钟。 </li><li>立即拍照S按使用荧光显微镜。 <ol><li> （分别为二十二分之四百七十零nm和四十二分之五百十纳米，激发和发射波长）取钙黄绿素的照片（活细胞）用GFP LED立方体/过滤器集。或者选择一个适当的激发和发射波长的荧光光谱查看器的帮助。钙黄绿素具有488nm的高峰期激发和507纳米的峰值发射。注:确保不使用GFP转染细胞与钙黄绿素或其他绿色荧光染料荧光染色。 </li><li>取的Hoechst 33342的照片使用DAPI LED立方体/滤波器44分之357纳米的激发波长和六十零分之四百四十七纳米的发射波长设置的。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。赫斯特33342具有347纳米的高峰期激发和483纳米的峰值发射。 </li><li>就拿乙锭同型二聚体的照片与RFP LED立方体/过滤器设置（激发和发射波长Ø˚F四十零分之五百三十一nm和四十零分之五百九十三纳米，分别）。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。乙锭同型二聚体具有530nm的高峰期激发和618纳米的峰值发射。 </li></ol></li></ol>

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作者宣称他们没有利益冲突。工作的任何部分已经或正在考虑公布或已在其他地方发表。

在支架表面起着其相互作用与体内周围组织，从而确定植入官能耐久性重要的作用。因此，支架的生物相容性，通过使用细胞（SCP1细胞系） 的体外测定，对支架镀时的研究。 与薄和光学透明支架功能良好的显微技术适合较差的非透明支架研究生物相容性。这主要是由于非透明支架防止未经显著失真15穿透光。为了部分解决这些问题，我们在此建立在/在?...

慢性背部疼痛是一种多因素疾病。自20世纪50年代的椎间盘退行性疾病的微创治疗选择的兴趣增加。直到今天，在脊柱的多节段的融合是最广泛使用的治疗方法。因为，这种方法常常导致在受影响的段1,2的移动性限制，关节造形术时代的勘探成为广泛的兴趣。总椎间盘置换髓核置换显著进步已成为治疗慢性背痛1一个很好的选择。尽管巨大的进步，没有一种方法已被临床评估。少刚性核植入物代表一个有前途的替代全椎间盘置换，只要该纤维环是完好3,4。然而，市场上的当前存在的核植入物通常伴有并发症像在椎体，错位，盘和叔垂直高度损耗变化他缺乏必要的相关机械刚性5。为了克服目前的缺点，提出针织钛线的新型核植入物已研制成功6。由于独特的编织结构，这种新开发的脚手架已经显示出杰出的生物力学性能， 如阻尼特性，孔径，承载能力和可靠性7。在归因于植入物的非透明性的（光学）分析技术旨在测试此新颖核植入物的生物相容性，描绘严重限制。 为了测试生物相容性，细胞-金属相互作用中起着突出的作用8-10。细胞和支架之间的相互作用是必要的稳定，从而为在主机系统内的更好的植入物的整合。然而，越来越长入深度可能改变支架的机械性能。旨在不变拟tigate所述支架表面是否提供了用于细胞附着，增殖和分化，或碱金属是否影响细胞生存力，它解决在非透明和不透明支架成像上/细胞的常见公知的问题是重要的。为了克服此限制几个荧光灯为基础的技术进行了探讨。公司提供大范围的荧光的可视化活细胞，细胞器，甚至是特定的细胞状态11。此实验的荧光团进行，以最好地适合我们的荧光显微镜与在线工具光谱观看者的帮助​​下选择。 上/在非透明针织钛支架贴壁细胞行为的分析开发策略涉及以下内容:osteochondro祖细胞的1）的荧光（绿色荧光蛋白/ GFP）标记，以允许在细胞的跟踪脚手架，2）测定存活率（线粒体chondrial活性）的细胞，以及3）可视化的细胞 - 细胞和所述支架内的细胞物质的相互作用。该过程具有的优点在于它可以很容易地转移到其它贴壁细胞和其他非透明或不透明的支架。此外，生存力和向内生长模式可以在几天进行监控，因此，它可以用有限数量的支架材料或细胞的使用。 本研究证明的成功使用我们的当前协议来测量细胞活力和可视化在生长上/ osteochondro祖细胞的图案在非透明针织钛支架。此外，开发的协议可能，以便确定所述支架的杂质，并检查清洗协议一起使用。

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">6/24/48 well plates, T25/ T75 culture flask</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">*</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 24 well plates</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">CELLSTAR 662 160</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 48 well plates</td>
			<td style="width:205px;">Corning Incorporated USA</td>
			<td style="width:144px;">3548</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 6 well plates</td>
			<td style="width:205px;">Falcon</td>
			<td style="width:144px;">353046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T25</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">690 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T75</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">658 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetic acid, purum &#8805; 99,0 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3738.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetone</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">5025.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Axioplan-2&#160;</td>
			<td style="width:205px;">Carl Zeiss, Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Biological safety cabinets</td>
			<td style="width:205px;">Thermo Scientific</td>
			<td style="width:144px;">safe 2020</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Calcein acetoxymethyl ester (calcein AM)</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">17783</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Cell Culture Incubtator</td>
			<td style="width:205px;">Binder, Tuttlingen, Germany</td>
			<td style="width:144px;">9040-0078</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Filter unit (0.22&#181;m)</td>
			<td style="width:205px;">Millipore, IRL</td>
			<td style="width:144px;">SLGP033RS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Centrifuges 5810 R And 5417 R</td>
			<td style="width:205px;">Thermo Fisher Scientific, NY</td>
			<td style="width:144px;">Megafuge 40R</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dimethylsulfoxid (DMSO)</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">4720.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dulbecco&#8217;s PBS without Ca &#38; Mg</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">H15-002</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethanol 99 %</td>
			<td style="width:205px;">&#160;SAV liquid prod. GmBH</td>
			<td style="width:144px;">475956</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethidium homodimer</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">46043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">EVOS Fluorescence imaging system</td>
			<td style="width:205px;">Life technologies</td>
			<td style="width:144px;">AMF4300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Fetal Bovine Serum (FCS)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:144px;">10270-106</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hemocytometer</td>
			<td style="width:205px;">Hausser Scientific, PA, USA</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Hoechst 33342</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">14533-100MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Knitted titanium nucleus implant</td>
			<td style="width:205px;">Buck co &#38; KG,Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">MEM Alpha Modification with Glutamine w/o nucleoside</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">E15-832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Omega microplate Reader</td>
			<td style="width:205px;">BMG Labtech,Germany</td>
			<td style="width:144px;">FLUOstar Omega</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Penicillin/Streptomycin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Resazurin sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">199303-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Sulforhodamine B sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">S1402-1G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Test tube rotator</td>
			<td style="width:205px;">Labinco B.V.,The Netherlands</td>
			<td style="width:144px;">Model LD-76</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">TRIS (hydroxymethyl) aminomethan</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">AE15.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Triton</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3051.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypan Blue 0.5 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">CN76.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypsin/EDTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">L11-004</td>
		</tr>
	</tbody>
</table>

imaging cell viability on non-transparent scaffolds &#8212; using the example of a novel knitted titanium implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

初步结果表明，所描述的新颖的核植入物不仅具有良好的阻尼特性，而且是生物相容与SCP-1的细胞。在植入物的生产过程中，它与强腐蚀性和有毒物质（润滑剂，媒染剂，电解抛光溶液）接触。用间接荧光染色技术的帮助下，我们能够可视化剩余的杂质，从而优化示出在支架上物质负载显著减少清洁协议; 图3示出了建立的清洁协议的效率。 <p class="jove_content...

Watch this Scientific Journal Video about Imaging Cell Viability on Non-transparent Scaffolds â€” Using the Example of a Novel Knitted Titanium Implant at JoVE.com

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

这里，我们提出一个基于荧光的成像技术来检测细胞生存力上的非透明钛脚手架以及检测的脚手架杂质影子。这个协议进行故障排除在非透明支架成像细胞 - 细胞或细胞 - 金属相互作用的缺点。

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

imaging-cell-viability-on-non-transparent-scaffolds-using-example

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.3K 次观看.  Eberhard Karls Universität Tübingen. 这里，我们提出一个基于荧光的成像技术来检测细胞生存力上的非透明钛脚手架以及检测的脚手架杂质影子。这个协议进行故障排除在非透明支架成像细胞 - 细胞或细胞 - 金属相互作用的缺点。 

视频: Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）:创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

科研

生物工程

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

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 ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案:二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生儿心脏支架:再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...