作者要感谢阿克伦大学为支持这项工作的经费。

1。目标蛋白的设计<ol><li>利用国家生物技术信息中心网站（http://www.ncbi.nlm.nih.gov/）获得获得目标蛋白考虑到利息和任何剪接变异体物种的氨基酸序列。选择对应的蛋白质所关注的活性区域的氨基酸序列。 注意：对于SEMA3A，氨基酸21-747被选中。融合蛋白在设计时，其中芽孢杆菌RNA，氨基酸1-90的顺序，加入NGF的氨基酸122-241神经生长因子。 </li><li>到N-末端添加如下序列：6X-His标签纯化（HHHHHH），烟草蚀纹病毒（TEV）蛋白酶切割位点以除去His标记的（ENLYFQG），生物素在K（GLNDIFEAQKIEWHE）标签序列的蛋白质的生物素化和柔性铰链与差距，让空间适当的蛋白质折叠（EFPKPSTPPGSSGGAP）。 注意：这些序列可以根据所期望的融合PROT变化EIN。 </li><li>发送完整的氨基酸序列，以一个公司的基因设计/优化所需的宿主物种和合成。亚克隆入选择使用的试剂盒使用的BamHⅠ-NotⅠ位点的载体，或通过利用商业服务。 </li></ol> 2。制作琼脂平板注：这是非常重要的抗生素，板，缓冲器等所有的股票都妥善保存（温度和时间），而且始终是自由蛋白酶（消毒）。 <ol><li>称取琼脂为1.5％（重量）和溶原肉汤（LB）在20克/升，并装在一个玻璃介质瓶。一个500毫升瓶装，使大约15板。 </li><li>容积添加超纯水，拌匀，和高压。 </li><li>让一瓶清凉的触感，并添加适当的抗生素。避免琼脂的过早凝固，但是，​​如果瓶子冷却过多，溶液开始凝结，再热在微波。 注：我们的质粒含有氨苄青霉素抗性根Ë。因此，所有步骤必须含有氨苄青霉素在100微克/毫升。 大肠杆菌克隆大肠杆菌 K12菌株需要四环素（12.5微克/毫升）的抗生素。 BL21细菌细胞不需要任何额外的抗生素。 </li><li>倒入25-30毫升溶液为90毫米的培养皿中，让时间来干。 </li><li>标签菜与适当的抗生素，并在4℃下，用封口膜或放在密封袋中存放颠倒。 注：板是好的约一个月。 </li></ol> 3。生物素标记质粒的克隆注：条件是在无菌条件下完成的。 <ol><li>为了沉淀降速质粒载体在6000×g离心3分钟，并加入10微升灭菌超纯水。 </li><li>除去50μl的化学感受态的大肠杆菌的高转化效率的细胞在-80℃下，并立即置于冰上5-10分钟。 </li><li>加入1μl的载体溶液在0.5-1 ng/5056，L细胞对50μLE.大肠杆菌细胞，然后将剩余的质粒在-80℃下</li><li>将装有冰块的矢量5分钟的细菌细胞。 </li><li>热休克在42℃水浴和地点的细胞回冰上2分钟30-45秒的细胞和载体的混合物。 </li><li>加入250μl与分解代谢物阻抑（SOC）的培养基（2％w / v的细菌用胰蛋白胨，0.5％w / v的细菌-酵母提取物，8.56 mM氯化钠，2.5mM的氯化钾，20 mM的MgSO 4干燥，20mM的葡萄糖超级最优肉汤水，超纯水，pH值= 7.0），并以250rpm摇动，在37℃30分钟。 注：SOC不应进行高压灭菌，但被过滤通过0.22微米的过滤除菌。 </li><li>干板10-15分钟电镀细胞之前。 </li><li>移液管25，50，和100微升的细胞溶液在含氨苄青霉素（100微克/毫升）和四环素（12.5微克/毫升）抗生素分离琼脂平板上。用玻璃珠均匀地分布在溶液琼脂平板。 </li> &lt;LI&gt;孵育板倒置于37℃下15-18小时。 </li><li>检查在平板上个别小菌落，并在4℃倒置存放，直到进一步的使用（ 图2A）。 <ol><li>如果没有菌落出现： <ol><li>检查的缓冲区和供应的新鲜度和不育。 </li><li>增加质粒加入到大肠杆菌的量大肠杆菌 K12菌株。 </li></ol></li><li>如果大菌落存在或有殖民地（ 图2B和2C）过度生长，用无菌接种环或板细胞在较低音量重新划了新的琼脂平板。 </li></ol></li><li>接种5ml的LB含有氨苄青霉素（100微克/毫升）和四环素（12.5微克/毫升）的抗生素与转化的大肠杆菌单菌落大肠杆菌细胞。长大了细胞过夜，于37℃在250-300转。 </li><li>次日，使用质粒分离试剂盒以从过夜GRO隔离目标矢量瓦特式和存储纯化的质粒在-80℃直至进一步使用。 <ol><li> 可选 ：使用在260和280 nm处得到的吸光度读数检查质粒的纯度和浓度。 </li></ol></li></ol> 4。质粒转化入表达宿主注：条件是在无菌条件下完成的。 <ol><li>为E.重复步骤3.2-3.10 大肠杆菌 BL21细胞并作如下改动： 注意：BL21细胞中，不需要任何额外的抗生素，因此只氨苄青霉素加入到琼脂平板上进行转化。 <ol><li>在0.5-2 ng/50从步骤3.12微升细胞加入2-5微升分离的质粒，以50微升E。大肠杆菌表达宿主细胞。 </li><li>热休克细胞为60-90秒。 </li><li>摇动含有溶液的SOC培养基，在250 rpm离心60分钟而不是30分钟。 </li></ol></li><li>采摘转化的细菌细胞的单菌落从培养皿用球面度异亮涂药棒，并置于含有5ml LB培养液用氨苄青霉素的合适浓度（100微克/毫升）的试管中。 </li><li>置于试管中摇床过夜，在37℃以250rpm。 </li><li>第二天早上，乘坐200微升的生长过夜，并把它添加到5ml的LB含有氨苄青霉素在100微克/毫升。 </li><li>冻结在剩余的细胞用250微升50％甘油（无菌），以750微升的文化，并保持在-80°C。 注：新的测试表现和放大程序可以通过使用无菌涂药棒获得的冷冻细胞刮接种LB与适当的抗生素来完成。 </li><li>置于试管中摇床在250-300 rpm下于37℃，直到光密度（OD）是在600nm的吸光度测定0.7-0.8之间。 </li><li>诱导的细胞用IPTG以1mM的最终浓度。 </li><li>振摇额外的4小时，在37℃，转速300rpm。另外电池还可以一夕动摇在18℃下在250-300转。这可以产生更高产量的质粒根据目标蛋白上。 </li><li>测试表达式十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析<ol><li>取500μl的培养和地点在1.5ml管中。降速为13-15×g离心5分钟。倒掉上清液并标记“可溶性”。重新悬浮颗粒与涡旋200微升上样染料（50微升2 - 巯基乙醇，950微升Laemmli样品缓冲液）的。 注：可溶于细菌粒料将用于确定是否将重组蛋白在细菌细胞中的细胞质区域。粒料可以被存储在-80℃下不直到需要加载染料。尚未转化的或诱导的一种控制细菌培养物可被视为可溶性以及用于比较。 </li><li>取1,000微升培养和地点在1.5ml管中。降速为13-15×g离心5分钟。倒掉上清液并标记“不溶性”。 注：不溶的颗粒将发生变性过程下面以释放在细菌细胞中的内含体中发现的蛋白质。粒料可以被存储在-80℃直到需要。还没有被转化或诱发的控制细菌培养可以被视为不溶性以及进行比较。 <ol><li>悬浮的不溶性沉淀物在200μl的BugBuster，并在室温下放置30分钟。 </li><li>再次降速样品在13-15×g离心5分钟，取50微升上清液，并将其添加到新的“不溶性”1.5 ml管。 </li><li>再加入50微升上样染料的这一新的样本。 </li></ol></li><li>煮沸可溶性和不溶性的样品5分钟以使蛋白质变性为SDS-PAGE分析。 </li><li>每个样品装入标准的阶梯电泳凝胶。 </li><li>以可视化的蛋白样品用stainin克的试剂，并按照公司的协议。 </li><li>确定蛋白质是否通过在SDS-PAGE凝胶（ 图比较这两个车道以及车道比较的可溶性和不溶性的控制通道（见注4.9.1和4.9.2）表示的水溶性和/或水不溶性组分2）。甲暗带应在可溶性或不溶性车道周围出现的蛋白质的分子量。这将决定在议定书6使用哪个分离步骤。 注意：如果有一个带在这两个区域中比在任一协议6中使用的隔离可以执行下，或该蛋白质可以分离两种方式，以确定哪个隔离方法产生更高产量的重组蛋白（图3） 。 </li><li>如果4小时和过夜感应进行，确定哪个感应方法具有最高的蛋白质生产的按比例放大处理（ 图2）。 </li></ol></li></醇&gt; 5。规模化发展过程及主要文化<ol><li>准备生长介质与85.7克了不起的肉汤和28.8毫升1.8升超纯水和高压灭菌的液体循环1小时50％的甘油。 </li><li>从在步骤4.5中创建冷冻细胞股票开始培养过夜。 注：条件是在无菌条件下完成的。 <ol><li>混批20毫升LB培养基中，并用氨苄青霉素的终浓度在无菌125毫升三角烧瓶中100微克/毫升。 </li><li>用消毒涂药棒取转化大肠杆菌的杀大肠杆菌细胞并涂抹滴加入到烧瓶中。删除涂药棒和插头量瓶中，用泡沫塞或棉花，盖上铝箔，以保持无菌。 </li><li>摇动过夜以250rpm在37℃下</li></ol></li><li>主要文化 注：条件是在无菌条件下完成的。 <ol><li>倒过夜培养成1.8升无菌生长介质，并在10加氨苄青霉素0微克/毫升和6〜8滴使用巴氏吸管无菌消泡剂204。 </li><li>地方文化在37℃水浴0.2毫米至曝气石过滤的压缩空气鼓泡成文化。 </li><li>一旦OD 600nm处达到0.7〜0.8，诱导与IPTG（1毫米），并允许感应根据从SDS-PAGE在步骤4.9可溶/不可溶的结果发生的无论是4小时，在37℃或隔夜在18℃。 </li></ol></li><li> E.收集大肠杆菌细胞<ol><li>在1升离心瓶（2瓶/文）15分钟，14,000×g离心降速细胞培养在4°C。 </li><li>倒掉上清液，并用薄锅铲，勺细菌沉淀成两个50 ml离心管。细胞可以再次通过离心沉淀几分钟。 注意：每个1.8升培养会导致两种细菌颗粒，1在每次50ml离心管中。 </li><li>店小球在-80℃直至蛋白分离。 </li></ol> &lt;/ LI&gt; </ol> 6。分离和纯化重组蛋白注意：如果蛋白质位于基于从步骤4.9 SDS-PAGE分析的可溶性区域，然后“原生隔离”将被使用，但对于不溶性蛋白质“外来隔离”程序将被执行。 <ol><li>细胞裂解<ol><li>外来<ol><li>删除转化大肠杆菌大肠杆菌沉淀在-80℃下冷冻，并添加20毫升裂解/洗涤缓冲液（ 表1），以每个离心管中。 </li><li>涡街流量计和摇动冷冻沉淀，直到它融化和溶解到缓冲液没有任何大块。孵育章动过夜。次日上午，将丸粒现在应该是由于该蛋白的从缓冲变性粘稠浆液。 </li><li>离心浆料在20500×g离心在室温下30分钟。将上清转移到一个新的管分离，弃去沉淀。 </li></ol></li><l我&gt;原生<ol><li>获得转化大肠杆菌大肠杆菌沉淀在-80℃冷冻箱。 </li><li>添加裂解缓冲液（ 表1）至离心管中，以达到30毫升的最终体积，并通过涡旋和摇动严格撞出冷冻颗粒。将沉淀置于冰上。 </li><li>用70％的乙醇中，在100％的振幅为1-2分钟洗超声波仪探针。然后重复用超纯水。 </li><li>超声细菌沉淀，而在冰浴5分钟（10分钟总时间）。 <ol><li>在30％的幅度与脉冲30秒on/30秒关闭的设置超声仪。 </li><li>鲍勃离心管起来，期间为了彻底打破了细胞沉淀超声间隔了下来。在总运行时间到底有应无可见的细菌沉淀留下粘性，粘浆。 </li><li>清洁不同蛋白质之间隔离的超声波仪的探针以及用于存储用70％的乙醇和超纯水作为步骤6.1.2.3中描述。 </li></ol></li&gt; <li>离心浆料在20500×g离心4℃30分钟。将上清转移到一个新的管分离，弃去沉淀。 </li></ol></li></ol></li><li>的Ni-NTA亲和层析<ol><li>外来<ol><li>加入1 ml的Ni 2 +-NTA树脂溶液到每个离心管（2毫升树脂总的1.8升培养物）并在室温下孵育至少1小时的章动器。 </li><li>浆倒入到亲和柱，并让溶液旋塞阀完全通过淌进废烧杯中。 </li><li>进行10次洗涤用10毫升裂解/洗涤缓冲液，每次洗涤。 <ol><li>在第一洗涤两次，加入10毫升的离心管中，以除去附加的树脂，然后倒入柱。额外的洗涤液，可以直接加入到柱。 </li><li>每次洗涤后，充分搅拌，用玻璃搅拌棒的树脂。一旦洗淌进废烧杯中，在未来10毫升能加入。 </li></ol></li><li>洗净后彻底博士的IPS通过，关闭活塞阀，排除废物烧杯和替换用50毫升离心管中的蛋白质集合。 </li><li>添加15毫升洗脱缓冲液（ 表1），搅拌式树脂，使溶液静置5分钟。打开活塞阀，并收集​​洗脱。一个额外的15毫升再次重复。 </li></ol></li><li>本地人<ol><li>按照外来亲和色谱以上（步骤6.2.1.1-6.2.1.4）除调整下列参数： <ol><li>倪孵育2在4°C +-NTA树脂溶液至少1小时的章动。 </li><li>使用适当的洗涤缓冲液（ 表1）。 </li></ol></li><li>加入5 ml洗脱缓冲液（ 表1）中孵育树脂5分钟。 <ol><li>打开活塞阀，并收集​​洗脱直到大部分的解决方案已经通过滴。 </li><li>添加90微升/孔的Bradford试剂以清除96孔板中。在每次洗脱允许10微升，使禄讯从柱滴入以及含有90微升Bradford试剂。 </li><li>继续加入5ml洗脱缓冲的时间，直到Bradford法不再检测蛋白（颜色去从深蓝到浅蓝清除）。这通常需要4-5洗脱体积。 </li></ol></li></ol></li></ol></li><li>透析/复性<ol><li>让外来的（复性）和原生透析缓冲区（ 表1），并存储在4°C。 注意：在透析缓冲液的pH值由目标蛋白的等电点（pI）确定。如拇指蛋白的规则应该被缓冲的至少一个完整的点高于或低于其等电点的值。 </li><li>传输本地和外来洗脱至透析管与相应的截留分子量（25,000截留分子量为NGF和50,000截留分子量为SEMA3A）。将每个样品的蛋白质在4小时各透析缓冲液1在4℃，然后换上了相应的缓冲区2晚上，在4°C。 注：蛋白质需要在每个缓冲区进行透析至少4小时的蛋白质的正确折叠和缓冲区交换发生。 </li><li>浓缩透析过的蛋白洗脱用离心机旋转浓缩器小于5毫升 注：蛋白质可进一步通过快速蛋白液相色谱（FPLC）纯化，并再次浓缩。 </li></ol></li><li>通过冷冻在一个预先称重的离心管中干燥10-50毫升样品测定蛋白质的浓度（毫克/毫升）。 <ol><li> 可选 ：确定消光系数，ε，所以蛋白质的该浓度可以在未来的隔离通过使用比尔-朗伯定律测定样品的280nm处的吸光度来确定：C = A 为280nm /εL，其中l是路径长度。 </li></ol></li></ol> 7。纯化蛋白的生物素化<ol><li>透析蛋白在10,000截留分子量透析卡带AGainst的10mM Tris（pH值= 8.0），改变缓冲液每隔4小时，共6的变化。 </li><li>重组蛋白（现在在10mM Tris缓冲液）转移至2ml微量离心管中进行生物素化。 </li><li>使用生物素蛋白连接酶试剂盒生物素化的蛋白质。 </li><li>使用10,000 MWCO透析盒3缓冲液透析用PBS（pH值= 7.4）的蛋白质改变一个天2天。 </li><li>确定使用的是定量试剂盒蛋白质的百分数生物素化。 </li><li>再次确定浓度在6.4和储存在4℃或分装储存在-20℃下长期贮存描述。 </li></ol>

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重组蛋白质工程是一个非常强大的技术，跨越许多学科。它是符合成本效益的，可调整的和相对简单的程序，从而允许生产定制设计的蛋白质的产量高。要注意，在设计和表达靶蛋白并不总是直截了当是很重要的。基础表达和重组蛋白的稳定性依赖于向量，E的具体选择大肠杆菌细胞株，肽标签添加和栽 ​​培参数。我们的具体设计标准采用完善的大肠杆菌大肠杆菌菌株的蛋?...

蛋白质覆盖很宽范围的生物分子，负责许多生物学功能，最终导致适当的组织形成和组织。这些分子引发数千信令控制的上调和/或下调的基因和其他蛋白质的通路，维持人体内部平衡。单个蛋白的干扰影响的信号，这样的整个网络，从而导致破坏性的病症或疾病的发作。在实验室工程个别蛋白质可为打击这些不良影响一个解决方案，并提供了一​​种替代小分子药物。在1977中，编码14个氨基酸的生长激素释放抑制因子序列的基因是用大肠杆菌产生的第一个工程化多肽之一大肠杆菌 1。在1979年后不久，胰岛素克隆在质粒pBR322，转化，表达和纯化2。此后，重组蛋白已扩大其影响力，水库的多个字段目录操作搜索，如生物材料，药物释放，组织工程，生物制药，农业，工业酶制剂，生物燃料等 （综述见参考文献3-8）。这主要是由于通用性通过加入特定应用化学部分或蛋白质序列为目的，包括该技术提供，但不限于，靶蛋白的识别，稳定和纯化。 通过重组DNA技术，重组蛋白可表达在多种真核和原核宿主系统，包括哺乳动物，植物，昆虫，酵母，真菌或细菌。每个主机提供了不同的优点，通常最好的系统是基于蛋白质的功能，收率，稳定性，整体成本，而且可扩展性来确定。细菌细胞往往缺乏真核宿主提供（ 即糖基化，二硫键桥接，电子翻译后修饰机制<html> TC）5。其结果是，哺乳动物和昆虫系统通常导致更好的真核生物蛋白的兼容性和表达，但是这些主机通常更昂贵和费时9。因此，E.大肠杆菌为宿主的青睐我们的表达系统，因为细胞在廉价的生长条件迅速扩大和遗传表达机制很好地理解5,9。此外，此系统很容易按比例放大用于生产目的和结果在功能性蛋白，尽管缺乏翻译后修饰10。 大肠杆菌大肠杆菌 K12菌株选择在本协议中的克隆，因为这株提供了基于高转化效率优异的质粒产量。此外，一个E。大肠杆菌 BL21菌株被用于表达，因为这样的宿主菌株含有T7 RNA聚合酶基因，它提供受控的蛋白表达和稳定性11。 帐篷“&gt;后宿主的选择，还必须注意在选择理想的表达载体中，以方便选择和控制蛋白质表达的影响。合成重组蛋白开始与下的噬菌体T7的转录和翻译信号的方向克隆的靶DNA序列，并表达诱导在含有T7 RNA聚合酶基因12的染色体拷贝的宿主细胞，这些载体中，从质粒载体pBR322衍生的（综述见参考文献13），被紧紧地由Studier和同事14最初开发的T7启动子控制并提供额外的通过包含lac操纵子和lac阻遏（LAC1）的控制15,16，对于重组蛋白质工程，该表达系统提供了通过将不同的目标DNA序列来调整所需要的蛋白质的特定氨基酸序列，或创建融合蛋白的能力由结合域S来自单一的蛋白质。此外，某些载体系列包括肽标签的修改被放置在N或C末端。我们的设计目的，组氨酸（His）标签加入到DNA靶序列进行纯化和一个15个氨基酸的序列biotinylatable被列入对生物素17,18。在这个协议中含有一个氨苄青霉素抗性基因的质粒，被选来进行我们的biotinylatable融合蛋白序列。表达是通过T7 lac启动子控制的该向量，并且很容易引起与异丙基-β-D-1-硫代半乳糖苷（IPTG）。 测试表达式（小规模培养物）被用于确定目标蛋白，它可以表示在任一的可溶性或不溶性形式配制的纯化程序的存在和溶解度。细菌细胞内的表达可溶性蛋白质会发生自发折叠，以保持其天然结构19。通常情况下，原生结构在热力学上是有利的。在许多情况下的主机的代谢活性，不利于目标蛋白，放置应力，导致不溶性的蛋白质的生产和不溶性蛋白质聚集体组成包涵体的形成系统。因而靶蛋白变性，使它们通常无生物活性20。两个测试表达式被按比例增加，并且分离步骤是将靶蛋白的溶解度来确定。一个额外的复性或再折叠步骤是必需的不溶性蛋白质。将所得的重组蛋白可以通过尺寸排阻色谱法进一步纯化。 在内部重组蛋白的生产提供了商业产品的成本优势，因为目标蛋白的毫克就可以每升主文化的隔离。大部分所需的设备可在一个典型的生物或化学实验室。蛋白质工程允许创建自定义的融合蛋白与添加的功能性，并不总是市售的。 图1描述了涉及工程重组蛋白的主要程序。用该表达系统，我们已经创建了许多biotinylatable蛋白质，如γ-干扰素，血小板衍生生长因子和骨形态发生蛋白21-23，但是我们将集中于两种蛋白质，我们设计的轴突导向，NGF（29 kDa的）和SEMA3A（91 kDa的）10（综述见参考图24）。生物素是一种常用的技术鉴定，固定化，并利用公知的生物素-链霉亲和相互作用25-27标记的蛋白质的分离。生物物理探针28,29，生物传感器30，和量子点31顷的，利用生物素-亲和共轭用K的D 10 -15米27的顺序上的高亲和力系统的一些例子。 大肠杆菌大肠杆菌生物锡连接酶BirA的，辅助的共价连接的生物素的生物素中发现的赖氨酸侧链的标签序列18,32。圈养生物素材料和生物分子产生了持续给药生长因子对细胞的多种组织工程应用21,33-35。因此，这些工程定制设计biotinylatable蛋白是一种功能强大的工具，它可以超越多的研究兴趣。

<table border="0" cellpadding="0" cellspacing="0" width="849">
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		<tr height="20">
			<td height="20" style="height:20px;width:299px;">1,4-dithio--DL-threitol, DTT, 99.5%</td>
			<td style="width:191px;">Chem-Impex International</td>
			<td style="width:121px;">127</td>
			<td style="width:239px;">100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">2-Hydroxyethylmercaptan &#946;-Mercaptoethanol</td>
			<td>Chem-Impex International</td>
			<td>642</td>
			<td>250 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid, glacial</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Bioshop</td>
			<td>AGR001.500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin sodium salt&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9518</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam 204</td>
			<td>Sigma-Aldrich</td>
			<td>A6426</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barstar-NGF pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BL21(DE3) Competent Cells</td>
			<td>Novagen</td>
			<td>69450</td>
			<td>1 ml; Expression Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BugBuster</td>
			<td>Novagen</td>
			<td>70922-3</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel filtration standard</td>
			<td>Bio-Rad</td>
			<td>151-1901</td>
			<td>6 vials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Bioshop</td>
			<td>GLY001.1</td>
			<td>1 L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Guanidine hydrodioride amioformamidine hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>152</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">His-Pur Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88222</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>EMD&#160;</td>
			<td>HX0603-3</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole&#160;</td>
			<td>Chem-Impex International</td>
			<td>418</td>
			<td>250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>Chem-Impex International</td>
			<td>194</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laemmli sample buffer</td>
			<td>Bio-Rad</td>
			<td>161-0737</td>
			<td>30 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Lauryl sulfate sodium salt, Sodium dodecyl surface</td>
			<td>Chem-Impex International</td>
			<td>270</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Broth&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NovaBlue Competent Cells</td>
			<td>Novagen</td>
			<td>69825</td>
			<td>1 ml; Cloning Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td>10 pack</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Chem-Impex International</td>
			<td>01247</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sema3A-pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886-1KG</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate diabasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136-500G</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5011</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Terrific Broth</td>
			<td>Bioshop</td>
			<td>TER409.5</td>
			<td>5 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracycline hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>667</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris/Glycine/SDS Buffer, 10X</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone, pancreatic</td>
			<td>EMD</td>
			<td>1.07213.1000</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract, granulated</td>
			<td>EMD</td>
			<td>1.03753.0500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:299px;">Name of Kits/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:121px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">&#160;&#196;KTApurifier10</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">28-4062-64&#160;</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Benchtop Orbital Shaker</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">SHKE4000</td>
			<td>MAXQ 4000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">BirA500</td>
			<td style="width:191px;">Avidity</td>
			<td style="width:121px;">BirA500</td>
			<td style="width:239px;">Enzyme comes with reaction buffers and biotin solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Casette</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">66380</td>
			<td>Slide-A-Lyzer (Extra Strength)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Tubing</td>
			<td style="width:191px;">Spectrum Laboratories&#160;</td>
			<td style="width:121px;">132127, 132129</td>
			<td style="width:239px;">MWCO: 25,000 and 50,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flow Diversion Valve FV-923</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">11-0011-70</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FluoReporter Biotin Quantification Assay Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:121px;">1094598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Frac-950 Tube Racks, Rack C</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fraction Collector Frac-950</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-00</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heated/Refrigerated Circulator&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:121px;">13271-102</td>
			<td>Model 1156D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heating Oven FD Series</td>
			<td style="width:191px;">Binder</td>
			<td style="width:121px;"></td>
			<td>Model FD 115</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">HiLoad 16/60 Superdex 200 pg</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">17-1069-01</td>
			<td style="width:239px;">Discontinued--Replacement Product: HiLoad 16/600 Superdex 200 pg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">J-26 XPI Avanti Centrifuge</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">393126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JA 25.50 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">363055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JLA 8.1 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">969329</td>
			<td>Includes 1 L polyporpylene bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JS 5.3 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">368690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laminar Flow Hood</td>
			<td style="width:191px;">Themo Scientific</td>
			<td style="width:121px;">1849</td>
			<td style="width:239px;">Forma 1800 Series Clean Bench</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microplate Reader</td>
			<td style="width:191px;">TECAN</td>
			<td style="width:121px;"></td>
			<td>infinite M200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">165-8004</td>
			<td style="width:239px;">4-gel vertical electrophoresis system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mini-PROTEAN TGX Precast Gels</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">456-9036</td>
			<td style="width:239px;">Any kDa, 15-well comb</td>
		</tr>
		<tr height="21">
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			<td>737-2512</td>
			<td>49 ml&#160;volume ECONO-Column</td>
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			<td style="width:121px;">D6943-01</td>
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			<td height="21" style="height:21px;width:299px;">PowerPac HC Power Supply</td>
			<td>Bio-Rad</td>
			<td style="width:121px;">164-5052</td>
			<td>250 V, 3 A, 300 W</td>
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			<td height="42" style="height:42px;width:299px;">Round Bottom Polypropylene Copolymer Tubes</td>
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			<td>50 ml&#160;tubes for JA 25.50 rotor</td>
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			<td height="21" style="height:21px;width:299px;">Spin-X UF Concentrators</td>
			<td style="width:191px;">Corning</td>
			<td style="width:121px;">431488, 431483</td>
			<td>&#160;20 and 6 ml; MWCO: 10,000 Da</td>
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			<td height="21" style="height:21px;width:299px;">Subcloning Service</td>
			<td style="width:191px;">GenScript USA Inc.</td>
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			<td height="21" style="height:21px;width:299px;">Ultrasonic Processor&#160;</td>
			<td style="width:191px;">Cole-Parmer</td>
			<td style="width:121px;">18910445A</td>
			<td>Model CV18</td>
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			<td height="21" style="height:21px;width:299px;">Vortex-Genie 2</td>
			<td style="width:191px;">Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model G560</td>
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expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration

重组蛋白质工程已使用大肠杆菌（大肠杆菌）表达系统今天近40年，和E。大肠杆菌仍然是最广泛使用的宿主生物体。该系统的灵活性允许添加部分，例如生物素标记（链亲和素相互作用），以及较大的功能性蛋白，如绿色荧光蛋白或樱桃红蛋白。此外，非天然氨基酸，如金属离子螯合剂，唯一的反应性官能团，光谱探针和分子赋予的翻译后修饰的融合，使更好地操纵蛋白质性质和功能性的。因此这种方法创建自定义的融合蛋白，提供用于研究各个领域的显著效用。更具体地，biotinylatable蛋白质序列已被纳入，因为生物素与亲和素和链亲和素之间的高亲和力相互作用的成许多靶蛋白。这除了有辅助提高检测标签蛋白的纯化，以及开辟道路的二次应用，如细胞分选。因此，生物素标记的分子表现出生物产业和生物医学领域的增加和广泛的影响。对于我们的研究目的我们设计含神经生长因子（NGF）和semaphorin3A（SEMA3A）功能区的重组生物素标记的融合蛋白。我们以前曾报道了这些生物素化的融合蛋白，以及与其他活性蛋白的序列，可以被拴在生物材料用于组织工程和再生的目的。该协议概述了工程biotinylatable蛋白质的基本知识在毫克的规模，利用T7 lac启动诱导型载体和E。大肠杆菌表达宿主，从改造规模和纯化启动。

<html> 克隆与表达测试 当被适当地进行化学镀，单个孤立的菌落形成，以增加拔除克隆转化的细菌细胞（ 图2A）的机会。然而，如果太多的细胞铺板，将板温育时间过长，在37℃或转化是有问题的，菌落可以覆盖琼脂平板上或形成细胞的更大的聚集体（ 图2B和2C）。在测试过程中表达，NGF和SEMA3A首次诱导4小时，在37℃和SDS-PAGE?...

Watch this Scientific Journal Video about Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration at JoVE.com

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

开发biotinylatable融合蛋白具有在研究各个领域的许多潜在的应用。重组蛋白质工程是直线前进的过程，是符合成本效益的，提供定制设计的蛋白质的产量高。

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

expression-isolation-purification-soluble-insoluble-biotinylated

Research

JoVE Journal

Bioengineering

33.4K 次观看.  University of Akron. 开发biotinylatable融合蛋白具有在研究各个领域的许多潜在的应用。重组蛋白质工程是直线前进的过程，是符合成本效益的，提供定制设计的蛋白质的产量高。 

视频: Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

明胶湿纺成型工艺促进组织再生

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...