这项工作由芮NSF的资助小型断路器-0950372和DMR-1105310题为“黑寡妇蜘蛛丝和力学性能蜘蛛胶彩衣的分子鉴定”，分别支持。

<img alt="图11" src="/files/ftp_upload/4191/4191fig1overview.jpg" /> 图形概述：纺纱过程的仿生学 仿生学的天然蜘蛛丝的生产途径：制造人造丝的路线此图像显示了主壶腹腺金球织布， 蜘蛛clavipes，天然蚕丝生产（白色文字）利用组件。尾地区综合大量的丝蛋白，被运送到壶腹，为纺丝原液的存储区域。这种集中的涂料是通过纺纱管解决方案体验离子交换和脱水纤维挤出前挤。在我们的实验室中使用的仿生过程是由红色文字表示。使用转基因细菌，蛋白层析纯化重组丝绸生产产生。下一步，纯化的蛋白是子如有冻干集中的材料。最后，蛋白质重新溶解在异丙醇和挤压成异丙醇浴从注射器针头。 1。质粒的构建和细菌细胞培养制备<ol><li>进行PCR所需的蜘蛛丝的基因和特异引物套。凝胶的提取和扩增的cDNA到原核表达载体如PBAD / TOPO ThioFusion（ 图1A）结扎。这个载体具有N-端硫氧还蛋白标记，以方便蚕丝蛋白溶解和C-末端6X蛋白质纯化的标签。主管大肠杆菌转化为结扎产品大肠杆菌细胞。 </li><li>选择包含重组克隆载体，并使用一个菌落接种到无菌的LB氨苄青霉素（ 图1B）补充200毫升的殖民地。增长轨道孵化器在200转摇培养过夜饱和，在37°C。 </li><li>结合200毫升的saturat用800毫升的新鲜LB培养编辑的文化。蜘蛛丝蛋白表达诱导4小时，加入阿拉伯糖0.2％（W ​​/ V）。确保下保持文化的抗生素选择。 </li><li>颗粒为10分钟，在4℃（ 图1C）细胞在16000 XG。为简单起见，沉淀在一个容器的整个体积。取决于离心管的容量，可能需要多个离心步骤。如果有必要，倒出后，纺纱机和重新沉淀的额外材料，以确保整个细胞沉淀在一个容器是液体体积。可以储存在-80°C，直到所需的细胞颗粒。 </li><li>前处理所花费的培养基，添加漂白剂或高压灭菌消毒。 </li></ol> 2。细胞裂解<ol><li>加入20毫升的1X裂解缓冲液1升的细胞沉淀（ 图2A）。确保完全悬浮细胞沉淀。 </li><li>加入溶菌酶至1毫克/毫升的浓度，进一步推动细胞裂解。这一步也可以添加在DNA酶消化染色体DNA，以帮助降低溶液的粘度。样本放在一个轨道摇床，轻轻摇动20分钟。 </li><li>超声，最多1分钟的解决方案。 </li><li>澄清10分钟的解决方案，离心16,000 XG 4°C间（ 图2B）。 </li></ol> 3。蛋白质纯化：镍NTA亲和层析<ol><li>去除上清转移到一个干净的25毫升层析柱。确保上清非粘性和明确的。如果解决方案是粘稠，混浊，添加更多的DNA酶或超声粉碎和/或重新沉淀上清液（重复步骤2.3-2.4）。 </li><li>加入1毫升镍NTA浆（0.5毫升珠）入列。紧紧保护盖和活塞，然后在摇杆设置到1小时的平衡，让6X他的标签绑定镍NTA珠（ 图3A）。 </li><li>直立上设置列站起来，并允许镍NTA珠定居约2分钟。淡蓝色层，可以看到在底部时，珠彻底解决。 </li><li>取下瓶盖，让溶液流经的活塞。收集此作进一步的分析解决方案（ 图3B）。 </li><li> 1X的洗涤缓冲液中加入20毫升，并允许珠定居。打开活塞，让溶液流过。收集这一解决方案作进一步的分析等分四个5毫升注：附加洗量，可用于减少污染的蛋白质，但是，有一个最终蛋白产量下降的可能性 。 </li><li> 1X洗脱缓冲液中加入20毫升，使树脂来解决。打开活塞，让溶液流过。收集这一解决方案，在4个5毫升作进一步的分析等分注：如果洗脱未完成的，额外的洗脱体积可以从收集的Ni-NTA树脂。 </li><li>样品可以存储在4°C或-80°C的短期或长期贮存，分别。 </li><li>大小分馏的样品，用SDS-PAGE分析。可视化的蛋白质与银或考马斯亮蓝R-250（ 图3C）。只有纯组分应注意以下步骤：可用于Western blot分析纯化蛋白的身份确认。 </li></ol> 4。透析和冷冻干燥<ol><li> 2天，至少有100倍的样本量（使用去离子水）对透析样品。每6小时换水解决方案，删除所有盐类注：重组丝蛋白的大小取决于分子量大小的透析管切断。 </li><li>重达6个1.5毫升空离心管，并记录他们的群众。它可能是有用的穿刺管重量为冻结干燥，以方便冻干（ 图4A）前盖的小孔或狭缝。 </li><li> AF特尔透析，1毫升的透析样品转移到每个6预先称重的离心管（ 图4B）。闪光冻结使用液氮和干下来的样品，用冷冻干燥机（ 图4C）。 </li><li>一旦干，每六个管转移到另一个1毫升的血液透析样品。重复，直到整个透析样品已干燥的离心管。 </li><li>冷冻干燥的样品可存放于-80°C长期保存。 </li></ol> 5。纺丝液制备<ol><li>权衡各干蛋白粉的离心管。减去空管的初始质量获得总干蛋白质量。 注意：根据蛋白质产量的不同，你可能需要净化纺纱过程中的其他材料。 </li><li>六氟异丙醇（异丙醇）计算量添加到每个管中获得200毫克/毫升500毫克/毫升，或20％至50％，每卷重量。添加适当吃进每个管（ 图4D）的异丙醇量注：异丙醇是高挥发性和毒性，吸管小心和盖帽管尽快。异丙醇应处理安全罩下方 。 </li><li>封口膜管和地方上的摇杆来溶解蛋白质。涡和离心机偶尔来促进溶解。这可能需要2天。 </li></ol> 6。注射器制备及设备安装<ol><li>装入至少25μL注射器溶成纺丝原液。确保有没有总量呈现，因为它可能会堵塞注射器注：此样品是令人难以置信的粘性，所以照顾时吸取。 </li><li>涂料以推动注射器垂直前，清除所有气泡（ 图5A） 注：气泡在纤维中创建的不一致。 </li><li>锁定泵的注射器。提高洋溢着95％的异丙醇高达400 mL烧杯使注射器的尖端刚刚打破了酒精的表面（ 图5B）。 </li><li>注射泵以15μL/ min的设置和启动程序。允许作为纺纤维，坐了20分钟，在异丙醇完全平衡注：这些纤维仍然可以使用MS / MS分析确认身份的蛋白质纤维。 </li></ol> 7。后旋抽奖及样品采集<ol><li>适用于双面胶带假脱机设备上梳两侧。创建假脱机设备从胶合金属梳子，以数字卡尺（ 图6A）。将后台打印设备的电机（ 图6B）。我们建议2转后台处理线程注意：更快的假脱机率变化的大量纤维质量和可重复性的结果。 </li><li>使用镊子轻轻抢纤维的一端，慢慢拉出来的异丙醇，附加到的阀芯梳武器的边缘（图6C）。接下来的几个步骤，应做到不停工，以防止干燥纤维。 </li><li>打开电机，轻轻地引导纤维上的假脱机设备注：后台打印过程中，不允许纤维加倍堆叠在彼此顶部。 </li><li>一旦整个光纤送入后台，脱离电机阀芯和申请胶水每个蚕丝纤维双面胶带的边缘（ 图6D）。这蜘蛛丝系上的仪器注：通过采用双面胶带胶水前，旋后绘图，它可以防止打滑整个光纤和选择性的伸展纤维的内部环节，使内卡尺武器。 </li><li>将阀芯的直线驱动器（ 图7A）。初始长度记录使用卡尺纤维的的注意，这是内部的纤维长度。它不包括长胶和包裹ARound阀芯。 </li><li>降低到75％的异丙醇浴阀芯。允许纤维平衡10分钟注：其他脱水的解决方案，可用于纺纱过程中，如甲醇，丙酮和硫酸铵。 </li><li>线性驱动器的速度设置为1.5毫米/秒。基于初始长度，计算所需的自旋后的拉伸比最终的长度。金额捉襟见肘，可以控制速度或长度的基础上，根据而成立的。例如，初始长度为15毫米和所需的自旋后的3倍拉伸比，最终的长度应该是45毫米。这也可以作为供电线性驱动器，1.5毫米/秒的速度在20秒计算。 </li><li>一旦所需的后旋抽奖是完整的，慢慢抬起阀芯的异丙醇。允许异丙醇液滴干燥1分钟，然后收集样本（ 图7B）。样品应装在卡片或铝箔帧测试PUR构成。 </li><li>如果需要进一步的后旋抽奖比率，降低到75％的异丙醇浴阀芯回。允许的纤维平衡10分钟，然后进行下后旋转拉伸比。 </li></ol> 8。拉伸试验<ol><li>允许采集机械测试前至少1小时的标准实验室环境的平衡。应记录温度和湿度，因为它们可能会影响纤维的力学性能。标准的温度和湿度条件下，应分别约为40％和25°C。 </li><li>胶的卡片或铝箔的边缘固定在框架上的纤维。注意帧开幕的大小。这是您的测试纤维的初始长度。这里显示一个1英寸（25.4毫米）的开放框架（ 图8A）。 </li><li>使用了100倍或更大倍率的光学显微镜，沿纵向纤维轴的直径测量。应至少有3个测量记录。倍率越高，并采取更多的测量的准确性，更好地。 </li><li>争取到tensometer机械装载架（ 图8B）的框架。削减的框架，使双方紧张仅通过光纤上运行。皮tensometer和收集数据。一个应变速率每秒2％的标准建议。 </li><li>利用收集到的数据，平均粒径，应力应变曲线可以绘制。 </li><li>破碎的纤维，现在可以安装在扫描电子显微镜（SEM）的形态分析和破发点直径测量的存根。可用于估计和检查光镜下测量的初始直径的纤维段离破发点。 </li></ol> 9。代表结果从第3步，在不同部位应进行SDS-PAGE分析蛋白质与银化或考马斯亮蓝R-250。从一个标准的Ni-NTA柱条件，可以得到洗脱组分纯度&gt; 90％（ 图9）。小污染的蛋白质可以进一步广泛透析去除。使用25μL纺丝原液在20％（W / V），至少30个不同的纤维样品可以收集从阀芯（假设使用13毫米的初始长度）连续伤口纤维。可以分析由tensometer测试（ 图10）的力学性能。根据重组丝蛋白用于纺纱过程中，最大的后旋平局比率将需要凭经验确定。在一般情况下，发表自旋绘制比例的4.0倍，可以实现没有光纤故障（ 图10）。纺纤维，后旋抽签之前或之后，可以用扫描电子显微镜可视化的超微结构（ 图11A，B）的分析。纺纤维也可以被用于力学性能测试，结果显示与低后旋转拉伸比样本组内变异（ 图10）。 <img alt="图1" src="/files/ftp_upload/4191/4191fig1.jpg" /> 图1。蜘蛛丝的细菌基因的表达。一）PBAD TOPO的/硫代载体含有蜘蛛丝的基因利益，转化大肠杆菌主管大肠杆菌细胞。 B）单菌落接种到200毫升的LB，一夜之间发展到饱和。接种之后，800毫升新鲜LB和表达诱导使用阿拉伯文化。 c）在感应结论，文化是由离心沉淀。 <a href="http://www.jove.com/files/ftp_upload/4191/4191fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4191/4191fig2.jpg" /> 图2。蜘蛛丝蛋白诱导后的细菌细胞裂解。一）第二十millili1X裂解缓冲液和DNase TERS添加到细胞沉淀置于轨道器和超声裂解细胞。乙）的细胞裂解液在离心机旋转上清清除细胞碎片，并收集上清。 <a href="http://www.jove.com/files/ftp_upload/4191/4191fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4191/4191fig3.jpg" /> 图3。蜘蛛丝绸重组蛋白用亲和层析纯化。一）细胞裂解液上清，镍NTA珠加入到层析柱和培养1小时。 b）在收集的穿透洗涤缓冲液和洗脱缓冲液20毫升，20毫升用于序列在5毫升的馏分收集。 c）不同的分数通过SDS-PAGE分析，纯样品含有靶蛋白被转移到一个透析袋，对DI水到透析完成。 <a href="http://www.jove.com/files/ftp_upload/4191/4191fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4191/4191fig4.jpg" /> 图4。纯化湿纺蜘蛛丝蛋白的制备。一）透析产品转移到预先称重的离心管分装在1毫升。乙）1毫升等分是Flash用液态氮冷冻。三）冷冻样品冻干，并增加更多的透析样品。 d）干质量计算，异丙醇添加到干粉生产了20％（W / V）纺丝液。 <a href="http://www.jove.com/files/ftp_upload/4191/4191fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4191/4191fig5.jpg" /> 图5。载入到玻璃注射器的湿纺纺丝液。 a）虽然持有syrinGE垂直中，的纺纱的涂料被被推到的注射器“列中的的页最上方，移除的空气气泡。乙类）加载的的注射器被附在注射器帮浦，到的，和成的95％的异丙醇浴的的之调低，，以便的小费被刚刚打破了的洗浴的表面。 <img alt="图6" src="/files/ftp_upload/4191/4191fig6.jpg" /> 如图6所示。作者：的合成的的蜘蛛到一个自定义的的缫丝装置上的真丝纤维的绕线机。 A）该假脱机的设备被构造从一个与所附的金属梳子的的数字卡尺。双双面胶带被应用到的梳子的两侧，，到附上的纤维的两端。乙类）的阀芯被附加到的缓慢的的的速度电机，使用一个短吻鳄剪辑。丙类）该纤维正在慢慢被拉从的酒精浴的，并周围的阀芯清盘。丁）胶水是应用于到每根光纤的段的的的边缘，，到按住他们在的地方。所示的从种不同的蛋白质纺的的两个不同的的纤维。 <img alt="图7" src="/files/ftp_upload/4191/4191fig7.jpg" /&gt; 图7。旋转后绘制的合成纤维，使用自制的设备。一）假脱机设备连接到线性驱动器安装，使用弹簧夹。 b）在后自旋抽奖步骤，阀芯被解除洗澡。异丙醇液滴蒸发前纤维收集。 <img alt="图8" src="/files/ftp_upload/4191/4191fig8.jpg" /> 图8。安装到卡片机械研究的人造丝纤维。一）收集的纤维装在卡片帧1“×1”的剪影。纤维最初举行的地方用双面胶带，然后用胶水固定。乙）的卡片框架是固定到tensometer上的。双方都再切，这样的张力纤维虽然只运行。 <img alt="图9" src="/files/ftp_upload/4191/4191fig9.jpg" /> 图9大小分馏纯化重组MaSp1 protei的。其次是银染的可视化氮组分用SDS-PAGE分析。描绘在kDa的蛋白质梯子。两个洗样本显示非特异性结合的珠子，而洗脱样品揭示了6集合的需要，以确保收回总蛋白。 <img alt="图10" src="/files/ftp_upload/4191/4191fig10.jpg" /> 图10。纤维应力应变曲线从TuSp1重组蛋白纺8种颜色显示受到不同的后旋平局比例，从2.5倍至6倍不等的纤维。纤维表明他们比组内变异;低后旋平局比率增加，纤维的强度增加，而可扩展性降低。 <img alt="图11" src="/files/ftp_upload/4191/4191fig11.jpg" /> 图11。从重组TuSp1蛋白质纺纤维的扫描电子显微镜图像。 A）在500倍放大倍率，平稳的外部表面可以看到。 B）在5000X，密室内的核心可以从天然纤维休息观察。

<ol>
	<li>Gosline, J. M., Guerette, P. A., Ortlepp, C. S., Savage, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+design+of+spider+silks:+from+fibroin+sequence+to+mechanical+function.">The mechanical design of spider silks: from fibroin sequence to mechanical function.</a> J. Exp. Biol. 202, 3295-3303 (1999).</li><li>Foelix, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of spiders. , (1996).</li><li>Vollrath, F., Knight, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+crystalline+spinning+of+spider+silk.">Liquid crystalline spinning of spider silk.</a> Nature. 410, 541-548 (2001).</li><li>Spiess, K., Lammel, A., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+spider+silk+proteins+for+applications+in+biomaterials.">Recombinant spider silk proteins for applications in biomaterials.</a> Macromol. Biosci. 10, 998-1007 (2010).</li><li>Stark, M., Grip, S., Rising, A., Hedhammar, M., Engstrom, W., Hjalm, G., Johansson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+fibers+self-assembled+from+recombinant+miniature+spider+silk+proteins.">Macroscopic fibers self-assembled from recombinant miniature spider silk proteins.</a> Biomacromolecules. 8, 1695-1701 (2007).</li><li>Lazaris, A., Huang, Y., Zhou, J. F., Duguay, F., Chretien, N., Welsh, E. A., Soares, J. W., Karatzas, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+Silk+Fibers+Spun+from+Soluble+Recombinant+Silk+Produced+in+Mammalian+Cells.">Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells.</a> Science. 295, 472-476 (2002).</li><li>Teule, F., Cooper, A. R., Furin, W. A., Bittencourt, D., Rech, E. L., Brooks, A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+production+of+recombinant+spider+silk-like+proteins+for+artificial+fiber+spinning.">A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.</a> Nat. Protoc. 4, 341-355 (2009).</li><li>Gnesa, E., Hsia, Y., Yarger, J. L., Weber, W., Lin-Cereghino, J., Lin-Cereghino, G., Tang, S., Agari, K., Vierra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+C-Terminal+Domain+of+Spider+Tubuliform+Spidroin+1+Contributes+to+Extensibility+in+Synthetic+Fibers.">Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers.</a> Biomacromolecules. , (2011).</li><li>Hayashi, C. Y., Shipley, N. H., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotheses+that+correlate+the+sequence,+structure,+and+mechanical+properties+of+spider+silk+proteins.">Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.</a> Int. J. Biol. Macromol. 24, 271-275 (1999).</li><li>Xu, M., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+protein+superfiber:+Spider+Dragline+Silk.">Structure of a protein superfiber: Spider Dragline Silk.</a> Proc. Natl. Acad. Sci. 87, 7120-7124 (1990).</li><li>Hayashi, C. Y., Blackledge, T. A., Lewis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+mechanical+characterization+of+aciniform+silk:+uniformity+of+iterated+sequence+modules+in+a+novel+member+of+the+spider+silk+fibroin+gene+family.">Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family.</a> Mol. Biol. Evol. 21, 1950-1959 (2004).</li><li>Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J. F., Duguay, F., Chretien, N., Welsh, E. A., Soares, J. W., Karatzas, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+silk+fibers+spun+from+soluble+recombinant+silk+produced+in+mammalian+cells.">Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.</a> Science. 295, 472-476 (2002).</li><li>Arcidiacono, S., Mello, C., Kaplan, D., Cheley, S., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+recombinant+spider+silk+expressed+in+Escherichia+coli.">Purification and characterization of recombinant spider silk expressed in Escherichia coli.</a> Appl. Microbiol. Biotechnol. 49, 31-38 (1998).</li><li>Menassa, R., Zhu, H., Karatzas, C. N., Lazaris, A., Richman, A., Brandle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+dragline+silk+proteins+in+transgenic+tobacco+leaves:+accumulation+and+field+production.">Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.</a> Plant Biotechnology Journal. 2, 431-438 (2004).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nat. Biotechnol. 19, 573-577 (2001).</li><li>An, B., Hinman, M. B., Holland, G. P., Yarger, J. L., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+beta-sheets+formation+in+synthetic+spider+silk+fibers+by+aqueous+post-spin+stretching.">Inducing beta-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching.</a> Biomacromolecules. 12, 2375-2381 (2011).</li><li>Elices, M., Guinea, G. V., Plaza, G. R., Karatzas, C., Riekel, C., Agullo-Rueda, F., Daza, R., Perez-Rigueiro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioinspired Fibers Follow the Track of Natural Spider Silk. Macromolecules. 44, 1166-1176 (2011).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nature Biotechnology. 19, (2001).</li><li>Kojic, N., Kojic, M., Gudlavalleti, S., McKinley, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+removal+during+synthetic+and+Nephila+fiber+spinning.">Solvent removal during synthetic and Nephila fiber spinning.</a> Biomacromolecules. 5, 1698-1707 (2004).</li><li>Jeffery, F., La Mattina, C., Tuton-Blasingame, T., Hsia, Y., Gnesa, E., Zhao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+Black+Widow+Spider+Silk-producing+Glands.">Microdissection of Black Widow Spider Silk-producing Glands.</a> J. Vis. Exp. (47), e2382 (2011).</li><li>Blasingame, E., Tuton-Blasingame, T., Larkin, L., Falick, A. M., Zhao, L., Fong, J., Vaidyanathan, V., Visperas, A., Geurts, P., Hu, X., La Mattina, C., Vierra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyriform+spidroin+1,+a+novel+member+of+the+silk+gene+family+that+anchors+dragline+silk+fibers+in+attachment+discs+of+the+black+widow+spider,+Latrodectus+hesperus.">Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus.</a> J. Biol. Chem. 284, 29097-29108 (2009).</li><li>La Mattina, C., Reza, R., Hu, X., Falick, A. M., Vasanthavada, K., McNary, S., Yee, R., Vierra, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+minor+ampullate+silk+proteins+are+constituents+of+prey+wrapping+silk+in+the+cob+weaver+Latrodectus+hesperus.">Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus.</a> Biochemistry. 47, 4692-4700 (2008).</li><li>Hsia, Y., Gnesa, E., Jeffery, F., Tang, S., Vierra, C., Cuppoletti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+Silk+Composites+and+Applications.">Spider Silk Composites and Applications.</a> Metal, Ceramic and Polymeric Composites for Various Uses. 2, 303-324 (2011).</li></ol>

没有利益冲突的声明。

这种方法纺成的合成纤维是机械上的同一量级的天然纤维相比。通过假脱机和自旋后抽签过程机械化，减少人为错误的数量，实验样品之间的变化有更多的控制，大大降低了。 我们的方法提供了潜力调查其他纺蜘蛛基因家族的其他成员的cDNA编码的重组蛋白纤维的力学性能。潜在的，它可以决定不同的蛋白质模块内素，或不同的素类型之间的机械作用，23它还允许蚕丝...

蜘蛛丝具有非凡的机械性能，包括高强度钢，芳纶和尼龙出执行数的人造材料，旋转1蜘蛛至少6-7种不同的光纤类型，显示不同的力学性能，数额不等的拉伸强度和可扩展性设计执行特定的生物任务。2研究的科学家正在迅速推行的蜘蛛丝作为下一代生物材料由于其优异的力学性能，其生物相容性，无毒，绿色材料的性质使用。由于“吃人的3,4和有毒的蜘蛛纲的性质，通过耕种收割蜘蛛丝是不是一个实际的战略，以满足工业规模生产所必需的要求。因此，科学家们转向了从合成纤维纺丝加上在体外重组丝蛋白的转基因生物的生产5-8全长重组蜘蛛丝蛋白表达本身的纯化蛋白质。一直技术上的困难，由于他们的基因序列的内在属性，其中包括他们的高度重复的性质和物理长度（15 KB），GC丰富的内容和偏见丙氨酸和甘氨酸密码子使用9-11迄今为止，大多数实验室都集中表达形式截断主壶腹丝蛋白的部分cDNA序列或人工合成的基因MaSp1或MaSp2 12-15纺纱合成蜘蛛丝是一个具有挑战性的过程，需要掌握知识，在若干学科，纺纱过程的复杂性没有得到充分暴露广大市民通过视频表示。事实上，只有少数遍布全球的实验室具备专业知识，表达蜘蛛丝的cDNA，净化丝蛋白，纺合成纤维和执行后的自旋战平，然后最后测试其生物材料的性能8。16,17纺合成纤维不同的方法包括湿法和干法纺丝以及静电方法16,18,19的所有程序，有一个共同的目标-一个协议，生产合成蜘蛛丝力学性能发展，对手自然线程大规模商业化生产进程。 在这里，我们描述的过程来生成实验室规模的人工蜘蛛丝，用湿纺方法。相对于其他纺纱方法，湿法纺丝纤维分析的最一致的结果。我们概括与表达中的细菌其纯化重组丝蛋白，其次这个过程的开始，然后纺纱，包括自旋后抽签的方法适用于“纺”的纤维，产生线程的描述蛋白质的准备步骤材料性能接近天然蜘蛛丝的质量。我们的方法论的y的设计紧密地模仿天然蚕丝纤维的纺纱工艺和倚重它吸引我们的ORB和芯织网的蜘蛛。20-22此外，丝绸生产的腺体结构和功能方面的专长，我们的结论与必要的步骤，以确定使用tensometer绘制应力 - 应变曲线，这让研究者计算的极限强度，极限应变，韧性纤维，合成纤维材料的性能。最后，但显著的价值，纺纱，后台处理和绘图仪器可以在家使用市售的部分，而不是购买定制的精致和昂贵的设备，建造。

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> PBAD / TOPO ThioFusion的表达试剂盒</td><td> Invitrogen公司</td><td> K370-01 </td><td></td></tr><tr><td>的快攻细胞裂解试剂，10X </td><td> Promega公司</td><td> V857C </td><td></td></tr><tr><td>镍-NTA琼脂糖</td><td> Qiagen公司</td><td> 30210 </td><td>包括对缓冲区的说明</td></tr><tr><td> ProteoSilver银染色套件</td><td> Sigma-Aldrich公司</td><td> PROTSIL1-1KT </td><td></td></tr><tr><td>自由贸易区冻干机</td><td> labconco </td><td> 7960041 </td><td>自由贸易区12Plus </td></tr><tr><td>六氟异丙醇（异丙醇） </td><td> Sigma-Aldrich公司</td><td> 52512 </td><td></td></tr><tr><td>注射器</TD&gt; <td>汉密尔顿</td><td> 7657-01 </td><td> 250微升</td></tr><tr><td>针</td><td>汉密尔顿</td><td> 7780-01 </td><td> 26S计，钝端可拆卸针</td></tr><tr><td>注射泵</td><td>哈佛仪器</td><td> 702208 </td><td> 11Plus </td></tr><tr><td>数显卡尺</td><td>卡雷拉</td><td> CP5906 </td><td> 0-150毫米范围</td></tr><tr><td>不锈钢钢钳</td><td>世界精密仪器</td><td> 501764 </td><td>迷你杜蒙＃M5S </td></tr><tr><td>马达</td><td>自然轧机</td><td> 7090529 </td><td> 12VDC，2 rpm的转速</td></tr><tr><td>线性驱动器</td><td>华纳电</td><td> 01-D024-0050-A06-LP的，符合IP65 </td><td> 24VDC，6英寸的范围</td></tr><tr><td>解剖显微镜</td><td>徕卡公司</td><td>徕卡MZ16 </td><td></td></tr><tr><td>数字microscOPE相机</td><td>徕卡公司</td><td> DFC320 </td><td>软件：徕卡应用套件v2.8.1 </td></tr><tr><td> vannas剪刀</td><td>世界精密仪器</td><td> 500260 </td><td></td></tr><tr><td> microtensometer </td><td>极光科学</td><td> 310C </td><td> ：5N双模式系统</td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

随着社会的进步和资源日益稀少，它正变得越来越重要，以培育新技术，新一代生物材料具有较高的性能工程师。这些新的结构材料的发展必须是快速，具有成本效益和涉及的加工方法和产品，环保和可持续发展。蜘蛛能纺出多种不同类型的纤维，具有不同的力学性能，提供丰富的新一代工程材料的来源，仿生学，对手最好的人造和天然材料。由于收集大量天然蜘蛛丝是不切实际的，人造丝生产有能力提供无限量供应的线程访问科学家。因此，如果纺纱过程可以简化和完善，人工蜘蛛纤维的防弹衣，应用范围广泛，手术缝合线的潜在用途，绳索和电缆，轮胎，乐器的弦，和航空航天技术的复合材料。以推动人造丝生产过程中产生纤维，显示在他们的物质属性从低自旋旋转方差，我们开发湿纺协议，整合重组蜘蛛丝蛋白的蛋白质的细菌，净化和浓度的表达其次是纤维挤压和机械旋转后处理。这是第一个可视化表示形式，揭示了一步一步的过程，旋转和分析实验室规模的人造丝纤维。它还提供了详细资料，以尽量减少从同一纺丝液纺纤维中引入变异。总的来说，这些方法将推动人造丝生产过程中，导致更高的质量超过天然蜘蛛丝纤维。

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

尽管优秀的机械和生化蜘蛛丝的性能，这种材料可以收获大量的常规手段。在这里，我们描述了一个有效的战略，以旋转人工蜘蛛丝纤维，这是一个重要研究蜘蛛丝作为下一代生物材料的生产和使用的调查过程。

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

synthetic-spider-silk-production-on-a-laboratory-scale

Research

JoVE Journal

Bioengineering

26.6K 次观看.  University of the Pacific. 尽管优秀的机械和生化蜘蛛丝的性能，这种材料可以收获大量的常规手段。在这里，我们描述了一个有效的战略，以旋转人工蜘蛛丝纤维，这是一个重要研究蜘蛛丝作为下一代生物材料的生产和使用的调查过程。

视频: Synthetic Spider Silk Production on a Laboratory Scale

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

空气过滤设备，包括非织造网格静电重组蜘蛛丝蛋白

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

科研

生物工程

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

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

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

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

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

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

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

实验室规模搅拌生物反应器的功率输入测量

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

热、压水固化重组蜘蛛丝的材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

基于可溶解微载体的贴壁细胞的闭路系统生产

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

作者聚焦：使用可溶解微载体推进细胞治疗生产 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

基于GMP级可溶性多孔微载体的大规模电池生产

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...