This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative.

1，登录<ol><li>上电生物反应器。点击任何地方打开登录页面。选择用户名并输入密码，然后单击“登录”。 </li></ol> 2，校准<ol><li>校准pH传感器（2点前高压灭菌）。 </li><li>检查pH传感器，并确认传感器尖端填充有电解质溶液。准备两个烧杯的pH标准溶液（电解液），pH值为4和7，并有可一洗瓶子蒸馏水。 </li><li> pH值电缆连接到pH传感器。导航到你好界面上的“操作”选项卡，然后点击“校准”。在校准溶液温度字段中输入缓冲区的温度。 </li><li>放置在缓冲区1（pH值为4）pH传感器，并在“零”字段中输入值。等待图形稳定和点击“校准1”按钮。用蒸馏水冲洗水的pH值传感器。 </li><li>在缓冲液代替传感器2（pH 7）中。输入缓冲器在“范围”字段2的值。等待图形稳定，并单击校准2按钮。 </li><li>点击“保存”，然后单击“关闭”。 </li><li>校准溶解氧（DO）的传感器。 </li><li>确保在DO传感器已被偏振，通过连接到系统的数个小时。导航到你好界面上的“操作”选项卡，然后单击校准。点击“执行”按钮。 </li><li>断开溶解氧传感器，并在“零”字段中输入0。等待图形稳定和点击“校准1”按钮。 </li><li>重新连接溶解氧传感器，并在“跨度”字段中输入100。等待图形稳定和点击“校准2”按钮。 </li><li>点击“保存”，然后单击“关闭”。 </li></ol> 3，高压灭菌器，并安装传感器和试剂容器<ol><li>校准，地点传感器和热井autoc后澡袋和高压锅30分钟，在121℃，15磅。 </li><li>高压灭菌消毒袋，用70％异丙醇（IPA）和转移袋生物安全柜（BSC）。取出容器的外包装。 </li><li>消毒包装内用70％IPA和血管转移到生物安全柜（BSC）。取出内包装和检查船舶和管道损坏在运输过程中造成的。 </li><li>安装pH和DO在前面的两个端口传感器。安装热井的左后卫端口。打开传感器帽。 </li><li>通过传感器端口引导传感器。紧紧地拧在传感器插入端口。 </li><li>转船出平衡计分卡。 </li><li>挂溶解氧和pH传感器电缆血管套管之外，并检查没有什么是中袖。容器先滑入袖子，脚上。 </li><li>仔细适应温度传感器放入容器热井。确保该容器的底部靠在加热器。 </li><li>删除从他们的行李中管套。匹配颜色编码的油管到相应的连接器和泵的生物反应器控制单元。 </li><li>通过按压连接器插入其气体出口安装在主气体管线。通过连接器顺时针扭进气口安装了微型气行。安装排气过滤器管： </li><li>打开过滤器烤箱。固定在U型通道排气过滤器，它的管子穿过两个挂钩的过滤器，并出炉。 </li><li>在管架安装管道通过冷凝器袋。关门。 </li><li>路线除了线A和B，两种介质线，后面的溶解氧传感器，并到板凳收获行旁边的生物反应器控制单元。 </li><li>将电缆连接到DO和pH传感器。 </li></ol> 4，增加中型和微型载体<ol><li>导航到“操作”选项卡，然后单击“控制泵”电脑接口上。 </li><li>形成了免缝未使用的介质除了线（1橙带）和焊接管或使用鲁尔接头介质瓶/袋源之间的勒连接。 </li><li>单击滑块以开启对媒体的泵。单击滑块打开媒体介质的另外所需的款额后泵。 </li><li>放置微载体珠中的 Ca 2 +，Mg 2 +的游离的PBS进行3小时，在室温。 </li><li>珠洗几次钙离子 ，镁离子的PBS。 </li><li>高压釜中进行15分钟，在115℃，15磅。 注：加3克/升（干重）在该实验中。 </li><li>泵在微载体已水合的，洗涤和灭菌成以相同的方式在介质中加入在步骤4.1反应器中。 </li></ol> 5，平衡和单点做校准时<ol><li>将控制器自动并输入所需的设定值。在这里，用搅拌设定值（SP）=每分钟15转，温度SP = 37.0℃，pH值= 7.2，DO = 100％。等待在parameters达到平衡。 </li><li>确认传感器是完全两极化。确认DO的现值已趋于稳定。 </li><li>导航到“操作”选项卡，然后点击“校准”。点击“执行”，然后单击“一个点”。 </li><li>在“跨越”字段中输入“100”。点击“校准1”按钮，单击“保存”，然后单击“关闭”： </li></ol> 6，启动运行<ol><li>导航到操作选项卡。单击“批量”。使用屏幕上的键盘或外接键盘来输入一个批次名称超过16个字符。 </li><li>点击屏幕键盘上的“隐藏”按钮。点击“开始批量”通过在覆盖点击“开始”进行确认。 </li></ol> 7，接种与细胞<ol><li>形成焊接一个未使用的介质除了线（1橙带）之间的无菌连接和电池瓶/袋源管或使用鲁尔接头。 </li><li>在介质的泵，以便向泵和容器之间的管箭头指向安装管子的硅氧烷部分。 </li><li>检查管夹处于打开状态和它的支管夹闭合。单击滑块以打开媒体水泵，点击“关闭”添加单元格后。 </li><li>导航到“操作”选项卡，然后单击“控制泵”。 </li></ol> 8采样<ol><li>接种和根据需要频繁的监测培养后，吸取从以下面的方式培养的样品： </li><li>导航到“操作”选项卡，并单击“取样品”。放置在采样泵，采样管，并按照屏幕上的说明操作采样活塞。 </li><li>执行至少每天显微镜观察并计数这些样品。 </li><li>当细胞达到所需的密度，在本C酶1.2×10 6个细胞/ ml，感染的细胞中加入了病毒接种物。 </li><li>在无菌条件下加入接种物，以20ml的注射器，并且该注射器连接到反应器上的备用加成端口之一。通过按压注射器柱塞介绍接种到反应器中。 </li><li>继续采样和文化分析的腺病毒的细胞内颗粒浓度。 </li></ol>

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	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells A Manual of Basic Techniques and Specialized Applications. , (2010).</li><li>Healthcare, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+Principles+and+Methods.">Microcarrier Cell Culture Principles and Methods.</a> GE Healthcare Data File 18-1140-62. , (2005).</li><li>Simaria, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+cell+therapy+bioprocess+economics+and+optimization+Single-Use+cell+expansion+Technologies.">Allogeneic cell therapy bioprocess economics and optimization Single-Use cell expansion Technologies.</a> Biotechnol and Bioeng. 111 (1), 69-83 (2014).</li><li>Lee, B., Fang, D., Croughan, M., Carrondo, M., Paik, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+novel+pneumatic+mixing+for+single-use+bioreactor+application.">Characterization of novel pneumatic mixing for single-use bioreactor application.</a> BMC Proc. 5 (S8), O12 (2011).</li><li>Eibl, R., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+in+cell+culture-based+upstream+processing.">Disposable bioreactors in cell culture-based upstream processing.</a> BioProcess Int. 7 (S1), 18-23 (2009).</li><li>Chaubard, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+for+viral+vaccine+production+challenges+and+opportunities.">Disposable bioreactors for viral vaccine production challenges and opportunities.</a> Biopharm Int Supp. , (2010).</li><li>Croughan, M. S., Hamel, J. F., Wang, D. I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Effects+on+Animal+Cells+Grown+in+Microcarrier+Cultures.">Hydrodynamic Effects on Animal Cells Grown in Microcarrier Cultures.</a> Biotechnol and Bioeng. 95 (2), 295-305 (2006).</li><li>DePalma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-use+Equipment+on+Cusp+of+Industrialization.">Single-use Equipment on Cusp of Industrialization.</a> Genet Eng Biotechnol News. 32 (1), (2012).</li><li>Baltz, R. H., Demain, A. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Industrial Microbiology and Biotechnology. , (2010).</li><li>Sousa, M. F., Giroux, D., Clemente, J., Lee, B., Carrondo, M. J., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impact of Bioreactor Design on the Performance of Microcarrier Cultures. , (2012).</li></ol>

著者D.吉鲁和Y桥村是PBS生物产生这篇文章中所使用的试剂和仪器的员工。

这种一次性使用的生物反应器系统是相对简单的使用，并提供实时分析的反应器监测和分析。这是非常适合以及哺乳动物和昆虫细胞培养与细胞密度达到30多万个细胞/ ml。除了​​A549细胞这个报告11中所述，我们已经发展SF-9昆虫细胞在生物反应器为好。由气动轮设置在温和混合减少细胞损伤。建立这个反应器时，有几个步骤是至关重​​要的。 pH值的第一，适当的校准和DO传感器是用于培养的最佳监测和加入试剂以调节pH或氧在该系统的重要。第二，试剂和种子的瓶子必须填写与鲁尔装在无菌的环境中进行，例如BSC。一旦试剂瓶被移出无菌环境中，连接到所述生物反应器进料管线必须作出谨慎，以避免微生物的污染。 <pCLASS =“jove_content”&gt;虽然这种生物反应器系统，非常适用于哺乳动物和昆虫细胞系是不适合细菌培养。系统不能提供所需的细菌细胞的快速混合和充氧。细菌生长的最好实现在搅拌式生物反应器。相比于其他一次性使用的生物反应器用于哺乳动物或昆虫细胞培养，此系统很容易使用，提供了足够的数据，运行的分析，且具有相似或更好的细胞生长比我们评估的其他一次性使用的系统。 单次使用的气动生物反应器系统有潜力满足众多的研究和生物治疗，疫苗等领域的临床应用中，干细胞，和个性化药物4。此外，该系统的灵活性，允许分批，补料，灌注，并根据转染生物反应器的应用5。最后，单次使用的一次性生物反应器系统具有POTENTiAl基合金，以满足大规模工业生产的需要，并坚持的指导方针以及国家和国际监管机构6-10建议。

哺乳动物细胞系，可分为基于它们的生长特性三类：细胞生长于悬浮液中的细胞生长为聚集体，以及细胞生长锚定到基底。虽然空气轮生物反应器中的该视频演示是能够生长的所有三种类型的细胞，该视频将演示用的生物反应器中生长贴壁依赖性细胞的微载体。锚地依赖性哺乳动物细胞能够生长的制造多细胞的目的 - 在细胞本身的产品。例如，人骨髓间充质干细胞，目前正在栽培的收获细胞，并注射到病变组织的目的。在此视频演示的气动生物反应器已被证明适用于生产诸如间充质干细胞的这种应用（塞拉等人 ，个人通信，2013）。 安克雷奇出发endent哺乳动物细胞通常生长小规模在2D培养容器中，如细 ​​胞培养板中，细胞培养瓶或滚瓶中，在那里它们粘附在经特殊处理的生长面1。当更多的单元被需要，该板或培养瓶可通过使用更多或更大的船只进行扩展。然而，对于更符合成本效益的栽培大量贴壁依赖性细胞，增加表面积为细胞附着可以通过使用小的固体颗粒称为微载体来实现。根据细胞的附着特性，有几种不同类型的微载体可商购的，如葡聚糖，肽或胶原包衣。微载体具有大的表面积与体积之比提供了更大的表面积用于细胞生长;和微载体可以保持在悬浮液中并搅拌，从而使这些细胞进行培养，以高密度，在生物反应器系统2。 biorea目前，该类型的其中贴壁细胞生长在微载体构建函数包括旋转烧瓶中，搅拌罐系统，其使用轴向叶轮保持悬浮液中的细胞涂布的微载体。 几种因素是很重要的成功培养细胞，包括氧张力，剪切应力，表面基质，和营养物和代谢物的浓度。利用生物反应器的允许生长的条件的实时监测和潜力显著降低生产成本1。有几个共同的生物反应器设计用于体外细胞培养，包括搅拌，旋转壁容器中，中空纤维，在摇杆平台袋生物反应器和流化床系统3。许多这些系统目前唯一的问题进行细胞培养和规模-up，如成本高，营养浓度梯度，流体剪切，细胞聚集，以及难以取样，监测和控制CEL升扩大规模。 各种粘附细胞系被用于生产病毒，要么在生产病毒疫苗或用于生产病毒载体用于基因治疗的应用。在这段视频中，采用一次性使用的气动（空气轮）生物反应器系统，我们证明人类肺癌细胞（A549）细胞在微载体生产的溶瘤腺病毒的培养。的气动生物反应器设计使用由气体喷入到反应器底部的浮力驱动的垂直搅拌轮。这种温和的搅拌方法限制流体剪切力，但仍保证了最佳培养基和细胞混合4。相比于搅拌槽反应器，该气动反应器具有低的壁剪切应力，即使以高音量气轮的生物反应器系统（ 图1）。而相比之下，搅拌罐生物反应器，该单次使用的反应器的垂直叶轮由气泡内的流导容器，其允许平缓而均匀的介质中的混合（ 图2）。

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 3&#160;</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Use Assembly</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Human Lung Carcinoma Cells (A549)</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:119px;">&#160;CCL-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose Medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA, 0.25%</td>
			<td style="width:124px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytodex 1 Microcarriers</td>
			<td style="width:124px;">GE</td>
			<td align="right">3781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifoam C</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">A8011</td>
			<td></td>
		</tr>
	</tbody>
</table>

cultivation of mammalian cells using a single-use pneumatic bioreactor system

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

在图3中，示出的参数为在生物反应器运行的开始。该图中示出了之前设定温度，溶解氧，pH和搅动的参数屏幕。一旦参数被设定，运行参数被连续地监视和调整可以维持所要求的条件。该软件会产生一个连续的读数，可轻松识别问题。在用2.5升含10％FBS，250 ppm的SAFC防泡沫的C 2毫米长谷氨酰胺和3g / L的微载体的DMEM该实验中，该系统是稳定的，以便该百分数的氧为50％，则温度是37℃，pH为7.2。该反应器在7×10 4个细胞/ ml（或10个细胞/微载体）的浓度接种A549细胞。选择用于此运行的参数导致了大多数附着于微载体中的2小时（ 图4）中的细胞。后12小时，细胞是魔平坦化并在微载体表面扩散（ 图5）的trating迹象。通过24小时，微载体具有相对均匀分布的细胞，用无微载体不含有细胞和细胞对微载体的没有大的团块（ 图6）。微载体定植的A549细胞的百分率是75％通过24小时和此后90％（ 图7）。将细胞持续48小时（ 图8）后，以几何级数增长至约1百万个细胞/毫升。感染溶瘤腺病毒（2×10 8的病毒颗粒/毫升），在50小时的剂量后，密度提高到1.2万个细胞/毫升，然后开始下降，随着裂解感染进展。有病毒接种〜10,000倍放大。 在以前的实验中，我们已经发现，监控和调整气体流量，以最大限度地提高细胞的生长（未发表数据）是至关重要的。快速生长的细胞会消耗氧气系统<html>溶解氧水平下降到零。当细胞继续增长，这是在一个非常慢的速度。关键是监视并调节氧气流量，以满足在对数生长期的细胞的需求。每次运行可实现最佳生长，通过比较pH值，溶解氧，温度和每日细胞计数分析。 <img alt="图1" src="/files/ftp_upload/52008/52008fig1highres.jpg" /> 气动生物反应器系统（PBS）与搅拌器式生物反应器测量壁面剪切力，在不同的反应器体积比较图1流体动力学 。 <img alt="图2" src="/files/ftp_upload/52008/52008fig2highres.jpg" /> 图2：气动气轮器叶轮。 <html> 52008 / 52008fig3highres.jpg“/&gt; 图3，气动砂轮航软件的屏幕截图参数来启动生物反应器的运行。 <img alt="图4" src="/files/ftp_upload/52008/52008fig4highres.jpg" /> 图4微载体定植A549细胞2小时后接种。（平均微载体直径〜180微米）。 <a href="https://www.jove.com/files/ftp_upload/52008/52008fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52008/52008fig5highres.jpg" /> 图5 12小时的培养开始后，在A549细胞被附着，压扁，并扩展到微载体上。5highres.jpg“目标=”_ blank将“&gt;请点击这里查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52008/52008fig6highres.jpg" /> 图6。微载体24小时培养后，涂上细胞。 <a href="https://www.jove.com/files/ftp_upload/52008/52008fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52008/52008fig7highres.jpg" /> 由A549细胞图7的百分比的微载体定植后接种。 <img alt="图8" src="/files/ftp_upload/52008/52008fig8highres.jpg" /> 可行的A549细胞在培养前的数量和交感染adenovir图8。我们注意，在病毒感染的步骤发生在50小时。

Watch this Scientific Journal Video about Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System at JoVE.com

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

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

培养哺乳动物细胞使用一次性使用气动生物反应器系统

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

cultivation-mammalian-cells-using-single-use-pneumatic-bioreactor

Research

JoVE Journal

Bioengineering

24.2K 次观看.  Johns Hopkins University. Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. 

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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

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

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

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

在三维自组装多肽水凝胶的人类神经祖细胞的培养

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

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

一个台式生物反应器的操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

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

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

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

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...