这个项目是支持部分由美国约翰霍普金斯大学，通过网关科学倡议办公室教务长。

<ol><li>开始操作容器的设置。常规的发酵罐中具有需要安装的（ 图1）包括以下组件: <ol><li> pH探头 - 用于pH在现场的文化测量。安装前校准探头和消毒用的容器。在磁头板安装探头。 </li><li>氧分压pO 2探测-用于在所述容器内发酵过程中溶解氧含量的测量。安装在磁头板。 </li><li>丰收管取样的文化。在磁头板安装此高度可调元件。 </li><li>气体分布器 - 驻留在容器的底部，并提供气体注入到培养。钩鼓泡后至发酵罐的气源，并在磁头板安装。 </li><li>叶轮的轴和叶轮 - 叶轮搅动培养，并维持培养的均匀性在容器中是至关重要的。 </li><li>废气冷凝器 - 用于通过冷却所述废气通路抑制在容器中的蒸发损失。 </li><li>试剂泵线，pH值控制或饲料添加。 </li></ol></li><li>清洁容器和磁头板用肥皂和水。软刷，建议擦洗容器和磁头板。漂白剂或清洁剂与氯不能用来清洁容器。 </li><li>如果媒体不热不稳定，将其添加到干净的容器中。只添加，直到最大工作容积就达到了。在这种情况下，最大工作容积为3.3升的5升总体积的容器。 </li><li>装入容器磁头板，确保O型密封圈正确就位。 </li><li>安装在排气冷凝器。 </li><li>安装消泡剂探入其10毫米端口。 </li><li>安装管道收获到其10毫米端口。把一块管在它的顶部和夹紧它关闭。 </li><li>安装管道和一个0.20微米的过滤器上的喷射入口，然后用镊子这一关。 </li><li>校准pH探头: <ol><li>聚集在小烧杯2基准缓冲器，一个洗瓶子，和一个备用娃sh的烧杯中。 </li><li>钩pH探头到发酵罐并打开发酵罐上。 </li><li>滚动到pH值的参数和使用菜单按钮，滚动到"CAL"选项，然后按"Enter"键。 </li><li>选择&#39;2&#39;2点校准。 </li><li>洗掉探头尖端和淹没在低参考缓冲器。在发酵罐中的值将趋于稳定。使用+和 - 键调整显示值相匹配的pH值的参考缓冲区的值。一旦它停止闪烁时，按下"Enter"键。 </li><li>关闭冲洗探针并将其插入到所述第二基准缓冲器。允许值稳定，然后使用键盘来使显示的值相匹配的高基准电压缓冲值。按下"Enter"键确认。 </li><li>关闭冲洗探头尖端和它从发酵罐断开。把保护帽连接上和在磁头板安装。 </li></ol></li><li>开宝2探头端部和检查，以确保有是足够的电解质以覆盖小费。如果没有，有些添加到水库和关闭它回来了。 </li><li>在磁头板安装在PO 2探头，确保高压釜盖放在保护其电气连接。将灭菌后进行校准。 </li><li>获得与汲取管和空气过滤器的瓶试剂饲料。添加泵管的浸管口，并连接另一端连接到发酵罐的进气口。 </li><li>在所有未使用的端口地方油管接钳油管关闭。 </li><li>在废气冷凝器安装一个0.45微米的过滤器。此过滤器可确保容器不会在高压釜加压。 </li><li>检查了低于媒体的级别的所有管夹关闭，以防止脱落媒体。 </li><li>把容器中的高压釜中进行25 - 30分钟，在121℃下，液体循环。注意:当它出来会很烫。 </li><li>在发酵罐底座安装船。 </li><li>胡克了pH值探头电缆。 </li><li>附上宝2探头电缆。 </li><li>钩鼓泡直至气体添加流量计。 </li><li>无菌条件下加入1 M氢氧化钠试剂的瓶汲取管和在基体泵压头主轴上安装泵头。 </li><li>把温度传感器热电偶套管上的磁头板。确保它去所有的方式，热电偶套管的底部。 </li><li>降低搅拌臂到血管叶轮耦合。 </li><li>确保发酵罐已打开。 </li><li>检查是否有水供应到发酵罐中。 </li><li>打开空气供应到发酵罐中。 </li><li>滚动到温度参数和设定温度为37°C。按&#39;回车&#39;按钮来启动温度控制。该容器将加热在15分钟以下。 </li><li>转气流，以每分钟或更少1容器体积的介质。为此设立，气体流量为3升/分。气泡应该出现在底部。 </li><li>滚动到搅拌parameter并设定为300rpm。按"Enter"键打开搅拌。 </li><li>一旦温度达到37℃时，滚动到pH值的参数，并将其设置为6.8。把pH控制在按&#39;回车&#39;键。 </li><li>设置搅拌至1,000 rpm的转速运行的最大速度。 </li><li>确保宝2探头极化正确显示正确的价值观。 2小时后，滚动到宝2参数，并使用"菜单"按钮，选择"校准功能"，然后选择1点校准。 </li><li> 15分钟后，使用光标的值设置为100，然后按Enter键。这将校准2宝。 </li><li>滚动到搅动，并设置为300rpm。 </li><li>使用"菜单"按钮，打开"级联"在鼓动菜单&#39;上&#39;。这将增加在努力砍气泡，并迫使更多的氧进入溶液作为增长的需求增加搅拌速度。 </LI&gt; <li>滚动到宝2参数并将其值设置为30。按&#39;回车&#39;按钮开始2宝。 </li><li>在PC上启动控制软件包。 </li><li>一旦探针校准，搅动是稳定为300rpm，温度为37℃，pH值接近6.8，现在是时候来接种。使用酒精，消毒，将用于接种的端口。 </li><li>绘制接种到一个注射器，并将其添加到灭菌的端口。关闭该端口。 </li><li>标志着接种在日志的时间。 </li><li>同样重要的是采取的试样，在灭菌时间。消毒收获管筒与醇的末端。 </li><li>打开夹子覆盖收获端口，并使用注射器抽取样本。这第一个样品通常被丢弃，因为它已经坐在管子。 </li><li>使用另一种注射器拉另一个样本。 </li><li>与第三注射器，推回空气通过管道以去除尽可能多的死区容积从行和夹紧线断开。 </li><li>确定细胞密度，pH，并记录该值。用微生物培养物，它每隔一小时记录的值是非常有用的。间隔是文化依赖。 </li><li>收获方法取决于意图的文化发展完成后。在这种情况下，样品的培养，最后一次接收端点值，细胞密度和湿细胞重量尽可能的pH值或测量值。打开一个指定的"必杀槽"与漂白剂或其他抗菌药物处置内容的磁头板。 </li></ol>

<ol>
	<li>Shuler, M., Kargi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioprocess Engineering Basic Concepts. , (2002).</li><li>Hambor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+Design+and+Bioprocess+Controls+for+Industrialized+Cell+Processing.">Bioreactor Design and Bioprocess Controls for Industrialized Cell Processing.</a> BioProcess International. 10 (6), 22-33 (2012).</li><li>Julien, C., Whitford, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor,+Monitoring,+Modeling+and+Simulation.">Bioreactor, Monitoring, Modeling and Simulation.</a> BioProcess International. 5 (01), S10-S17 (2007).</li><li>Fike, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+Supplementation+Strategies+for+Biopharmaceutical+Production.+Part+1:+Identifying+a+Formulation.">Nutrient Supplementation Strategies for Biopharmaceutical Production. Part 1: Identifying a Formulation.</a> BioProcess International. 7 (11), 46-52 (2009).</li><li>Jana, S., Deb, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+efficient+production+of+heterologous+proteins+in+Escherichia+coli.">Strategies for efficient production of heterologous proteins in Escherichia coli.</a> Appl Microbiol Biotechnol. 67, 289-298 (2005).</li><li>Wang, Z., Ly, M., Zhang, F., Zhong, W., Suen, A., Hickey, A. M., Dordick, J. S., Linhardt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+K5+fermentation+and+the+preparation+of+heparosan,+a+bioengineered+heparin+precursor.">E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor.</a> Biotechnol. Bioeng. 107, 964-973 (2010).</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>Infors, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Minifors operating Manual and user guide. , .</li></ol>

作者答:木兰是ATR Biotech公司雇员产生在这篇文章中使用的试剂和仪器。

搅拌罐生物反应器在生物技术行业标准，并已被用于超过40年的1。小搅拌罐一直重要的比例增加，按比例缩小，应变优化，表征和工艺开发。它也可以具有在个体化用药2的发展中起重要作用。在小规模的生物反应器是最相似的原位条件下细胞的生长，因为它可以被监控并在整个运行优化。大多数情况下，最初的实验中所使用的摇瓶中进行，但在小规模的生物反应器的条件从摇瓶显著差异。在一个实验中，我们发现，对于大肠杆菌的最佳生长条件大肠杆菌 ，并在摇瓶中生产的绿色荧光蛋白（GFP）的未翻译到搅拌罐（未发表数据）。 生长的细胞在大规模的其它方法包括滚瓶，单U本身摇摆平台生物反应器3，以及较大的一次性使用的生物反应器具有工作体积从50到5,000 L。每个方法提供规模化的挑战，但已发现在生产中的地方。单次使用的摇摆平台生物反应器是相似的搅拌槽，并提供稳定的环境。其不同之处在于混合的搅拌釜的发生是由于摇摆运动，以产生波，以防止细胞沉淀，并提供氧气。流体力学此方法是不同的从搅拌罐，并限制最大音量，以1,000 L的差别会影响细胞的生长和产品生产。其它一次性使用的系统相结合的搅拌罐用一次性反应器中，以提供具有最小的基础结构和相关联的开销的平台，而对于高通量的能力，生物处理4。 台式生物反应器的新用户可能会遇到麻烦确定初始设定值pH值，PO 2和温温度，然而，发表的研究可以对这些信息5，6，7，8，9引用。与在特定的细菌培养物时，建议以相同的速度作为摇瓶中，并在同一给定的温度，开始搅拌。从前面的摇瓶培养的运行pH值也可以用来作为一个起点。设定宝2的值是比较困难的，通常是凭经验确定。然而，50％的PO 2开始是建议起点。

基本发酵技术是用于生长培养物的简单摇瓶法的延伸。它的前身，以控制生长环境，以便在一个更完整，更定量的方式生活文化的愿望。分批培养摇瓶通常是由温度控制不精确的限制。温度均匀度在培养摇床或温暖的房间是充满变数，有时偏离5°C以上的预期设定值。由于摇瓶通常搅拌以固定的速度，耗氧量和气体交换是有限的。一旦环境提供氧气被耗尽，大多数文化无法茁壮成长。有在摇瓶没有pH控制。在许多情况下，如果文化不被原料的限制，它变成酸性，以损害的点到文化和呼吸减慢显着。最摇瓶培养物也可作为"批处理"，这意味着它们在室温或接近的培养的开始inoculatio只喂一次运行Ñ​​。经过这一初步的碳源被消耗，将培养停止生长。在某些情况下，它的代谢可能会移动并开始消耗其它代谢物在培养液中，有时会改变所得到的生物量或蛋白质的特性。摇瓶通常也须在37°C在温暖的文化环境中，每24小时的量通常为10％介质蒸发损失这个损失改变培养的密度，并禁止该系统的较长期的操作。最后，用户可能会遇到的搅拌后，从介质起泡。泡沫在培养上面的顶部空间的出现会限制气体交换，并进一步抑止增长。 基本发酵系统旨在解决所有的这些限制。仔细的温度控制在发酵容器通过使用叶轮的搅拌和加热夹套来实现。通常插入加热这件夹克和冷却的容器和反馈控制的传感器结果在温度控制在±0.1℃下围绕设定值。台式发酵罐一般是通过泵通过液体试剂除了提供pH值的控制。 pH值在努力保持环境最佳的细胞生长持续监测。适当的通风是通过上述混合叶轮或通过空气或补充氧气直接加入培养物的输液维持。与剪切敏感的培养，补充氧气是主要的机制维护的在培养的氧气水平。氧在溶液中的测量通常是由极谱探针，其通常不是可用于在摇瓶中使用来实现。另外，也可以连续地或周期性地加入进料至容器保持增长以线性或指数方式。出口气体的冷凝器提供的废气流冷凝冷的表面为气相，从而维持培养物体积和密度。定期加消泡剂的表面活性剂是促动通过在培养的导电率探头，降低泡沫的表面上，并允许气体交换。 的容器中，与所有的探针，管件，叶轮，收获的管道，管，组装和灭菌的标准高压釜中。经过最后的探头校准和稳定经营环境，文化加入到容器中。该系统可以被用来向培养的方式，比用摇瓶法更加量化和精确的表征。温度，pH，氧含量，饲料消耗，液体的蒸发和泡沫水平的紧密控制所有向更高的生物量和更好的蛋白质产量。

<table border="0" cellpadding="0" cellspacing="0" width="356">
	<colgroup>
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:117px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">LB broth</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">L3022</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">ampicillin</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">A1593-25G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">LB broth</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">L3022</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">antifoam 204</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">A8311</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;"> </td>
			<td style="width:117px;"> </td>
			<td style="width:117px;"> </td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">1 M Sodium Hydroxide</td>
			<td style="width:117px;">Sigma</td>
			<td>38215-1EA-R</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">Reference Buffer, pH 4.00</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">B5020</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">Reference Buffer, pH 7.00</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:117px;">B4770</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">pO2 probe electrolyte</td>
			<td style="width:117px;">Broadley James</td>
			<td>AS-3140-C30-0025</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">0.45 micron filter</td>
			<td style="width:117px;">Cole Parmer</td>
			<td style="width:117px;">EW-02915-22</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">0.22 micron filter</td>
			<td style="width:117px;">Cole Parmer</td>
			<td style="width:117px;">EW-29950-40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">Luer Lock syringe, 10 mL</td>
			<td style="width:117px;">Cole Parmer</td>
			<td style="width:117px;">EW-07940-12</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">Minifors Bioreactor</td>
			<td style="width:117px;">Infors HT</td>
			<td style="width:117px;">B-Pack 5.0</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">Air Admiral air pump</td>
			<td style="width:117px;">Cole Parmer</td>
			<td style="width:117px;">EW-79202-00</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:123px;">1175 PD Chilling circulator</td>
			<td style="width:117px;">VWR</td>
			<td style="width:117px;">13721-204</td>
		</tr>
	</tbody>
</table>

operation of a benchtop bioreactor

发酵系统用于提供最佳的生长环境为许多不同类型的细胞培养物。通过发酵得到小心控制温度，pH值，特别是溶解的氧的浓度的能力使得它们必须有效地大规模生长和发酵产物的表达。此视频将简要地描述在摇瓶发酵的优点。它还将确定一个典型的台式发酵系统的重要组成部分，并给予基本的指令上设置了探头的容器和校准。观众将熟悉的灭菌过程，并展示了如何接种生长培养基与文化的容器。操作时，取样和收获的基本概念也将被证明。简单的数据分析和系统清理也将被讨论。

生物反应器可以使用或不使用IRIS软件来运行。然而捕获数据，最好是使用该软件。除了细菌之前，将pH和氧传感器必须进行校准时，叶轮速度设定和设定温度。在图2中，数据输出为生物反应器运行呈现。将温度设定为30℃，搅拌速度为200rpm。该参数可以是不同的每个实验，但除了细菌之前，系统应该处于稳定状态。在图3中，示出了在氧浓度的变化与另外的细菌培养物。设定点对于本实验是在37℃，搅拌器在200rpm下，O 2在70％以上。在时间19:20，进料泵提供了菌种培养在0.1的OD造成立即在O 2水平下降。所述生物反应器来响应变化的O 2水平与增加的气流和叶轮速度。 Ŧ他设置点的增加是在级联（步骤35）设置。将反应器pH值进行实时监测，pH值下降，然后就证明了随着时间的推移将pH线波动（ 图4）增大。在整个运行可以分析和参数调整后续实验。 <img alt="图1" src="/files/ftp_upload/50582/50582fig1.jpg" /> 图1。台式生物反应器与标记和连接所有部分。该图显示了搅拌罐反应器在运行开始。在反应器的部分被标记为与包括传感器，叶轮，温度计，进料和取样瓶，排气口，空气压力计，加热夹套，冷凝器塔，和控制面板。 <img alt="图2" src="/files/ftp_upload/50582/50582fig2.jpg" /> 图2。生物反应器SOF的屏幕图像tware在运行开始时，在一个搅拌罐运行开始时，将温度设置在30℃（黄线），叶轮转速为200rpm（红线），pH为6.5（绿线），和PO 2在57.2％（蓝线）。 <a href="https://www.jove.com/files/ftp_upload/50582/50582fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50582/50582fig3.jpg" /> 图3。生物反应器中的软件的运行为氧时屏幕图像而减小。当培养是在活跃的生长阶段，它消耗的氧气和氧气在反应器中的量减少（蓝线）。通过增加叶轮转速，更多的氧气可以被添加到文化（红线）。 <a href="https://www.jove.com/files/ftp_upload/50582/50582fig3large.jpg" target="_blank">点击这里查看大图</a> 。 图4。文化的生物反应器软件显示随时间变化的pH值的 pH值屏幕图像的连续监测，并随着时间的推移它减少乳酸的产生，然后会随着碱加入到生物反应器（蓝线）。 <a href="https://www.jove.com/files/ftp_upload/50582/50582fig4large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Operation of a Benchtop Bioreactor at JoVE.com

Operation of a Benchtop Bioreactor

发酵罐是用来增加培养产量生物工程化细胞和生产率。在摇瓶筛选多种微生物或动物细胞培养人选后，下一个合乎逻辑的步骤是增加了选择的文化的生物质发酵罐。该视频演示了一个典型的台式生物反应器系统的设置和操作。

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 ...

Research

JoVE Journal

Bioengineering

88.7K 次观看.  Johns Hopkins University. 发酵罐是用来增加培养产量生物工程化细胞和生产率。在摇瓶筛选多种微生物或动物细胞培养人选后，下一个合乎逻辑的步骤是增加了选择的文化的生物质发酵罐。该视频演示了一个典型的台式生物反应器系统的设置和操作。 

视频: Operation of a Benchtop Bioreactor

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

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 ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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

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

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

培养哺乳动物细胞使用一次性使用气动生物反应器系统

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

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 ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

增强分子化合物输送到细胞的声透流体装置的组装和操作

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

开源工具包:用于神经记录的台式碳纤维微电极阵列

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...