The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.

1.细胞系和维护<ol><li>得到βTC6细胞（或其他期望的粘附的哺乳动物细胞系）。在用于研究制备，培养，传代，并根据供应商的说明冷冻保存的细胞。 </li></ol> 2.组装连续进料系统注:连续给料系统设计在下面的方法是根据文献中描述的17类似的系统- 19,21,47 - 49。这里使用的系统的组件的详细情况的先前出版物3所述。 <ol><li>收集所需馈送系统，该系统由五个主要部件的组件:一个介质贮存器，蠕动泵，搅拌生物反应器，废物储存器，和定制设计管道/采样集。 <ol><li>使用1升的玻璃瓶中，用2升玻璃瓶废物储存器，并且stirre建立介质容器使用的250毫升容积的玻璃反应器（现成版本）D的生物反应器。 </li><li>使用数字蠕动泵或类似的泵用8通道泵头来控制媒体交流。 </li><li>从制造硬塑料高压灭菌的容器和修改的生物反应器盖与不锈钢管直通端口通过无菌过滤器，以提供通风，并允许水库和生物反应器之间的介质输送。此外，与购买不同厂商的流通口盖专业化。 注:该生物反应器盖可能包含可选的仪器和监控探头（如氧气检测仪）额外直通端口。 </li><li>附上从多孔玻璃曝气管具有40微米至60微米的平均孔径范围制造的流出管（OT），和40％的孔密度，以修改后的单边带盖子。或者，选择基于文化的特殊要求另一流出管。 注:选择OT孔径仅去除培养基和细胞碎片，留下在培养细胞聚集体（如更多细节在第6和出版物3描述）。 </li></ol></li><li>组装从聚偏二氟乙烯（PVDF）管连接器和耐用蠕动泵管高压灭菌灌注管套。该部分的长度是依赖于不同的部件之间的距离。 注意:选择各种管线时，确保长期的实验，耐用性和可靠性，应特别小心。 </li><li>装配油管从三个部分设置:馈送线，废线，和一采样线。 <ol><li>组装使用两个管道直径（L / S 14和L / S 13）的进料管线。使用L / S 14用于主油管，将跨越从生物反应器至介质储存器的距离，并且在使用适当的PVDF适配器泵部的管道长度的中间插入L的短长度/ S 13。 </li><li>使用标准的PVDF软管带刺TUBI纳克适配器加入的L / S 14油管两片在较短泵部（L / S 13）管的两侧。 注意:可替换地，管的整个长度可以是直径较小的L / S 13油管，并且当泵截面完整性是有问题的整个管道长度可以被替换。 </li></ol></li><li>组装使用直径较大（L / S 16）管垃圾处理第二管道部分，并在中间的泵送段插入L / S 14管材短节。这是因为馈送线组件使用的PVDF软管倒钩的接头，或者可替换地使用所需的泵管大小的连续长度，并根据需要替换以相同的方式完成的。 注意:如果同一泵和泵头部被用于馈电电路和废电路，所述废物去除管道线的泵部分必须比馈送线的泵部更大的直径。这确保了去除泵速度比进给速度更快，并且将避免效IAL在培养物体积大的变化，并减少溢出的风险。 </li><li>组装管道组的最后一个组成部分:一个样本收集组件。 <ol><li>此过程描述组装取样口系统定制;可替代地，无菌取样口可以从各种供应商处购买。 </li><li>从三个短（〜6厘米）L / S 14油管具有T型PVDF连接器连接在一起的长度，并在两个管道长度的两个小软管夹构造集。 </li><li>附加气体放空无菌气体过滤器，以被夹持长度之一，而另一个是用于连接到一个无菌取样注射器（灭菌气体过滤器可能需要在生物安全柜中添加以下灭菌许多无菌过滤器是不高压灭菌）。 </li><li>第三端连接到安装在该生物反应器的盖的不锈钢样品连接器。 </li><li>高压灭菌取样组件而连接到前开始在生物反应器实验（参见下面的步骤3）。 </li><li>使用此装配需要，而不会干扰连续进料过程，从生物反应器中收集样品不育。 </li></ol></li></ol> 3.高压灭菌器所有材料<ol><li>准备高压灭菌的所有生物反应器组件。 </li><li>收集灭菌单个组件。介质和废物储（从步骤2.1.1组装），250毫升旋转瓶中（从2.1.1组装），必要的修正盖（距离2.1.3组装），流出管（在2.1.4中描述），三管套（在组装根据需要持续喂养）第2.2 through2.5），流出管（。 <ol><li>组装旋转烧瓶中的所有组件，并确保流出管的旋转烧瓶内牢固地附着（在2.1.4节中所述），并附加了自定义组装采样口（2.5节），或其他取样口的盖子的顶部。 </li><li>包裹自动整个旋转烧瓶装配釜包装材料，和高压表明磁带。要确保包装不透气。注:铝箔或类似的材料可被用于覆盖在生物反应器盖的每个接入端口的各个端部，以保持连接器的无菌展开时和当非连接到培养箱内管套/结束。 </li><li>组装平台和废物储层的改性盖子，然后附加到各自的玻璃瓶中。包括他们用铝箔和高压灭菌指示胶带。 </li><li>单独包装的管套（2.2节 - 2.5）与釜包和磁带，以协助总装。铝箔或类似的材料可用于包装每个管道组以保持连接器的不育的个体端部展开时/结束。 </li><li>高压灭菌使用标准的干釜周期都包裹项目（ 例如 ，"万有引力"〜设定与在121℃下15磅30分钟以上）。 注意:不要将无菌高压灭菌前，过滤器，除非它们被证实是安全的高压锅。 </li></ol></li></ol> 4.球状体形成注:这种技术是类似于在文献17中描述- 19,21,50 - 52，用于其它哺乳动物细胞培养物。以下灭菌所有程序应在层流罩来完成，并使用无菌手套，以维持细胞培养无菌条件。 <ol><li>在所有条件下，培养和扩大β-TC6细胞标准粘附培养物（由供应商所描述），直到足够的细胞数量时，获得种子生物反应器的期望数目。 <ol><li>组装与流出管的连续进料的生物反应器， 如图1和第2所述，和取样口连接到采样盖子。注:流出管和取样口组件应该被添加到bioreact或连续馈送之前球体的形成，并应直到连续馈送启动拉升出培养基。 </li><li>通过高压灭菌器修改SSB组件（第3节中所述）。 </li><li>附加通风口灭菌（0.22微米或更小的无菌过滤器）在旋转瓶的盖子，和中和废物容器中的适当位置。 注意:这是重要的，以防止蒸气锁，并允许培养箱（5％CO 2）的环境和生物反应器之间的气体交换。气体交换是必要使用碳酸氢盐缓冲介质时保持正确的pH值。 </li></ol></li><li>通过使用0.25％（重量/体积）胰蛋白酶0.53毫摩尔EDTA溶液温和胰蛋白酶消化收集细胞，在通过机械搅拌进行2辅助室温- 3分钟，并以约1.3×10 6细胞/ ml的密度接种到生物反应器在200毫升培养基中。 <ol><li>移动指定烧瓶出保育箱的和成生物安全柜。 注:所有细胞培养和操作应在使用适当的无菌技术一个生物安全柜来完成。 </li><li>通过抽吸从瓶中取出培养基。 </li><li>吹打5毫升磷酸盐缓冲盐水的（不含Ca ++或Mg ++），进烧瓶中，并在表面上横跨细胞漂洗洗涤细胞，然后吸出PBS中。 </li><li>添加3ml胰蛋白酶-EDTA溶液，以将收集的每种烧瓶（通常为约10倍175厘米2 T-烧瓶）。 </li><li>允许收集的细胞在室温下进行2孵育 - 在生物安全柜3分钟。 </li><li>轻轻搅动用手从烧瓶表面松开的细胞，并通过加入6毫升培养培养基（血清）的向烧瓶收集细胞，并将细胞转移到50毫升管中。当一个50ml管中充满，用另一种50ml管中，直到所有需要的细胞已收集（大约将需要每5的T-175烧瓶1个50毫升管集）。 </li><li>轻轻离心（约50×比重）所收集的细胞悬浮液以沉淀细胞。 </li><li>吸出剩余的培养基而使粒料完好无损。 </li><li>通过重新悬浮颗粒培养基（含血清）中使用移液管，并且所有丸粒转移到同一管收集在一个单一的管中的细胞。 </li><li>算使用标准血细胞计数用锥虫蓝染色，如在文献53中描述的细胞密度。 </li><li>加总细胞的期望数量的无菌单边带每个条件（1.3×10 6个细胞/ ml是这个示范针对性的起始细胞密度）。 </li><li>添加培养基（与所需的葡萄糖浓度，在这种情况下，我们使用在生理范围内介质葡萄糖水平）使用移液管，以达到所需的总培养物体积（使用200毫升培养体积为这些研究），以SSB。 注意:对于这些连续补SSB研究了铜<html> ltures用培养基（100毫克/分升）的变形的"低血糖"版本接种。这使培养物在所期望的目标葡萄糖浓度开始，而不是与一个"高葡萄糖"介质开始并等待葡萄糖消耗带来的血糖水平下降到生理范围开始喂养。在这些研究中饲养的所有其他介质中使用的标准的"高葡萄糖"介质（500毫克/分升）。 </li><li>与细胞移动到SSB细胞培养孵化器内的轰动板。 </li></ol></li><li>培养中的细胞的生物反应器，而不在37℃下3天喂养，5％的CO 2，相对湿度100％和70 rpm的搅拌速率以允许球体形成。 注意:应在为期三天的球体形成期没有观察到显著的增殖。 </li><li>球体形成后，用分所需的培养条件下的生物反应器中的球体。 </li></ol>乐"&gt; 5。连续投喂文化和调整进给速度<ol><li>高压釜或气体灭菌的所有组件，并且在使用无菌技术的生物安全柜组装（步骤2 - 4）。 </li><li>球体形成后，从孵化器中取出的SSB和组装用于连续进料系统的部件在生物安全柜中。 <ol><li>第一填充所需培养基的新鲜培养基贮存，然后连接到馈送线的一侧。同时确保新鲜培养基水库正常使用无菌过滤器排出。 </li><li>进料管线上的生物反应器的进料流入口的一侧连接以无菌的方式。无菌过滤器应在进料管线和反应器入口端口进入所述生物反应器前过滤介质之间进行安装。 </li><li>连接废液管到生物反应器的流出管和介质废物储存，并确保废物贮存器正确地与一个与不孕放空Ë过滤器。 </li></ol></li><li>移动部件从生物安全柜（这可能需要两个人来进行所有的容器和管道），并把所有的组件出长期培养。 <ol><li>把SSB到搅拌板使用球体的形成（37℃，5％CO 2，100％湿度和70的转速）相同的培养参数培养箱的内部。注意:这可能是必要的暂时断开从生物反应器的管段，如果线路需要通孔在培养箱的侧面通过在门上的垫片运行，而不是进行。这可以在无菌的方式通过包装管套的端部用铝箔前高压灭菌（步骤3），并等待到进料和废料管段的生物反应器一侧连接至所述生物反应器是培养箱内进行。该管线可以到位，并且铝箔可以培养箱内取出，并迅速附着到生物反应器。 </li><li>用完通过培养箱进入口的管子套其余端（没有连接到生物反应器盖的那些）（传递），或者通过在孵化门衬垫的凹口。 </li><li>培养箱（在附近的生物安全柜，如果可能的话）之外，快速连接的进料管线到新鲜培养基水库，且废线向培养基中废物储存器，并确保该废物储存器被正确用无菌过滤器排出。注:要做到这一点在无菌的方式，从管道和容器连接器为快速删除铝箔和连接到各容器尽可能（特别是如果这方面需要的生物安全柜以外的地方完成）。 </li><li>把附近的小冰箱里的新鲜中型水库（充满希望的饲料中）。检查，以确保进料和废线能够退出培养箱和没有防止在每个门从关闭进入电冰箱。它也是进给升临界INES没有被挤压通过任何冰箱或孵化器的门关闭​​。 注意:用于这些研究的培养基是由供应商为这种细胞类型所建议的标准的高葡萄糖（500毫克/分升）的培养基。任何高葡萄糖培养基可以根据细胞的类型是培养的具体需要一起使用。补料培养基的葡萄糖浓度应该相当高（2倍或更多）比作为馈送系统依靠增加高葡萄糖培养基进料速率，以保持足够的葡萄糖水平在培养物，同时保持合理的进料速率的培养所需的浓度。 </li><li>接下来，废物中型水库可以在一个方便的位置摆上了台面孵化器之外。 </li><li>接下来，将进料和废线入泵头确保正确的方向，以使供电线将从新鲜培养基储到单边带泵送介质的"泵部"，和废线将泵从SSB和废中型水库中。 </li></ol></li><li>打开泵上的所需进料速率。 <ol><li>计算使用文献3并在下面所提供的代表性的结果部分中所述的调整的进料速率方程的进给速率。 </li><li>使用具有生长预测沿着最近的细胞计数信息（在步骤5中描述的过程），并且该介质葡萄糖测量来估计，以维持在培养所需要的葡萄糖浓度所需要的进给速率。 注意:细胞生长率和葡萄糖消耗率就需要在做这些程序来获得。 </li><li>设置基于计算出的进料速率泵的转速。 </li></ol></li><li>重复进给速率的计算和调节泵的转速为每个采样点（对于所描述的方法，每三天）。 </li></ol> 6.细胞计数，活力和葡萄糖浓度测量 &lt;OL&gt; <li>从每三天，或根据需要每个生物反应器收集样本。 </li><li>从使用上述专门取样口连续供给的SSB培养物采取培养样品。 <ol><li>离开连续加入SSB生物反应器内，附加一个无菌注射器（用5ml的注射器体积为这些研究）的采样口的未过滤的连接。 </li><li>向下移动至取样管进入培养基。 </li><li>松开管部将注射器，并撤销所述期望的总样品体积。 </li><li>移动至取样管上，以便它在培养不再淹没，并继续撤回注射器，直到空气被除去。 </li><li>钳哺养注射器管，取下装有样品的注射器，并预留。 </li><li>预负荷与空气的第二新鲜无菌注射器，并连接到采样口的无菌过滤器的一面。 </li><li>松开管要的注射器过滤器侧取样端口，然后通过经由无菌过滤器轻轻排出空气中的注射器清除取样端口。这确保了采样端口将清楚任何残留的介质。 </li><li>再夹紧管将过滤器，断开无菌注射器和丢弃。 </li></ol></li><li>采取包含从培养的细胞样品的注射器，它排出到10ml管中。已知体积的子样品可以直接从该管作出。 </li><li>用于细胞计数，去除细胞的已知体积和转移到微离心管中，并进行1轻轻离心 - 2分钟（约50×重力）。 </li><li>将上清转移到单独的管，用于葡萄糖的测量，并重新悬浮在相同体积的胰蛋白酶-EDTA溶液中的沉淀（0.25％（重量/体积）胰蛋白酶，0.53毫摩尔EDTA），并使用标准血细胞计数器与一式三份计数如文献53中所述台盼蓝染色。 <ol><li>添加培养基（血清）到将样品稀释为必要的。 </li><li>计数细胞，并通过记录活（未染色）细胞计算细胞活力，和死（染色）细胞独立地（存活率（％）=活细胞/总细胞）。 </li></ol></li><li>测量使用血糖仪和单次使用试条在一式三份培养基葡萄糖水平。 <ol><li>采取如由血糖仪指示代替血液培养基中描述的葡萄糖的测量。 </li><li>浸测试条在被测试的介质（从上面数样品收集），并重复对重复的期望数目（三次重复的建议）。 </li><li>测试既为葡萄糖浓度的新鲜培养基和废物网上平台。 注:对于一些米，测量低于20毫克/分升（米可检测阈值），可以观察到与表输出一个错误。 </li></ol></li></ol> 7.球体沉降速率测量<ol><li>为了确保CEL升集群不是由连续馈送系统中除去，通过观察在一大直径的塑料吸管其沉降测量β-TC6球状体的沉降速率。 </li><li>培养β-TC6细胞SSB生物反应器中，如上所述，以形成球状体。 </li><li>在培养3天后，取球体悬浮液和放置30毫升样品中的50ml管中。 </li><li>轻轻吸管上下使用宽直径为25毫升吸管分配均匀悬浮，然后在移液管（ 如20毫升标记）已知水平与悬架停止移液，然后记录时间为所有的球体来解决5厘米。 </li><li>重复此过程足够的时间达到统计置信（通常N&gt; 3）。 注:对于生物研究，p值小于0.05被认为是比较测量时是统计学显著。用于报告错误均值的标准差和双尾未配对学生t检验被用来比较对所呈现的数据的条件。 </li></ol>

The authors have nothing to disclose.

生成哺乳动物细胞产品的生产生物战剂和细胞治疗需要大规模55文化和哺乳动物细胞的监测- 58。此外，这些应用要求定义和验证的培养条件。简单地增加使用研究技术不能满足所有这些要求细胞的体积。造成波动的营养物质和废物积聚产品中手动修改降低细胞的质量，生存能力和产量。利用生物反应器的是公认的微生物的生物制药应用的商业文化，和类似的策略已经应用?...

为了产生大量可行的和功能性人细胞移植，的培养条件调节是必要的。营养物质的消耗，与代谢废物堆积沿主要贡献者，以减少电池产品的质量1衰老和代谢改变- 3。此过程说明使用搅拌生物反应器具有调整速率灌注料系统相结合，以调节整个培养期间在生理范围4中的葡萄糖球体培养的哺乳动物细胞的方法。这些研究的目的，在生理范围被定义为100 200毫克/分升之间。相同的方法可用于调节其它营养物质和代谢废物如乳酸盐。 在小体积的静态培养物（1 - 30毫升）在实验室环境通常用于维持和分化为experime细胞系ntal目的。细胞传代与根据需要定期完全培养基变化进行的。最"常规"培养基具有高的葡萄糖浓度（在这些研究中使用的DMEM 450毫克/分升），以允许无养分限制的风险较不频繁的介质的变化。然而，这种分批输送方法仍然需要频繁操纵，在细胞环境中引入了可变性，并增加污染5的风险- 9。搅拌的生物反应器（SSB）提供更好的混合，降低处理3,10 - 20，但像静态的文化，需要有助于营养物质和废物产品级别潜在的破坏性波动手动媒介的变化。单边带培养物的灌注馈送减少这些问题通过连续输注和去除介质，但是在营养水平由于细胞生长大的变化仍是一个问题。使用调整后的喂养岭德从基于估计的细胞所需养分使用的计算可以提供优化细胞生存力所需的稳定的细胞环境和功能21 - 24。 有大量文献描述的哺乳动物细胞的可扩展SSB文化专为多能干细胞的培养和拓展方法25 - 32，与其他专注于胰岛（测试版）单元17,33,34，或生产生物制品24， 35 - 38。许多这些研究的细胞类型，可以在球体培养物中生长，并为所使用的细胞类型特异性的程序应之前实施的连续进料系统进行优化。在这个演示中，采用灌注饲养方法，扩大成长为在搅拌反应器39的球状体β细胞系- 43。本文所描述的方法提供了一种简单的实现饲养的基础上脱机葡萄糖测量来实现有针对性的培养条件下利率调整。用这种方法调节进料速率，以保持生理葡萄糖水平被示出为增加细胞产量。哺乳动物细胞是依赖于一个关键营养素，葡萄糖，用于能源生产，所以使用该细胞系的代表许多培养的哺乳动物细胞44的模型。此外，这条线例示的β细胞，这是慢性高含量葡萄糖45的敏感的进一步复杂性。在这项研究中，β-TC6允许细胞以形成培养球体近似体内胰岛的平均尺寸。灌注生物反应器系统17 - 19,21,46调整为葡萄糖消耗的进料速率，导致在保持生理条件和较高的细胞产量无生存力的改变。

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Name of Reagent/ Equipment</td>
			<td style="width:249px;">Company</td>
			<td style="width:153px;">Catalog Number / Link</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BTC-6 Cells</td>
			<td style="width:249px;">ATCC, Manassas, VA</td>
			<td style="width:153px;">CRL-11506</td>
			<td>Mouse Insulinoma cell line (adherent cell type)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS No CA, No Mg</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">14190-144</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144">https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Dulbecco&#39;s Modified Eagles Medium</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;"></td>
			<td>See below for product numbers&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM High Glucose (500mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11965-092</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11965092">http://www.lifetechnologies.com/order/catalog/product/11965092</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM Low Glucose (100mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11885-084</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11885084%20(note%20that%20this%20medium%20already%20contains%20pyruvate)">http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">L-gultamine</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25030081</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product">http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium Pyruvate</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">11360070</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Heat Inactivated Porcine Serum</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">10082147</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/10082147">http://www.lifetechnologies.com/order/catalog/product/10082147</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypsin-EDTA</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25200056</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T-150 Tissue Culture Treated Flasks</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">430825</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuAire Cell culture incubator</td>
			<td style="width:249px;">Princeton, MN</td>
			<td style="width:153px;"></td>
			<td>US Autoflow , Any water-jacketed CO2 regulating cell culture incubator could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Sorvall RT 7 (Any similar benchtop centrifuge may be used)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Refrigerator</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Any laboratory refrigerator could be used (a small table-top version was used for these studies)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-1L</td>
			<td>Any vendor could be used http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences) 
			&#38;categoryname=</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">2L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-2L</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">250 ml stirred bioreactors&#160;</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">4500-250</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stir Plate</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">11-496-104A</td>
			<td>Any incubator safe stir-plate can be used, any vendor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tissue Culture Dishes 100mm Diameter</td>
			<td style="width:249px;">Nunc, Rochester, NY (Fisher Scientific)</td>
			<td style="width:153px;">1256598&#160;</td>
			<td>Any vendor could be used (ordered through Fisher Sci)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">FALCON 50 ml Conical Tubes</td>
			<td style="width:249px;">Falcon, San Jose, CA</td>
			<td style="width:153px;">1256598</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Delran Plastic Used for Custom Parts</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any material of choice could be used, but Deran is chosen because it is autoclave safe, non-reactive, and easy to machine, http://www.mcmaster.com/#acetal-homopolymer-sheets/=rjrcac</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stainless Steel Pipe for custom lids</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any vendor could be used, http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Delran Bioreactor Lids for Continuous Feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Not aware of any vendors producing a similar product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Glass Bottle Lids for Continuous feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Some vendors (eg. Fischer Sci, Corning) make similar products in the links below</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Masterflex Digital Peristaltic Pump</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">EW-77919-25</td>
			<td>Any precision peristaltic pump could be used, http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_ 
			Cartridge_Pump_System_115_230 
			_VAC/EW-77919-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PVDF Tubing Connectors (various)</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">see link</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium 
			_Luer_Fittings/55889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 16</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-16</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 14</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-14</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 13</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-13</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-13</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Millipore Millex GP PES membrane 0.22ul sterile syringe filter (used for venting, and medium filtration)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">SLGP033RS</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">25ml Graduated Pipette</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-678-11</td>
			<td>Any vendor could be used, and various sizes may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pipetter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-681-15E</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Hemocytometer</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">02-671-6</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypan Blue</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">15250-061</td>
			<td>Any vendor, or similar product could be used, https://www.lifetechnologies.com/order/catalog/product/15250061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Inverted Light Microscope</td>
			<td style="width:249px;">Leica</td>
			<td style="width:153px;"></td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra Blood Glucose Meter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-293&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra-Strips</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-292&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
	</tbody>
</table>

nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

中血糖水平和波动限制在标准SSB培养细胞扩增 血糖水平在静态的文化和文化SSB波动在整个培养周期3。这些波动加强与在21天的培养期增加细胞数量并分别在静态和SSB文化几乎相同。这些意见在我们以前的出版物3中。葡萄糖水平可以是超生理的培养期间这两种方法的持续时间。由于这种长期接触可能会抑制细胞生长54，连续供料系统的开?...

Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

nutrient-regulation-continuous-feeding-for-large-scale-expansion

Research

JoVE Journal

Bioengineering

7.7K 次观看.  University of Minnesota. Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

视频: Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

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 ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...