我们感谢吉尔·奥斯本在乳化过程中的铺路工作以及劳伦·威尔金森的技术支持。我们感谢IgorLaçik博士，Timothy J. Kieffer博士和James D. Johnson博士的投入和合作。我们感谢魁北克省魁北克省，JDRF，ThéCell，中心区域自然科学和工程研究理事会（NSERC），人类胰岛移植和β细胞再生中心，加拿大干细胞网络，迈克尔·史密斯健康研究基金会，法国自然保护协会和COST 865财务支持。

1.准备藻酸盐溶液，CaCO 3悬浮液和酸化油<ol><li> 准备过程缓冲区和介质。 <ol><li>制备用于使用10mM HEPES，170mM NaCl产生高浓度藻酸盐珠粒的典型工艺缓冲液。将pH调节至7.4。 </li><li>使用含有10％胎牛血清（FBS），6mM谷氨酰胺，100U / mL青霉素和100mg / mL链霉素的Dulbecco&#39;s Modified Eagle培养基（DMEM）制备典型的培养基。 </li></ol></li><li> 将藻酸盐原液制备为珠粒最终所需浓度的1.17倍。 <ol><li>首先称重适量的藻酸盐粉末。例如，为了获得具有50:50 LVM和MVG藻酸盐的5％藻酸盐的最终浓度，通过组合0.583g LVM藻酸盐粉末和0.583g MVG藻酸盐粉末来计量总共1.17g藻酸钠。 </li><li>将20 mL过程缓冲区放入autoc在搅拌盘上搅拌约200转/分钟。逐渐向溶液中加入藻酸钠粉末。 </li><li>将溶液以低速搅拌过夜。如有必要，将烧瓶固定到搅拌板上。 </li><li>如果溶液不完全溶解，将瓶子固定在旋转混合器上，并在37℃下继续混合24小时。 </li><li>通过在小于半满的容器中将溶液高压灭菌30分钟来灭藻藻酸盐溶液。在取出溶液之前让温度降至60°C以下。 </li><li>如果需要，通过在高压灭菌之后不久将藻酸盐溶液过滤至达到室温，同时粘度保持足够低的情况下除去颗粒。 </li></ol></li><li> 以最终所需浓度的21倍制备碳酸钙悬浮液用于内部凝胶化。 <ol><li>称量CaCO 3粉末。例如，广告d 1g CaCO 3至20mL工艺缓冲液以获得24mM CaCO 3作为最终胶凝剂浓度。 </li><li>将CaCO 3悬浮液高压灭菌30分钟。 注意:由于碳酸氢二氢二氧化碳的平衡，CaCO 3浓度会随时间而变化。避免使用相同的库存CaCO 3悬浮液进行10多次封装程序。 </li></ol></li><li>高压灭菌用于乳化过程的搅拌釜。在使用前，清除旋转瓶中剩余的冷凝水痕迹。 </li><li>在乳化过程之前，准备酸化油。溶解44μL醋酸每11 mL矿物油放置在一个50mL锥形管中。 注意:一个常见的错误是醋酸的不完全溶解。避免吸取少量乙酸，并确保通过反复涡旋将乙酸完全溶解在油中。 注意:乙酸是一种有毒的<html> d挥发酸。在通风橱下处理此试剂，并将乙酸/油溶液保持在密闭容器中直到乳化步骤。请参阅此试剂的MSDS信息了解更多信息。 </li><li>在进行细胞包封之前，让所有的溶液达到室温。 </li></ol> 2.通过乳化和内部凝胶化产生藻酸盐珠<ol><li>将10 mL轻质矿物油放入旋转式烧瓶中，并以低转速开始搅拌（ 如本影像中使用的旋转烧瓶为250 rpm）。 </li><li> 如果用于该过程的细胞来自贴壁培养物，则应用胰蛋白酶来悬浮细胞。通过加入含FBS的培养基或胰蛋白酶抑制剂结束反应，并收集样品用于细胞计数。 <ol><li>使用血细胞计数器或自动细胞计数器确定台盼蓝染色后的细胞浓度和活力，如前所述ass ="xref"&gt; 32。 </li></ol></li><li>以300×g离心细胞7分钟，并在所需培养基中洗涤一次以进行细胞固定。确保该培养基含有乳化剂，如FBS或牛血清白蛋白（BSA）。例如，使用含10％FBS的DMEM。 </li><li>将细胞沉淀重新悬浮在相同的培养基中，以获得珠粒中所需的最终浓度的10.5倍。 </li><li>加入1.1 mL 10.5倍浓缩的细胞储备液至9.9 mL藻酸盐溶液。然后，加入550μLCaCO 3悬浮液，并用无菌刮刀搅拌混合，以确保CaCO 3均匀分布。 </li><li>使用注射器立即将10.5mL的藻酸盐，细胞和CaCO 3混合物转移到搅拌油中。 注意:对于高粘度溶液，可以缓慢吸出并喷射混合物以避免气泡。在一些情况下，优选在将混合物加入烧瓶中以避免搅拌停止藻酸盐夹带在叶轮轴上。 </li><li>在将藻酸盐，细胞和CaCO 3混合物加入到油中之后，立即增加搅拌速度。 注意:对于本文使用的5％LVM:MVG藻酸盐，应用1025rpm的转速以产生适合于体外培养的约900μm直径的珠粒。如先前的出版物25,32所述，较高的转速和较低的藻酸盐浓度将导致更低的平均珠粒直径。叶轮和容器几何形状的微小变化以及藻酸盐性质可以极大地影响平均珠径。对于容器结构或藻酸盐批次的每个变化，应该产生一个将表面积平均珠粒直径与转速相关的标准曲线32 。 </li><li>启动计时器并将藻酸盐乳化12分钟。 </li><li>加入10 mL t他用油和乙酸溶液酸化乳液，溶解CaCO 3 ，从而将藻酸盐物理交联成胶凝珠。允许该酸化步骤8分钟。 </li></ol>珠回收<ol><li>将搅拌速度降至400 rpm。加入40 mL与10％培养基混合的工艺缓冲液以增加pH并引起相转化。 </li><li> 1分钟后停止搅拌，并将混合物转移到50mL离心管中。用额外的20 mL培养基冲洗旋转瓶，并将其加入管中。在吸出油相之前，尽可能多地从旋转瓶中吸出水相，以尽量减少与油相的珠珠接触。 注意:在此步骤和使用大口径移液管（ 例如 25毫升移液管），以避免损坏珠子。对于较小的体积，珠悬浮液也可以用切割移液管吸头进行操作。要获得这样的提示，用剪刀剪切移液管尖端的末端ile佩戴眼睛保护。 </li><li>以630×g离心管3分钟以加速珠沉淀和相分离。 </li><li>通过用巴斯德吸管吸出去除油和多余的水溶液。 </li><li>用介质洗涤珠子至少一次。过滤40μm尼龙细胞过滤器上的珠粒悬浮液，并从过滤器下方吸出与珠粒相关的过量液体。使用无菌刮铲将珠子转移到已知体积的培养基中。 注意:根据目标最小珠粒直径，此步骤可使用较小或较大孔径的过滤器。然而，推荐重力过滤，而不是通过亚微米孔径过滤器进行压力驱动过滤，以避免损坏珠粒。 </li><li>通过体积位移（加入珠子后的体积减去加入珠粒之前的体积）测量珠体积并填充培养基以获得所需的珠粒:总体积比（通常为1:5）或1mL珠粒4 mL培养基。从这一点上，始终使用大口径移液器，以避免损坏珠子。 </li><li>将包封的细胞转移到T型瓶中用于体外培养或移植实验。 </li></ol> 4.质量控制与应用注意:为了确保包封的细胞和珠粒质量，应该量化该过程之后的珠粒度分布和细胞存活。通常进行反转凝胶以从珠内回收细胞用于进一步分析。 <ol><li> 为了评估珠粒尺寸分布，用甲苯胺蓝-O染色珠粒。 <ol><li>将0.5 mL珠粒放入含有10％完全培养基的4 mL过程缓冲液中。 </li><li>加入500μL在工艺缓冲液中制备的1 g / L甲苯胺蓝溶液。 </li><li>在旋转振荡器上的锥形管中以50rpm孵育60分钟。 </li><li>加入含有10％完全培养基的5 mL过程缓冲液将珠粒和溶液转移到培养皿中，并在低倍数显微镜上或使用手持式数码相机拍摄图像。如果需要，在用手持相机拍摄图像之前，将培养皿放在灯箱上，以增强对比度，避免阴影。 </li><li>执行图像分析以如前所述32量化珠粒度分布，例如使用图像分析免费软件36 。 </li></ol></li><li> 为了定性评估细胞存活，使用同源二聚体染色细胞以鉴定死细胞和钙黄绿素AM以鉴定活细胞。 <ol><li>将1体积的珠添加到含有10％完全培养基的4体积的过程缓冲液中。 </li><li>加入适量的钙黄绿素AM和乙二胺二聚体储备液，分别得到4μM和2μM浓度。通过向一些珠粒样品中添加一种试剂来产生单一染色剂对照。</li><li>在冰上孵育珠子20分钟。 </li><li>将溶液放置在载玻片和盖玻片之间，使用间隔物如O形环，以避免压缩珠粒。 </li><li>继续进行荧光显微镜检查。使用单一污渍控制来评估荧光渗透。根据与钙黄绿素AM（494/517 nm）和与DNA结合的乙炔同二聚体（528/617 nm）相关的激发/发射波长，选择合适的荧光滤光片。 </li></ol></li><li> 为了从珠中回收细胞用于进一步分析，使用柠檬酸盐或另一种螯合溶液使藻酸盐脱胶。 <ol><li>在pH 7.4下制备含有55mM柠檬酸盐，10mM HEPES和95mM NaCl的去脂溶液。 </li><li>将该溶液与10％培养基混合，然后向9mL混合物中加入1mL藻酸盐珠粒。 </li><li>将冰珠上的冰珠75g搅拌20分钟。 注意:细胞现在可用于分析或从去角质的藻酸盐溶液中去除通过离心离心。例如，消毒后的细胞活力可以通过台盼蓝染色定量。或者，可将细胞离心并洗涤，随后进行mRNA，DNA和/或蛋白质取样和分析。 </li></ol></li></ol>

作者没有什么可以披露的。

在内部凝胶化反应期间的各种步骤（ 图2中所示 ）可以限制整个动力学。对于大于〜2.5μm的碳酸钙颗粒，碳酸盐溶解速率已被证明是速率限制26,44 。导致内部钙释放的酸化步骤也被证明是影响细胞存活的关键过程变量32 。因此，导致内部凝胶化的条件对于珠粒质量以及细胞存活是至关重要的。根据珠粒的最终应用，可以选择不同的最佳工艺条件。例如，对于其中珠的长期稳定性至关重要的移植应用，可能需要较高的pH降（ 例如使用10mM HEPES来缓冲藻酸盐溶液）。另一方面，较低的pH下降（ 例如使用60mM MOPS来缓冲藻酸盐溶液）对于体外应用可能是优选的。 除了调整酸化的水平和持续时间之外，还可以设想其它的工艺修改。例如，可以使用其它搅拌容器构型，藻酸盐浓度，工艺缓冲液，有机相和酸化剂。如前所述，通过改变乳化步骤中的搅拌速度可以容易地调节平均珠粒度。此外，较长的乳化时间可导致较小的珠粒尺寸，但可导致细胞活力降低。 Poncelet 等人的出版物25,26,45和Hoesli 等人 31,32已经测试并讨论了其中的一些选项。 上述协议略有变化可以产生深远的影响珠生成，珠粒质量和细胞存活。 表1提供了解并解决可能发生的问题的故障排除指南。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 问题 </td><td> 典型原因 </td><td> 建议的解决方案 </td></tr><tr><td rowspan="2">在藻酸盐或酸性油添加期间，叶轮停止旋转</td><td rowspan="2">磁场不足以充分混合或叶轮轴不对准</td><td>确保旋转烧瓶在磁性板上位于中心位置，并在加入藻酸盐混合物之前以最佳位置贴在板上。测试非常高的搅拌速率，然后在加入藻酸盐混合物之前降低到所需的搅拌速率。 </td></tr><tr><td>确保叶轮轴良好的调整和稳定。对于贝尔lco旋转瓶，确保通过旋转瓶盖两侧的螺栓将叶轮牢固地固定在适当位置。确保叶轮运动受径向限制（叶轮不应摆在轴上）。 </td></tr><tr><td>没有珠子</td><td>藻酸盐不正确乳化</td><td>确保藻酸盐微滴在乳化过程中是可见的。如果没有，请确认油相密度和容器几何尺寸23与本协议中描述的类似。不能提供足够的局部能量耗散的致密油相或容器几何形状可能不允许适当的藻酸盐乳化。 </td></tr><tr><td rowspan="2">没有珠子</td><td rowspan="2">内部凝胶化没有发生</td><td>常见的错误是乙酸在油相中不完全溶解。确保酸性油混合物充分涡旋，并且没有观察到乙酸管底。 </td></tr><tr><td>如果叶轮没有到达油，可以在加入藻酸盐之前加入更多的油。但是，乙酸的量应根据下列公式进行调整: <img alt="公式3" src="/files/ftp_upload/55280/55280eq3.jpg" /> 哪里<img alt="方程式4" src="/files/ftp_upload/55280/55280eq4.jpg" /> 1是修改之前的油/水相比（例如，在10mL油+ 10mL酸油中的10.5mL藻酸盐为0.525） <img alt="方程式4" src="/files/ftp_upload/55280/55280eq4.jpg" /> 2是修改方案后的油/水相比，Dow是两相之间的乙酸分配系数。该分布系数可以通过文献测量的实验或近似值来测量。例如，庚烷与水46之间的乙酸分配系数为0.011。 </td></tr><tr><td>没有珠子或珠粒是机械不稳定的</td><td>藻酸盐溶液零剪切粘度低于0.004Pa·s或高于112Pa·s </td><td>由于藻酸盐批次的批次间变化很大，建议测量不同浓度的藻酸盐溶液的粘度。可能需要高粘度批次与低粘度批次的组合才能达到目标粘度。对于这里使用的5％LVM和MVG混合物，零剪切粘度为3.3 Pa·s。应当注意，具有太短古罗糖醛酸封闭长度或短链长度的藻酸盐可能不允许凝胶形成。 </td></tr><tr><td>珠子太大或太小</td><td>乳化过程中的搅拌速度对于藻酸盐浓度或使用的粘度来说太低或太高</td><td>通过增加搅拌速度或加入挡板来增加湍流会降低胎圈的尺寸。藻酸盐浓度，因此，容器和叶轮几何将都会影响所获得的珠粒尺寸。 </td></tr><tr><td>观察到许多破碎的珠粒</td><td>粗糙的珠粒处理，内部凝胶化步骤中的损伤或珠粒强度不足。 </td><td>一旦发生内部凝胶化，对珠粒的机械损伤会导致珠粒破裂。在酸化步骤之前立即降低搅拌速度，并确保使用25 mL移液管或1000μL带有切割尖端的移液器轻轻处理珠粒。如果在酸化期间即使仔细处理和减少搅拌也观察到损坏的珠粒，珠粒强度可能不足。这可能是由于古洛糖醛酸含量不足或不完全凝胶化（如酸化不足）。 </td></tr><tr><td>低细胞活力</td><td>初始过程pH过高或最终pH太低</td><td>通过限制pH降低和酸化时间可以增加哺乳动物细胞过程的存活</tr><tr><td>较小的珠子中的细胞活力较低</td><td>藻酸盐溶液缓冲能力不足</td><td>由于这些珠的较大的表面/体积比，预期在较小的珠子中比在较大珠粒中更大的pH降低。由于添加到乳液中的乙酸是有限的，因此相对于CaCO 3浓度，在较小的珠粒中预测出较高的乙酸浓度。增加藻酸盐溶液缓冲能力（例如使用60mM MOPS而不是10mM HEPES）可以增加较小珠粒32中的细胞存活。可以通过过滤或沉降选择性地去除具有高电池损耗的小珠，而不会产生大的体积损失。 </td></tr><tr><td>一世不完全CaCO 3溶解</td><td>酸化不足或酸化时间不足。请注意，如果珠稳定性足够，此问题可能不成问题。 </td><td>根据本方案32 ，在较大的珠子中观察到不完全的CaCO 3溶解。 CaCO 3溶解的程度可以根据CaCO 3晶粒尺寸，过程中的pH下降，pH下降的持续时间以及所用的藻酸盐浓度而变化。只要珠的机械稳定性足以满足所需的应用，该问题就不被认为是有问题的。为了增加珠粒交联，确保CaCO 3颗粒足够小（〜2.5μm）。超声处理CaCO 3悬浮液可以帮助破坏聚集的颗粒26 。在非常低或高的藻酸盐浓度下，可能需要调节CaCO 3浓度。最后，螯合剂或溶液含量低二价离子的水平将导致凝胶中Ca 2+的逐渐损失。如果观察到珠粒机械稳定性逐渐丧失，则验证珠粒存储或培养缓冲液含有&gt; 2mM Ca 2+ 。 </td></tr></tbody></table> 表1:故障排除指南。问题清单，可能的原因和潜在的解决方案。 哺乳动物细胞和移植应用的体外培养的乳化和内部凝胶化过程的潜在限制包括:（1）瞬时暴露于低pH值的需要，（2）需要除去残留油，（3）多分散珠粒度分布，（4）藻酸盐液滴形成期间的潜在剪切应力和（5）与外部胶凝珠粒47相比，内部胶凝珠的孔隙率更高。 已获得MIN6和βTC3的高细胞存活和功能过程优化32 。然而，该方法可能不适合于更多的pH敏感细胞。从珠粒中完全消除油也可能是具有挑战性的，食品和药物管理局（FDA）批准的油类型是最终临床应用所必需的。对于免疫隔离应用，多分散珠粒度分布也可能有些问题，其中期望完全细胞包封和长期细胞存活。尽管小珠粒度可导致细胞突出48 ，但是大珠粒可阻碍营养物扩散和/或细胞功能35,49 。另一方面，乳化过程提供了一个有趣的机会，通过在单个过程中产生小和大珠子来研究珠粒尺寸对移植后细胞存活的影响。通过改变durin的搅拌速度可以容易地调节平均珠粒度g乳化步骤（ 图4 ）。最后，内凝胶珠的较高孔隙率对免疫隔离也可能是有害的，其中分子如抗体和细胞因子应从珠粒中排除。然而，乳化过程还允许从非常浓缩的藻酸盐溶液产生珠粒，从而降低藻酸盐珠粒孔径。 乳化和内部凝胶化过程是用于希望扩大细胞封装或不能进入封装剂的实验室的基于喷嘴的封装剂的有意义的替代方案。该方法是使用非常稀的或非常浓缩的藻酸盐溶液将细胞固定在藻酸盐珠中的稳健和简单的方法。可以用固定化细胞进行的一些随后的实验是确定藻酸盐基质对细胞命运决定的影响，或者确定珠在多大程度上提供移植之间的屏障d细胞和宿主免疫系统。乳化和内部凝胶化方法提供了大批量包装主要胰岛的新方法。该方法还允许细胞包封在具有降低的孔隙率的高度浓缩的藻酸盐珠中，从而减少抗体进入包封的细胞。因此，乳化和内部凝胶化方法提供了产生临床相关数量的包封的治疗细胞的新手段。

已经广泛研究了哺乳动物细胞包封作为保护移植细胞免于免疫排斥的手段1或为固定的细胞培养2,3,4提供三维支持。藻酸盐珠粒中的胰岛包囊已经用于逆转异种5,6或异种7,8,9,10,11 和 12啮齿动物中的糖尿病。包封胰岛移植治疗1型糖尿病的临床前和临床试验正在进行中13,14,15 。适用于移植应用或大规模应用通常使用体外固定化的细胞生产，基于喷嘴的珠发生器。通常，藻酸盐和细胞的混合物通过喷嘴泵送以形成落入含有二价阳离子的搅拌溶液中的液滴，导致液滴的外部凝胶化。同轴气流16,17 ，喷嘴振动18 ，静电排斥19或旋转导线20有助于在喷嘴尖端形成液滴。 常规珠发生器的主要缺点是其有限的生产量和溶液粘度的有限范围，这将导致足够的珠形成21 。在高流速下，离开喷嘴的流体分解成小于喷嘴直径的液滴，减小了尺寸控制。多喷嘴珠发生器可用于提高生产量，但是喷嘴之间流动的均匀分布和&gt; 0.2Pa的溶液的使用是有问题的。最后，由于所使用的喷嘴的直径在100μm和500μm之间，而15％的人胰岛可以大于200μm23，因此预计所有的喷嘴基装置都会对胰岛造成一定的损害。 在这个视频中，我们描述了通过在单个乳化步骤中形成液滴而不是逐滴地包封哺乳动物细胞的另一种方法。由于珠粒生产在简单的搅拌釜中进行，所以该方法适用于小型（〜1 mL）至大规模（10 3 L范围）的珠粒生产，设备成本低24 。该方法允许使用具有短（ 例如 20分钟）珠生成时间的宽范围的藻酸盐粘度来生产具有高球形度的珠粒。这种方法最初由Poncelet 等人开发湖25,26 ，用于固定DNA27，包括胰岛素29的蛋白质28和细菌30 。我们最近已经将这些方法适用于使用胰腺β细胞系31,32和原发性胰腺组织32来哺乳动物细胞的包封。 该方法的原理是在矿物油中产生由藻酸盐液滴组成的油包水乳液，随后是藻酸盐液滴的内部凝胶化（ 图1 ）。首先，将密封剂（ 例如，细胞）分散在含有细晶粒钙盐的藻酸盐溶液中，在初始工艺pH下具有低溶解度。然后将藻酸盐混合物加入到搅拌的有机相中以产生乳液，通常在a表面活性剂。在哺乳动物细胞包封的情况下，存在于血清中的成分可以作为表面活性剂。接下来，通过加入分解成水相的油溶性酸来降低pH以便溶解钙盐。矿物油/水分配系数&lt;0.005 33的乙酸应预先溶解在油中，然后加入到油相中混合的乳液中，并快速分配到水相34中 。 图2说明在酸化和内部凝胶化步骤期间发生的化学反应和扩散。最后，通过相转化回收包封的细胞，通过离心加速分离，重复洗涤步骤和过滤。然后可以采用珠和细胞取样进行这些步骤，用于质量控制分析， 体外细胞培养和/或包封细胞的移植。 图1:封装哺乳动物细胞的基于乳化的方法的示意图。首先通过在矿物油中乳化藻酸盐，细胞和CaCO 3混合物（示意图中的步骤1和2）来生产珠粒，通过加入乙酸引发内部凝胶化（步骤3）。然后通过加入水性缓冲液将凝胶状珠粒与油分离以引发相转化（步骤4），然后离心和抽吸油（步骤5），然后过滤（步骤6）。最后，将过滤器上收集的珠子转移到细胞培养基中进行体外培养或移植。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55280/55280fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <imgalt ="图2"class ="xfigimg"src ="/ files / ftp_upload / 55280 / 55280fig2.jpg"/&gt; 图2:在内部凝胶化过程中发生的反应和扩散步骤。 （1）将乙酸加入到有机相中，并通过对流输送至藻酸盐液滴。 （2）乙酸分成水相。 （3）在水的存在下，酸解离并扩散到深蓝色所示的CaCO 3颗粒。 （4）H +离子与CaCO 3中的Ca 2+离子交换，释放Ca 2+离子。 （5）钙离子扩散直到遇到未反应的藻酸盐，导致藻酸盐链的离子交联。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55280/55280fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 与传统的基于喷嘴的细胞封装剂相反，具有宽的珠粒度分布由于在搅拌乳化中液滴形成的机理，该方法由该过程引起。对于应用的一个子集，这种珠粒度分布可能是有问题的。例如，更大部分的细胞可能在较小珠粒的珠粒表面暴露。如果关注营养（ 如氧气）的限制，这些限制可能会在较大的珠粒中加剧。搅拌乳化法的优点是通过改变乳化步骤中的搅拌速度可以容易地调节平均珠粒度。也可以利用宽的珠粒度分布来研究珠粒尺寸对封装的细胞性能的影响。 通过乳化和内部凝胶化的哺乳动物细胞包封是没有配备珠粒发生器的实验室的有意义的替代方案。此外，该方法还给用户减少处理时间，或生成非常低或非常高的藻酸盐浓度的珠粒ations。 下面概述的方案描述了如何将细胞包封在10.5mL在10mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲液中制备的5％藻酸盐溶液中。藻酸盐由移植级LVM（低粘度高甘露糖醛酸含量）和MVG（中等粘度高古洛糖醛酸含量）藻酸盐的50:50混合物组成。使用终浓度为24mM的碳酸钙作为物理交联剂。轻质矿物油构成有机相，而乙酸用于酸化乳液并引发内部凝胶化。然而，藻酸盐类型和组成以及所选择的过程缓冲剂取决于期望的应用32 。已经使用各种藻酸盐类型（参见表格材料）来制备具有该方案的珠粒。

<table border="0" cellpadding="0" cellspacing="0" width="990">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Reagents and consumables</td>
			<td style="width:164px;"></td>
			<td style="width:223px;"></td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LVM alginate (transplantation-grade)</td>
			<td style="width:164px;">Novamatrix</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">Referred to as &#34;alginate #1&#34; in the results.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">MVG alginate (transplantation-grade)</td>
			<td style="width:164px;">Novamatrix</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">Referred to as &#34;alginate #2&#34; in the results.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Alginate (cell culture-grade)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">A0682 (low viscosity) or A2033 (medium viscosity)</td>
			<td style="width:348px;">A2033 is referred to as &#34;alginate #3&#34; in the results.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">DMEM</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:223px;">11995-065</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Fetal bovine serum, characterized, Canadian origin</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">SH3039603</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Glutamine</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:223px;">25030</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Penicillin and streptomycin</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">P4333-100ML</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">HEPES, cell culture tested</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">H4034-100G</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">NaCl</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">S271-1</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:256px;">Fine-grain CaCO3</td>
			<td style="width:164px;">Avantor Materials</td>
			<td style="width:223px;">1301-01</td>
			<td style="width:348px;">After preparing the CaCO3 suspension, sonicate and use within one month.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Light mineral oil</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">O121-4</td>
			<td style="width:348px;">Sterile filter through a 0.22 &#956;m pore size membrane prior to use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Glacial acetic acid</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">A38-500</td>
			<td style="width:348px;">Handle with caution: refer to MSDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Sterile spatulas</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">CLS3004-100EA</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Sterile nylon cell strainers, 40 &#181;m</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">08-771-1</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td>
			<td style="width:164px;">Sarstedt</td>
			<td style="width:223px;">86.1252.001, 86.1253.001, 86.1254.001 and 86.1685.001</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Pasteur pipettes</td>
			<td style="width:164px;">VWR</td>
			<td style="width:223px;">14673-043</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Toluidine Blue-O</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">T3260</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Equipment</td>
			<td style="width:164px;"></td>
			<td style="width:223px;"></td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:256px;">100 mL microcarrier spinner flasks</td>
			<td style="width:164px;">Bellco</td>
			<td style="width:223px;">1965-00100</td>
			<td style="width:348px;">The impeller configuration with recent models may not be suitable for adequate emulsification. A blade able to sweep the oil down to 0.5 cm from the bottom of the flask can be custom-made from a Teflon sheet.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Magnetic stir plate with adjustable speed</td>
			<td style="width:164px;">Bellco</td>
			<td style="width:223px;">7760-06005</td>
			<td style="width:348px;">The rotation speed should be calibrated (e.g. using a tachometer) prior to use.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:256px;">Cell counter</td>
			<td style="width:164px;">Innovatis</td>
			<td style="width:223px;">Cedex AS20</td>
			<td style="width:348px;">This system is now sold by Roche. This automated cell counter can also be replaced by manual cell enumeration after Trypan blue staining using a hemocytometer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">LED light box</td>
			<td style="width:164px;">Artograph</td>
			<td style="width:223px;">LightPad&#174; PRO</td>
			<td style="width:348px;">This item can be replaced by other types of illuminators.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:256px;">Handheld camera</td>
			<td style="width:164px;">Canon</td>
			<td style="width:223px;">PowerShot A590 IS</td>
			<td style="width:348px;">A variety of handheld cameras can be used to capture toluidine blue-o stained bead images. A ruler should be placed next to the Petri dish containing the beads prior to acquiring images.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:256px;">Fluorescence microscope with phase contrast and adequate fluorescence filters</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:223px;">IX81</td>
			<td style="width:348px;">Several microscopy systems were used to image the beads. The results shown here were obtained with an IX81 microscope equipped with GFP and TRITC fluorescence filters. To capture entire beads, 4X to 20X objectives were used depending on the agitation rate. Live/dead staining images were typically captured with 20X to 40X objectives.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Image aquisition software</td>
			<td style="width:164px;">Molecular Devices</td>
			<td style="width:223px;">Metamorph</td>
			<td style="width:348px;">A variety of image acquisition software can be used to acquire phase contrast and fluorescence images.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Image analysis freeware</td>
			<td style="width:164px;">CellProfiler</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">A variety of image analysis software can be used to identify beads as objects and analyze bead size (e.g. ImageJ).</td>
		</tr>
	</tbody>
</table>

mammalian cell encapsulation in alginate beads using a simple stirred vessel

藻酸盐珠粒中的细胞包封已被用于体外固定化细胞培养以及体内免疫隔离。已经广泛研究胰岛包囊作为增加同种异体或异种移植物中胰岛存活的手段。藻酸盐封装通常通过喷嘴挤出和外部凝胶化来实现。使用这种方法，在喷嘴尖端形成的含细胞的藻酸盐液滴落入含有二价阳离子的溶液中，这些二价阳离子在它们扩散到液滴中时会引起离子型藻酸盐凝胶化。在喷嘴尖端形成液滴的要求限制了可以实现的体积通量和藻酸盐浓度。该视频描述了可扩展的乳化方法，以0.5％至10％的藻酸盐包封哺乳动物细胞，其中70％至90％的细胞存活。通过这种替代方法，含有细胞和碳酸钙的藻酸盐液滴在矿物油中乳化由于pH降低，导致内部钙释放和离子型藻酸盐凝胶化。目前的方法允许在乳化20分钟内生产藻酸盐珠粒。封装步骤所需的设备包括可供大多数实验室使用的简单搅拌容器。

<html>在乳化和内部凝胶化过程结束时，应回收与初始藻酸盐和细胞混合物体积相似的珠粒体积。珠子应该是高度球形的，几乎没有缺陷（ 图3 ）。珠子应该足够坚固，以通过大口径移液管来承受移液。在高藻酸盐浓度下，可以在珠粒中观察到包封的油或气滴。这可能是由于在乳化步骤（油/水/油双重乳液）期间在藻酸盐液滴中捕获油。在我们手中，这些珠代表小珠体积的小部分（&lt;5％），并且由于其较低的密度，在珠回收步骤期间抽吸油时，大部分这些珠被除去。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55280/55280fig3.jpg" /> 图3 :含有通过乳化和内部凝胶化方法获得的包封的βTC3细胞的代表性的5％藻酸盐珠粒含有5×10 6个 βTC3细胞/ mL珠粒的5％藻酸盐珠粒的相差图像<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55280/55280fig3large.jpg" target="_blank">请点击此处查看更大的版本的这个数字。</a> 获得的珠的尺寸将不均匀 - 这是搅拌乳液的固有特征（ 图4A ）。在湍流中的乳化期间，压力波动起到破坏液滴37,38的作用，抵消分散相39中的表面张力和粘性力。如果分散相的粘度远高于th，粘性力是不可忽略的e连续相。假设最大局部能量耗散率与容器中的平均能量耗散比成比例，最大稳定液滴直径d max （如果&gt;&gt;比Kolmogorov尺度）可以计算如下: 39,40,41,42 <img alt="公式1" src="/files/ftp_upload/55280/55280eq1.jpg" /> 其中ρc是连续相的密度， ε是容器中的平均能量耗散率， σ是两种流体之间的界面张力， μd是分散相的粘度， C是比例常数。当表面张力大于粘性力时，右侧简化为C ，而如果粘性力占主导地位，则简化为: 在搅拌容器中，湍流流量下的能量输入与每单位体积的功率输入成比例， 即 N i 3 D i 2 ，其中N i是搅拌速率， D i是叶轮直径。因此，结合公式1和给定常数D i ，如果表面张力占主导地位，则预测d max与N i -1.2 （ 图4B ）成比例，而如果其中的粘性力与N i -0.75成比例珠主要作为主要的保持力。 图4B和4C表明界面效应决定了d低海藻酸盐浓度的大小，而粘性力在较高的藻酸盐浓度（ 例如材料表中所述的藻酸盐＃3为5％）时更为显着。由于实验确定d max是不切实际的，所以已经使用了直径或表面积力矩平均直径（ d 32 ）的高分位数值（ 例如第95 位分位数），因为它们通常与d max 43成比例。 在封装单元之前，建议生成d 32作为N i的函数的曲线（ 图4B ）。该曲线将有助于选择足够的N i以获得所需的平均珠粒度。对于不同批次的油或藻酸盐也可能需要新的标准曲线，或者如果是藻酸盐浓缩液修改。取决于血管几何形状和藻酸盐浓度，应该可以实现相对较宽的平均珠粒直径范围。例如，使用上述方案，可以使用2％（ 图2C在Hoesli 等人 32 ）至5％藻酸盐（ 图4 ）获得200μm和&gt; 1mm之间的d 32值。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55280/55280fig4.jpg" /> 图4 :珠粒尺寸分布。 （ A ）甲苯胺蓝-O染色5％藻酸盐珠。 （ B ） d 32作为1.5％和5％藻酸盐珠粒的搅拌速率（ N i ）的函数，使用50:50的藻酸盐＃1和＃2的混合物（参见材料表）。 5％algi的单点显示珍珠（ C ） d 32作为2％和5％藻酸盐＃3的N i的函数（参见表材料）。误差棒代表样品（N&gt; 150珠）内的珠粒尺寸的标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55280/55280fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 来自乳化和内部凝胶化过程的细胞存活主要取决于细胞经历的pH降低的程度和持续时间。使用本视频中描述的过程，测量76±2％βTC3细胞存活31 。当将分散​​的细胞以10 5细胞/ mL藻酸盐包封至10 7个细胞/ mL藻酸盐接种密度时，所有珠子应含有细胞。当封装细胞簇如胰腺细胞时，珠子的一个子集可以预期被排除年。 图5显示了使用该方法获得的典型活细胞/死细胞染色结果。更高的细胞存活可以通过增加过程缓冲能力和减少酸化步骤的持续时间来实现。例如，通过使用60mM MOPS（3-（N-吗啉代）丙磺酸）作为过程缓冲液并通过将总处理时间减少到4分钟32获得90±2％MIN6细胞存活。然而，由于在较低pH 31下释放的Ca 2+的量增加，所产生的珠子与10mM HEPES过程缓冲液相比，显着高于60mM MOPS工艺缓冲液。短MOPS过程和较长的HEPES过程都应产生足够稳定的珠子， 体外培养或移植超过2周。然而，珠子可以在具有低钙或高螯合剂浓度的溶液中完全膨胀或降解（例如，，无钙磷酸盐缓冲盐水溶液）。 对于在体外生长的包封的MIN6细胞， 在体外固定化细胞扩增〜5-7天后形成可见的细胞聚集体 ，并且可以在2周后收获〜150μm直径的球体。藻酸盐固定化细胞的生长速率预期低于非固定化培养物，特别是对于具有较高藻酸盐浓度或较高古罗糖醛酸含量的凝胶。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55280/55280fig5.jpg" /> 图5 :过程后立即封装细胞。 （ A ）包膜细胞的活细胞（绿色，钙黄绿素AM）和死细胞（红色，乙锭同二聚体）染色。 （ B ）相同珠粒的相位图像。将MIN6细胞封装在2％使用12分钟乳化，8分钟酸化和如本方案所述的10mM HEPES缓冲液的藻酸盐珠粒。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55280/55280fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel at JoVE.com

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

该视频和手稿描述了一种基于乳液的方法，以将哺乳动物细胞包封在0.5％至10％的藻酸盐珠中，其可以使用简单的搅拌容器以大批量生产。包封的细胞可以在体外培养或移植用于细胞治疗应用。

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 ...

mammalian-cell-encapsulation-alginate-beads-using-simple-stirred

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

19.3K 次观看.  McGill University. 该视频和手稿描述了一种基于乳液的方法，以将哺乳动物细胞包封在0.5％至10％的藻酸盐珠中，其可以使用简单的搅拌容器以大批量生产。包封的细胞可以在体外培养或移植用于细胞治疗应用。 

视频: Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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 ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

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

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

实验室规模搅拌生物反应器的功率输入测量

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