Name: mammalian cell encapsulation in alginate beads using a simple stirred vessel
Uploaded: 2017-06-29T13:00:00.000+00:00
Duration: 10 min 20 s
Description: Watch this Scientific Journal Video about Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel at JoVE.com

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...

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="http://ecsource.jove.com/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="http://ecsource.jove.com/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><tbody><tr><td>&lt;strong&gt;Reagents and consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LVM alginate (transplantation-grade)</td><td>Novamatrix</td><td>Non-applicable</td><td>Referred to as &quot;alginate #1&quot; in the results.</td></tr><tr><td>MVG alginate (transplantation-grade)</td><td>Novamatrix</td><td>Non-applicable</td><td>Referred to as &quot;alginate #2&quot; in the results.</td></tr><tr><td>Alginate (cell culture-grade)</td><td>Sigma</td><td>A0682 (low viscosity) or A2033 (medium viscosity)</td><td>A2033 is referred to as &quot;alginate #3&quot; in the results.</td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Fetal bovine serum, characterized, Canadian origin</td><td>Thermo Fisher Scientific</td><td>SH3039603</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Sigma</td><td>P4333-100ML</td><td></td></tr><tr><td>HEPES, cell culture tested</td><td>Sigma</td><td>H4034-100G</td><td></td></tr><tr><td>NaCl</td><td>Thermo Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>Fine-grain CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Avantor Materials</td><td>1301-01</td><td>After preparing the CaCO&lt;sub&gt;3&lt;/sub&gt; suspension, sonicate and use within one month.</td></tr><tr><td>Light mineral oil</td><td>Thermo Fisher Scientific</td><td>O121-4</td><td>Sterile filter through a 0.22 &amp;mu;m pore size membrane prior to use.</td></tr><tr><td>Glacial acetic acid</td><td>Thermo Fisher Scientific</td><td>A38-500</td><td>Handle with caution: refer to MSDS.</td></tr><tr><td>Sterile spatulas</td><td>Sigma</td><td>CLS3004-100EA</td><td></td></tr><tr><td>Sterile nylon cell strainers, 40 &amp;micro;m</td><td>Thermo Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td><td>Sarstedt</td><td>86.1252.001, 86.1253.001, 86.1254.001 and 86.1685.001</td><td></td></tr><tr><td>Pasteur pipettes</td><td>VWR</td><td>14673-043</td><td></td></tr><tr><td>Toluidine Blue-O</td><td>Sigma</td><td>T3260</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mL microcarrier spinner flasks</td><td>Bellco</td><td>1965-00100</td><td>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><td>Magnetic stir plate with adjustable speed</td><td>Bellco</td><td>7760-06005</td><td>The rotation speed should be calibrated (e.g. using a tachometer) prior to use.</td></tr><tr><td>Cell counter</td><td>Innovatis</td><td>Cedex AS20</td><td>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><td>LED light box</td><td>Artograph</td><td>LightPad&amp;reg; PRO</td><td>This item can be replaced by other types of illuminators.</td></tr><tr><td>Handheld camera</td><td>Canon</td><td>PowerShot A590 IS</td><td>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><td>Fluorescence microscope with phase contrast and adequate fluorescence filters</td><td>Olympus</td><td>IX81</td><td>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><td>Image aquisition software</td><td>Molecular Devices</td><td>Metamorph</td><td>A variety of image acquisition software can be used to acquire phase contrast and fluorescence images.</td></tr><tr><td>Image analysis freeware</td><td>CellProfiler</td><td>Non-applicable</td><td>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，?...

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

Research

JoVE Journal

Bioengineering

19.4K 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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

医学

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

化学

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

生物学

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 ...

生物工程

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

使用电喷雾技术合成载有黑籽油、海藻酸盐基的 pH 敏感磁珠

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

制造淀粉样蛋白-β-分泌阿尔金酸微珠,用于模拟阿尔茨海默氏病

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...