笔者想感谢肯特州立大学（合作研究补助金和再生医学倡议肯特州立支持 - ReMedIKS）本项目的资金支持。

注意:使用3臂星形嵌段共聚物的三维LCE泡沫状制剂下面的步骤示于图1。用于核磁共振（NMR）表征，光谱在在Bruker DMX 400MHz的仪器室温记录在氘代氯仿（CDCL 3）和内部在7.26引用残余峰。傅里叶变换红外（FT-IR）光谱使用利用衰减全反射模式的Bruker矢量33 FT-IR光谱仪记录。对于以下协议中的每个步骤中，穿适当的个人防护服（PPE）是重要的。 1.α氯ε己内酯的合成（单体）（根据杰罗姆等中的程序。28） <ol><li>在开始合成之前，净化27克的3-氯过苯甲酸，如下所示: <ol><li>在800毫升的蒸馏水中，添加1.28克钠磷酸盐一水合物和8.24克磷酸氢二钠七水合物。将pH调节到7.4（用氢氧化钠或盐酸）和储备30毫升的该溶液;这是缓冲溶液。 </li><li>在35毫升乙醚使用分液漏斗，溶解3-氯过苯甲酸。洗用10mL的缓冲溶液中的有机溶液（在步骤1.1.1制备。）。重复洗三次。 </li><li>加入3克硫酸钠直接将有机溶液;这种干燥剂从有机溶液中吸收水。 </li><li>过滤该溶液以除去干燥剂。通过在850毫巴和40℃下使用旋转蒸发器在减压下浓缩滤液。 </li></ol></li><li>在超声浴中搅拌溶解18.5克在150毫升无水二氯甲烷纯化3-氯过苯甲酸的;这个过程通常需要20分钟。将分液漏斗内的解决方案。 </li><li>内双颈，圆底烧瓶中，利用在氮气下磁力搅拌溶解13.1克2-氯环在15毫升无水二氯甲烷中。不断搅拌。 </li><li>适合的含3-氯过苯甲酸溶液（来自步骤1.2）的分液漏斗中的两颈烧瓶中，在步骤1.3。冲洗用氮气系统。调整分液漏斗中的开口中，使得氯过苯甲酸溶液逐滴落入2-氯环溶液（1滴每隔一秒），并继续搅拌下氮气的混合物96小时。 </li><li>将反应混合物冷却至-20℃1个小时，沉淀出米氯苯甲酸（ 米 -CBA）副产物。 </li><li>过滤米氯苯甲酸（ 米 -CBA）中，用硫代硫酸钠，碳酸氢钠，和氯化钠的饱和溶液洗涤剩余的溶液。 </li><li>使用旋转蒸发器在850毫巴和40℃在减压下除去溶剂。净化浅黄色，粘稠液体肥皂剂量下，在2.3乇和减压96℃ID通过蒸馏进行。 </li><li>监视使用以下1个 H-NMR峰的合成的成功。 1 H-NMR（400MHZ，CDCL 3，δ[ppm的]）:4.75-4.68（M，1H，CH-ClCO），4.37-4.26（M，1H，C H 2 O），4.18-4.05（M，1H ，C H 2 O），和2.06-1.58（M，6H，-C H 2 - ）6,27。 </li></ol> 2.合成α-三臂星型嵌段共聚物（SBC-αCL）通过开环共聚（Sharma 等人6和Amsden 等人 29） <ol><li>合成前，通过用2％（V / V）1H，1H，2H，2H- perfluorooctyltriethoxysilane的甲苯溶液填充它，并搅拌约24小时硅烷化的20mL安瓿。用异丙醇冲洗，并通过在140℃下将其放置在烘箱中30分钟干燥。 </li><li>添加30.64克蒸馏ε己内酯，0.5g的α氯代ε己内酯，和0.25毫升甘油的安瓿。混合使用涡旋1分钟。 </li><li> 4.90克D的L-丙交酯添加到安瓿中并用氮气吹洗。放置安瓿在烘箱中在120℃下以熔融D，L-丙交酯;这个过程通常需要约2小时。使用旋涡，以确保所有的内容都充分混合，并添加锡的66微升再次混合（II），2-乙基己酸（SN（10月）2）安瓿。 注:D，L-丙交酯将在此过程中冷却下来，将需要在烘箱中熔化被重新加热。 </li><li>使用涡流最后一次剧烈混合并用氮气冲洗。 </li><li>用橡胶塞封闭安瓿。放置连接到真空管的针（房子真空通常是足够了）通过橡胶塞。打开真空和，使用火焰，熔融玻璃的长颈，慢慢扭转直到克姑娘崩溃本身。小心不要融化橡皮塞。一旦安瓿是火焰密封，将其置于砂浴，烘箱，或适当的加热元件在140℃下48小时。 </li><li>取出安瓿，让它冷却室温。 </li><li>打破在密封标记的安瓿，并通过加入10毫升二氯甲烷溶解高粘度液体。将溶液转移到分液漏斗中。 </li><li>制备含100毫升冷甲醇的烧瓶中（在一定温度下用干冰/丙酮浴中冷却周围-78℃）。固定在烧瓶的顶部的分液漏斗中（步骤2.7）。调整分液漏斗中的开口，使得大约两滴落入每隔一秒（逐滴）。 </li><li>通过过滤收集白色沉淀物（使用纸过滤器），并在50和60℃之间的真空烘箱中干燥。 </li><li>监视使用以下1 H NMR和FT-IR峰合成的成功。 1 H-NMR（400MHZ，CDCL 3，δ[ppm的]）:5.29-5.03（M，COCħ] C H 3），4.43-4.25（M，CH-CL）4.24-4.12（M，C H 2 O），4.11-4.03（T，J = 4.6赫兹，C H 2 O），3.80-3.68（M，] C H] C H 2），3.09-2.64（宽峰，S，O 1H），2.39（T，J = 4.5赫兹，α-1H），2.33 （T，J = 5.1赫兹，α-H）和1.77-1.25（M，C H 2，C H 3）; FT-IR（1 /λ[-1]）:2932（S），2869（M），1741（S），961（S），866，和735（M）6，27。 注意:为了制备更亲水的3D LCE，制备线性嵌段共聚物（LBC）代替SBC，按照以上描述的开环共聚（ROP）过程。 <ol><li> 0.3克聚乙二醇（PEG），3.15克ε己内酯，1.0g的α氯代ε己内酯，和5.0g D的L-丙交酯添加到硅烷化的小瓶组合。 </li></ol></LI&gt; </ol> 3.α-CL-三根臂SBC的合成修饰至αN3 -三臂SBC（SBC-αN3）（根据Sharma 等人。6） <ol><li>在一个圆底烧瓶中并在氮气下，溶解5克SBC-αCL在30mL无水N，N"二甲基甲酰胺。 </li><li>添加0.22克叠氮化钠，并允许在室温下过夜反应。 注意:叠氮化钠是有毒的;穿戴合适的个人防护服（PPE）。 </li><li>除去N，使用旋转蒸发器在11毫巴和40℃下减压N"二甲基甲酰胺。溶解在30毫升甲苯的混合物中。三次离心溶液在2800×g离心15分钟以除去形成的盐。使用旋转蒸发器在77毫巴和40℃在减压下蒸发甲苯。 </li><li>监视使用1 H NMR和FT-IR峰的叠氮取代的成功。 注意: <sup&gt; 1个H NMR谱是类似于亲SBC-αCL，具有峰在3.90 ppm的相关的叠氮基团的外观的除外; FT-IR（1 /λ[-1]）:2928，2108（S，叠氮化物），1754（S），1450（S），960（M），865（S），733（S），和696 （M）。 </li></ol> 4.胆固醇合成5- Hexynoate（LC物部分）（根据Sharma 等人 6和Donaldson 等 。30） <ol><li>在一个圆底烧瓶中，混合3克-5-己炔酸和130毫升的二氯甲烷中。冷却至0℃，用冰浴。 </li><li>在另一圆底烧瓶中，混合8.28克的N，N" -dicyclohexylcarbodiimide，10.3克胆甾醇和0.2克4-二甲基氨基吡啶。 </li><li>转移-5-己炔酸溶液滴加到含有胆固醇混合物的烧瓶中，并维持最终的混合物在0℃下1个小时。 </li><li>允许混合物升温至室温过夜。 </LI&gt; <li>使用级415纸过滤器通过过滤除去所得到的二环己基脲沉淀并丢弃它。 </li><li>使用旋转蒸发器在850毫巴和40℃在减压下浓缩滤液。溶解在150毫升己烷中收集到的残余物。 </li><li>使用旋转蒸发器在335毫巴和40℃在减压下蒸发溶剂。添加350毫升乙醇的油状残余物，收集最终产品。洗灰白色固体用乙醇形成，并且在50℃真空下干燥该固体产物。 </li><li>使用以下1 H NMR和FT-IR峰监测合成。 1 H-NMR（400MHZ，CDCL 3，δ[ppm的）:5.39（D，J = 4.7赫兹，1H，C H = C），4.70-4.58（M，1H，CH-OCO），2.44（T， J = 2.5赫兹，2H，C H 2 CO），2.34（M，2H，C H 2 -C H =），2.31（M，2H，C H 2 C H 2 -COO），2.28（S，1H， HC≡C），2.27（D，J = 2.3赫兹，2H，≡CCH 2），2.07-1.06（M，2H，C H 2，C 2H），0.94（S，3H，C H 3），0.89 （D，J = 1.8赫兹，3H，C H 3 C H）和0.88（D，J = 1.8赫兹，6H，C H 3 -C 2 H 4），0.67（S，3H，C H 3）。 FT-IR（1 /λ[-1]）:2,830-2,990（宽和强峰），2104（M），1695（S），1428（M），1135（M），999（S），798 （S），和667（一个或多个）。 </li></ol> 5.合成修饰α-N 3 -三臂SBC为α-胆固醇基三根臂SBC（SBC-αCLC）经由叠氮化物-炔烃休斯根环加加成反应（"点击"反应）获取SBC-澈（根据Sharma 等人。6） <ol><li>在一个圆底烧瓶中，溶解1个摩尔当量的SBC-αN3（1.5克）在100毫升新蒸馏的四氢呋喃（THF）中。添加1.2摩尔equivalen胆甾-5- hexynoate（1.94克），铜（I）的0.1摩尔当量的碘化（0.06克）吨和三乙胺的0.1摩尔当量（0.03克）。搅拌在氮气下在35℃下该混合物过夜。 </li><li>使用旋转蒸发器在357毫巴和40℃在减压下蒸发溶剂。 </li><li>溶解在80mL二氯甲烷中，并离心5分钟的残余混合物在2,800×g下在室温下，以除去未反应物及副产物。 </li><li>使用以下1 H NMR和FT-IR峰监测合成。 1 H-NMR（400MHZ，CDCL 3，δ[ppm的）:7.54（S，CH = C-三唑），5.43-5.34（M，C = CH胆固醇），5.10-5.06（M，COCHCH 3），4.68 -4.55（M，O-CH胆固醇），4.24-4.19（M，CH 2 O），4.18-4.13（T，J = 5.0赫兹，CH 2 O），4.11-4.05（T，J = 4.4赫兹，CH 2 O），2.47-2.41（T，J = 4.9赫兹，COCH 2），2.31-2.25（M，COCH 2），2.07-1.02（M，CH 2 </sUB&gt;，CH 3），1.05-1.03（S，CH 2，CH 3），0.96-0.92（D，J = 3.3赫兹，CH 2，CH 3），0.91-0.87（DD，J = 1.9赫兹，J = 1.8赫兹，CH 3），和0.71-0.68（S，CH 3）。 FT-IR（1 /λ[-1]）:3260（S），2920（S），1710（S），1460（S），1370（S），1240（M），1190（S），733 （S），和668（一个或多个）。 </li></ol> 6. 2,2-双（1-己内酯-4-基）丙烷（交联剂，BCP）的合成（根据Gao 等 27和Albertsson 等人 31） <ol><li>在一个圆底烧瓶中，制备含有10.8克的1,2-双（4-羟基 - 环己基）丙烷和52毫升乙酸的溶液中。 </li><li>在50ml乙酸和8ml的蒸馏水制备含11克三氧化铬的溶液中。此溶液逐滴添加到在步骤6.1制得，同时保持17至20℃（ 例如间的混合物的温度，在该溶液水浴）;此滴加过程花费2个小时。一旦该过程完成后，使该溶液搅拌约30分钟。 </li><li>加入50毫升的2-丙醇。在室温下搅拌该溶液过夜。 </li><li>集中使用在137毫巴和40℃的旋转蒸发器在减压下暗紫色溶液。加入300毫升的蒸馏水中以沉淀;沉淀物应该是浅紫色。 </li><li>滤液用415级纸过滤粗产物。用〜250mL的蒸馏水或直到固体变为白色洗涤固体材料。 </li><li>溶解在15毫升苯中的固体材料在40℃，并让它再结晶在25℃下。 </li><li>添加8.34克溶于干燥的二氯甲烷，并包括6.0克3-氯过苯甲酸在75毫升二氯甲烷中的至烧瓶中的溶液干燥二酮。 </li><li>回流，在40℃的溶液中24个小时。 </li><li>将反应混合物冷却至-20℃10分钟以沉淀米 </EM&gt;氯苯甲酸副产物。 </li><li>通过过滤（使用纸过滤器）除去米氯苯甲酸和在减压下浓缩该溶液。 </li><li>用200ml 2-庚酮洗粘胶粗产物，并在50℃下在真空下干燥沉淀物过夜。 </li><li>使用以下1 H NMR和FT-IR峰监测合成。 1 H-NMR（400MHZ，CDCL 3，δ[ppm的]）:4.42-4.37（DD，J = 14.2，7.4赫兹，2H，C H 2 OC = O），4.21-4.15（T，2H，J = 11.3赫兹，C H 2 OC = O），2.80-2.75（DDT，J = 14.3，6.5，1.6赫兹，2H，C H 2 COO），2.63-2.57（TT，J = 13.3，2.1赫兹，2H，CH- 2 COO），2.04-1.93（M，4H，-C H 2 CH 2 OC = O），1.70-1.56（M，4H，-C H 2 C H 2 COO），1.48-1.38（M，2H， - ] C H C（CH 3）2），和0.84（S，6H，C H 3 C-）。 </li></OL&gt; 7.创建三维多孔弹性体脚手架使用任一种二异氰酸酯（HDI）或2,2-双（1-己内酯-4-基）丙烷（BCP）27作为交联剂（根据Gao 等人27） <ol><li>准备使用0.75克SBC-αCLC的通过加入0.25毫升HDI的（或0.45毫升BCP的）0.24毫升蒸馏水ε己内酯单体的三臂弹性体混合物中。添加锡（10月）2的60微升。如果使用BCP代替HDI，使用涡旋混合SBC-αCLC和BCP并将它们放置在烘箱中在140℃，直到完全BCP熔化并溶解（此步骤可能需要长达2小时）。一旦BCP已经溶解，把它从烤箱中，并且在锡（10月）2，并涡旋。 </li><li>通过切割1×4cm的金属片制备一个"牺牲"镍泡沫模板。从短端部中的一个辊它使得最终辊近似为1×1cm的金属片（参见图4）。 </li> &lt;LI&gt;把镍泡沫在玻璃小瓶或铝箔包和倒在步骤7.1制得完全覆盖泡沫2分钟该混合物。用巴斯德吸移管除去过量的混合物。在80℃下留在烘箱中过夜。 </li><li>剥离铝箔或打碎玻璃。使用刀片，剃弹性体周围的金属泡沫，以暴露金属镍。 </li><li>制备1M的铁（III）氯化物（的FeCl 3）在100毫升水中的溶液。放置泡沫在烧瓶中，并添加70毫升的FeCl 3溶液。搅拌三天在室温下，每天更换的FeCl 3溶液。每次更改之前，轰动离子水，泡沫0.5小时。 注:蚀刻工艺三天后内完成。为了确保蚀刻工艺完成后，进行触觉压缩试验，直到泡沫手感柔软。到触觉压缩试验泡沫电阻表示残留金属模板的存在。 </li><li>冲洗ELAST奥马尔泡沫在真空乙醇和地点烘箱中过夜，在40℃。 </li><li>表征使用扫描电子显微镜（SEM），示差扫描量热法（DSC），机械压缩试验，和热重分析（TGA）27的材料。 注意:为了制备更亲水的3D LCE，与LBC（确保该LBC还包含一个LC结构部分）之后，在步骤7.5中描述的步骤进行更换SBC。 </li></ol> 8.播种弹性体支架的与SH-SY5Y成神经细胞瘤细胞和培养利用无菌操作技术<ol><li>通过用1mL 70％乙醇洗涤的两次消毒弹性体。 10分钟进行紫外线照射，并用1毫升70％乙醇洗涤。用1mL无菌水和1mL的磷酸缓冲盐水（PBS）冲洗两次。置于24孔培养板中的弹性体。 </li><li>制备细胞生长培养基为含有90％的Dulbecco改良Eagle培养基（DMEM）supplem SH-SY5Yented有10％胎牛血清（FBS）和1％青霉素 - 链霉素（青霉素 - 链霉素）。 </li><li>计数使用血细胞计数细胞后，制备悬浮在100微升生长培养基的（种子溶液）1.5×10 5 SH-SY5Y细胞。添加在弹性体顶部的解决方案，确保形成液滴。 </li><li>孵育在37℃和5％CO 2接种的弹性体2小时以促进细胞粘附。添加0.5毫升生长培养基。在37℃用5％CO 2孵育再次。 </li><li>改变介质隔日用1毫升的PBS洗涤后。 </li></ol> 9.构建体，弹性体的显微成像<ol><li>固定上生长，用4％多聚甲醛（PFA）的PBS 15分钟的弹性体的细胞。用3mL的PBS清洗三次漂洗，每次5分钟，并放置弹性体与具有附接的盖玻片培养皿固定的细胞。 注意:PFA是有毒的。穿戴合适的个人防护服（PPE）。 </li><li>站用4&#39; ，在500微升PBS中的10分钟，6-二脒基-2-苯基吲哚（DAPI）的0.1％的固定样品中和冲洗用1毫升的PBS两次5分钟。 </li><li>立即图像使用共焦显微镜用DAPI荧光，获取跨越样本图像栈的弹性体。 注:在这里，用20倍的目标和60X的目标是获得的图像栈。 </li><li>分析使用ImageJ 32光学共焦图像栈。 </li></ol>

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作者什么都没有透露。

液晶弹性体，最近被研究作为生物相容性细胞支架由于其刺激的反应。他们已经被证明是作为细胞支架的理想平台。然而，准备和设计新的LCE支架时要记住的一个重要因素是孔隙度。可浸出的固体23或气体的引入并不总是导致均匀的孔隙率或完全互连的孔隙。使用可刻蚀出的金属模板，不仅提供了机会，有一个更加有组织和定期的内部结构，但也允许孔径大小和密度的选择。这直接关系到先前用于石墨烯泡沫22的制备中的金属模板。金属模板之前还LCE交联提供了机会，形成的形状，从而允许的可能性繁多建立定期孔隙德精密和复杂的架构签约酷似内生环境。泡沫LCES使用这种方法制备的允许和，更重要的电池材料的改进的研究，空间细胞 - 细胞相互作用，二维（2D）环境中不可能的技艺。 2D细胞支架不允许细胞自由地与邻近细胞相互作用。此外，细胞通常生长在单层（或直接在彼此之上），而无需完全访问用于生长和空间，更重要的是，对于相互作用。特定空间相互作用的细胞类型，例如神经元和神经胶质细胞，设计构建体作为潜在的植入物或设计为反映活体系统长期实验平台时是极为重要的。 我们的模块化，使用金属模板无溶剂合成LCE，这里提出，有助于通过选择单体7的适当中央节点和比率通过调节疏水/亲水平衡调节细胞粘附<sup&gt; 27。这是相关的，以避免额外的步骤，其包括添加基质胶层的细胞接种，通常是为了促进细胞附着。量和中央节点的大小，以及作为交联剂，在最后的LCE中发挥关键作用，因为它们影响LC弹性体的热和机械性能。此外，这还允许生物降解率的调整，因为之前提出的，作为LCE降低6，以适应组织的完全再生。目前，我们有持续的实验重点研究如何交联剂，中央核心，并更换2d的类型，L-丙交酯L-丙交酯影响弹性特性，细胞黏附，增殖和单元格对齐方式。 此协议的局限性是:（1）有限的多种市售金属（镍）泡沫和它们有限的范围在支柱的尺寸，（2）的的要求，即LCE或任何其它聚合物/弹性体材料必须在化学抗蚀金属蚀刻（在此，一个的FeCl 3的蚀刻）的条件下，以及（3）该小区对准似乎仅限于这里报告的液晶改性弹性体的事实（父非LCE弹性体没有表现出任何明显的细胞排列）。 总之，已经表明以前的SmA LCES可以使用我们的模块化合成方法来制备。附加生物相容的LC部分可以被并入以探索它们的机械性能和对细胞增殖，特别是细胞对准新液晶性的效果。已知的是，机械性能，特别杨氏模量的值，是用于细胞粘附，增殖和细胞对齐是至关重要的。这里的结果表明，的SmA LCES的孔隙率可以通过使用金属模板的被调谐到提供到控制孔径的有效方式和以调节LCE形状（ 即，整体形态）。浸渍镍模板进入SCB聚合物混合物中，然后通过蚀刻的Ni模板提供了新的LCE形态的简单的方法。这里所描述的泡沫状LCES提供细胞（这里，SH-SY5Y，一个标准单元的模型来研究神经元功能）具有用于生长和交互的更逼真，3D环境。允许多种细胞类型，以分散在弹性体密切模仿内源性神经环境，提供更复杂的三维组织培养实验有趣的可能性多层增长。使用可调和动态LCES的模仿原生架构铺平了道路，为下一代电池型号为纵向和临床相关细胞研究的方式。

有设计用于细胞研究和用于组织再生瞄准细胞附着和增殖1，2，3，4，5应用生物和生物相容的合成材料的几个例子。已经有生物相容的材料，被称为液晶弹性体（LCES）的几个例子，这可能具有各向异性分子订货6,7对外部刺激作出响应。 LCES是刺激响应材料，与光学功能性和液晶8的分子排列，9结合弹性体的机械和弹性性能。 LCES可以响应于外部STIM经历形状的改变，机械变形，弹性行为，和光学性能ULI（ 即 ，热，胁迫，光等 ）10，11，12，13，14，15，16。早先的研究已经表明，液晶（LCS）能够感测单元4，17的生长和取向。这是可能然后假设LCES可以适合于生物学和医学相关的应用程序，包括细胞骨架和对齐。我们以前曾报道近晶生物相容的，可生物降解的，铸塑，和薄LCES膜设有一个"瑞士奶酪型"多孔形态6，18的制备。我们还制备了球状形态向列生物相容性LCES作为支架用于细胞生长19 &lt;SUP&gt;，20。我们的工作的目的是调整材料的机械性能匹配的利息21组织的。此外，这些研究集中于理解弹性体 - 细胞相互作用，以及当所述弹性体受到外界刺激的细胞应答。 的主要挑战是在部分以定制LCES以允许通过弹性体基质细胞附着和渗透的孔隙率和更好的质量传输。这些薄膜6的孔隙率允许细胞渗透通过本体的基体，但不是所有的毛孔呈完全互连的或具有更规则的（均匀的）孔径。然后，我们就呈球状形态的生物相容性向列LCE弹性体的报道。允许这些向列型弹性体的附着和细胞的增殖，但孔尺寸只有10-30微米，这阻止或不等限于使用这些弹性体与更广泛的各种细胞系19，20。 通过Kung 等前期工作。与石墨烯泡沫的使用"牺牲"模板金属形成为，所获得的石墨烯泡沫具有由所选择的金属模板22决定的非常规则的多孔形态。这种方法提供了孔隙率和孔径的完全控制。与此同时，所述金属模板的延展性和柔韧性允许之前泡沫制备不同的模板的形成形状。其它技术，如材料浸出23，气体模板24，或电纺纤维25，26也为多孔材料的制备中的潜力，但它们更耗时的，并且，在一些情况下，孔尺寸被限制为只有几微米。泡沫样的3D LCES使用金属模板允许更高的小区负载制备一种改进的扩散率;共培养;并且，最后但并非最不重要的，更好的质量运输管理（ 即营养物质，气体和废弃物），以确保全组织发展27。泡沫状3D LCES似乎也改善细胞对准;这是最有可能在相对于LC吊坠感应细胞生长和细胞方向。 LC部分的LCE内的存在似乎增强与LCE支架内相对于单元格位置细胞对齐。细胞中的LCE的支柱内对齐，而没有观察到明显的取向，其中所述支柱接合在一起（结）27。 总的来说，我们的LCE细胞支架平台作为细胞支持介质提供了机会，以调整弹性体的形态和弹性性质和具体指导（个别）的细胞类型的对齐创建一个有序，空间安排ØF细胞类似于生命系统。除了提供能够维持和引导长期细胞生长和增殖的支架，还LCES允许动态实验，其中细胞定向和相互作用可能在运行中修改。

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			<td height="21" style="height:21px;width:291px;">1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:132px;">L16606</td>
			<td style="width:153px;">Silanizing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-bis(4-hydroxy-cyclohexyl)propane</td>
			<td>TCI</td>
			<td><a href="http://www.tcichemicals.com/eshop/en/us/commodity/B0928/">B0928</a></td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-chlorohexanone&#160;</td>
			<td>Alfa Aesar</td>
			<td>A18613</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-heptanone&#160;</td>
			<td>Sigma Aldrich</td>
			<td>W254401</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-propanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>278475</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-chloroperbenzoic acid, m-CPBA</td>
			<td>Sigma Aldrich</td>
			<td>273031</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-dimethylaminopyridine</td>
			<td>Alfa Aesar</td>
			<td>A13016</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole, DAPI&#160;</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Nuclear Stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-hexynoic acid&#160;</td>
			<td>Alfa Aesar</td>
			<td>B25132-06</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid</td>
			<td>VWR</td>
			<td>36289</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td>Sigma Aldrich</td>
			<td>34850</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol 200 proof ACS Grade&#160;</td>
			<td>VWR</td>
			<td>71001-866</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzene</td>
			<td>Alfa Aesar</td>
			<td>AA33290</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#949;-caprolactone&#160;</td>
			<td>Alfa Aesar</td>
			<td>A10299-0E</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>VWR</td>
			<td>BDH1109</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma Aldrich</td>
			<td>C8503</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromium(VI) oxide</td>
			<td>Sigma Aldrich</td>
			<td>232653</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper (I) iodide</td>
			<td>Strem Chemicals</td>
			<td>100211-060</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D,L-Lactide&#160;</td>
			<td>Alfa Aesar</td>
			<td>L09026</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>320269</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethyl ether&#160;</td>
			<td>Emd Millipore</td>
			<td>EX0190</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>270547</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium, DEME&#160;</td>
			<td>CORNING Cellgo</td>
			<td>10-013</td>
			<td>Cell Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Alfa Aesar</td>
			<td>33361</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde&#160;</td>
			<td>SIGMA Life Science</td>
			<td>F8775</td>
			<td>Fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum, FBS&#160;</td>
			<td>HyClone</td>
			<td>SH30071.01</td>
			<td>Media Component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper, Grade 415, qualitative, crepe</td>
			<td>VWR</td>
			<td>28320</td>
			<td>Filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Central node (3-arm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexamethylene diisocyanate, HDI</td>
			<td>Sigma Aldrich</td>
			<td>52649</td>
			<td>Crosslinker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron(III) chloride&#160;</td>
			<td>Alfa Aesar</td>
			<td>12357</td>
			<td>Etching agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl alcohol</td>
			<td>VWR</td>
			<td>BDH1133</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Alfa Aesar</td>
			<td>L13255</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N&#39;-dicyclohexylcarbodiimide</td>
			<td>Aldrich</td>
			<td>D80002</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>270547</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nickel metal template</td>
			<td>American Elements</td>
			<td>Ni-860</td>
			<td>Foam template</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neuroblastomas cells (SH-SY5Y)</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin&#160;</td>
			<td>Thermo SCIENTIFIC</td>
			<td>15140122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol 2000, PEG</td>
			<td>Alfa Aesar</td>
			<td>B22181</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide&#160;</td>
			<td>VWR</td>
			<td>97064-646</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>AMRESCO</td>
			<td>865</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>S-374</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>239313</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran</td>
			<td>Alfa Aesar</td>
			<td>41819</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiosulfate de sodium</td>
			<td>AMRESCO</td>
			<td>393</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tin(II) 2-ethylhexanoate</td>
			<td>Aldrich</td>
			<td>S3252</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluene&#160;</td>
			<td>Alfa Aesar</td>
			<td>22903</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>471283</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>Cell Detachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV1000</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of biocompatible liquid crystal elastomer foams as cell scaffolds for 3d spatial cell cultures

在这里，我们提出了一种一步一步制备3D，可生物降解的，泡沫状细胞的支架。这些支架是由交联的星形嵌段共聚物设有胆固醇单元作为侧链的侧基，从而导致近晶A（SMA）液晶弹性体（LCES）制备。泡沫状的支架，使用金属模板制备，设有相互连接的微通道，这使得它们适合作为三维细胞培养物的支架。金属泡沫的和弹性体的结果中相比于传统的多孔模板膜，但质量传输的还更好的管理（ 即，营养物，气体，废物促进不仅更高的细胞增殖的三维细胞骨架的规则结构的综合性能等 ）。所述金属模板的性质允许泡沫的形状（ 即，轧辊或薄膜）的易操作和用于不同的孔径为不同细胞研究的支架的制备，同时保留互连线模板的泰德多孔性。蚀刻过程中不影响弹性体的化学成分，并保持其生物相容性和生物降解性质。我们发现，这些碟状LCES，培养广泛的时间段时，使临床相关的和复杂的组织结构的研究，同时促进细胞的生长和增殖。

此报告显示多孔3D LCE作为使用镍金属模板细胞培养的支架的制备方法。将所得到的三维LCE演示了一个复杂的相互关联的信道的网络，其允许容易细胞浸润，以及更合适的质量传输27。研究发现，细胞能够充分渗透相互连接的渠道网络，并且也能够在LCE内对齐。在此，金属镍泡沫（99％的Ni，860密度g / cm 2）被选择以下的类似方法以前由Kung 等报道的石墨烯泡沫制剂。 22。金属泡沫聚合物注射成形，完全覆盖所有的金属支柱。金属泡沫的大小和密度的选择是很重要的，因为它赋予了低碳经济的最终形态，可以在模拟感兴趣的组织环境有所帮助。 D，L-丙交酯（ 图2）。最终的产物是一种疏水性3-臂SBC。与图4个6个臂，通过与季戊四醇和二季戊四醇代替甘油节点作出的SBC分别之前已经报道18。为更亲水的SBC，中央节点与低聚环氧乙烷代替（见协议音符）。然而，利用低聚环氧乙烷的结果替换甘油以线性嵌段共聚物27。我们使用从尤尼斯等人修改的合成。 29。修改后的版本允许使用经修饰的ε-CA的与在两个不同的位置，α和γ羰6卤基prolactone。一旦SBC形成，修饰的ε己内酯块遭受与叠氮化物基团的卤原子的位移。由叠氮基的卤素基团的位移由2100厘米的外观确认- 1个带使用衰减全反射（ATR）FT-IR（ 图3）。叠氮基稍后用于共价连接的LC结构部分（胆固醇hexynoate）使用炔 - 叠氮化物的Huisgen的环加成反应（也被称为"点击反应"）的星型嵌段共聚物，具有侧面获得最终星型嵌段共聚物α-链胆固醇侧链单元（SBC-CHOL）。三唑环的形成是由2100厘米的消失证实- 1间带和中的1 H NMR谱在7.30 ppm的观察到单峰的外观（参见图3）。我们最近报道了卤素基团其α的位置的（γ-Cl）的对官能ε-CL 6的羰基的效果（α-Br）或γ。应当指出的是，代替中央节点中与4-臂和6臂的中心核的SBC共聚物因为中央节点兼作引发剂和本征交联剂18具有对所得到的LCES的机械特性的效果。 一旦SBC-澈已经制备和完全表征，使用金属模板的交联过程是用于制备泡沫的最后一步。该SBC-澈用六亚甲基二异氰酸酯（HDI，交联剂），ε己内酯，和Sn（辛）2混合（ROP催化剂，参见步骤7.1）。双 - 己内酯（BCP）的交联剂用HDI代替，如先前报道。金属泡沫被切割并成形为期望的˚FORM（ 图4）。用于制备泡沫体，即原生孔隙（LCEF PP）和伯/仲孔隙率（LCEF P + SP）两条路径，已有报道。这里，LCEF P + SP，或"浸渍"方法，提出，其中，所述镍模板在聚合物混合物中快速浸渍。金属泡沫置于由铝箔或含有该聚合物混合物的闪烁小瓶的容器内，与所有金属支柱完全浸没在聚合物混合物中。过量的聚合物混合物小心除去。该聚合物覆盖的镍模板在80℃下放置过夜，仍然在容器内，在烘箱中。交联是在催化剂的存在下进行约2小时。交联过程之后，将聚合物覆盖的镍模板是从它的容器通过剥离铝箔或打碎玻璃容器中取出。过量的聚合物小心地从该聚合物覆盖的镍TEMPL剃吃以露出镍模板的边缘，并放置在容器内用饱和的FeCl 3溶液（ 图5）。几个小时后，将镍几乎完全去除，并且只有LCE泡沫漂洗用去离子（DI）水，以消除镍/的FeCl 3溶液。的LCE泡沫再次置于用饱和的FeCl 3溶液的容器内，并用DI水漂洗两次。在蚀刻过程完成且整个镍模板是完全消失后，LCE泡沫示出了具有中空支柱（ 图6）互连的通道网络。 图6示出了使用扫描电子显微镜（SEM）观察到的内部LCE泡沫形态。该LCE泡沫支撑是中空的，整体形态规则是在镍泡沫模板队伍。 此前，使用BCP所需的几个步骤，以保持它在SOLU灰（ 即熔化），因为BCP开始作为溶液几度冷却尽快再结晶（从140℃至室温，添加Sn的（10月）2;参见步骤7.1）。这使得金属泡沫和聚合物混合物交联之前挑战的操作。使用HDI的作为允许使用BCP的情况相比更快的交联时间的交联剂。 BCP是在室温下是固体，用140℃的熔点，而HDI是在室温下为液体。交联剂的选择直接影响的准备时间和，如在我们的情况下，降低交联温度为140〜80℃，还减少了弹性体的制备。 的LCE泡沫体使用压缩变形（ 电影1）进行测试。在LCE大小减少70％，观察到，与对总体尺寸没有影响。当从LCE压缩的版本，它完全恢复其原厂高原肺水肿和大小。据信，在液晶部分的在LCE材料中存在是重要的，在相同条件下所制备的弹性体没有胆固醇ε己内酯块没有恢复其原始形状，并且折叠成自己。 一旦获得LCE泡沫，他们准备使用标准细胞培养技术成神经细胞瘤（SH-SY5Y）接种。 LCE泡沫在70％乙醇中洗涤两次，并用PBS细胞接种之前三次漂洗。在细胞接种的2-3天，观察细胞附着到3D LCE网络的墙壁。充分研究的LCE网络内细胞附着和膨胀，将细胞在30天之后（ 图7）是固定的。将细胞固定，用DAPI染色，并用共聚焦显微镜成像。发现所述细胞已经增殖，附接，并扩展到广泛覆盖3D LCE支架网络。进一步以上，详细的分析显示细胞核的伸长率，在大多数情况下不影响所述3D LCE支架网络的弯曲部分。细胞伸长也可以与细胞排列和最有可能呈现的LCE的近晶A相特性的结果。这是以前在14天后27生长C2C12细胞中观察到。在这项研究中，我们表明，细胞继续增殖超过14天;这并不仅仅局限于C2C12细胞。使用LCE泡沫的可扩展到几乎所有的细胞系。细胞的三维LCE泡沫状支架生长从更高质量传输效率获益相比2D支架。在2D支架，细胞通常生长在层中，在彼此的顶部上。细胞在顶层成长是具有完全访问所有的营养物质，气体和废物清除（大众交通）唯一的。较低层中的细胞是无法访问的营养物质，并有一个较高程度的小区D的eath在这种情况下。内3D支架，细胞具有更有效（相对于2D支架）获得营养物，生长因子，和气体，从而允许长期细胞和组织再生的研究。使用3D LCE泡沫状支架细胞废物管理（除去废物）也比在2D支架更有效。所述LCES的孔隙率（和/或其他的结构特性）允许快速质量传输和小区负载增加相比较少孔（或其它）的矩阵，允许媒体和基体的中心区域蜂窝接入。这LCE平台是可调的，以支持多种类型小学和永生化细胞系，包括肌肉，神经，皮肤，等等的多细胞培养系统的发展，以及。从本质上讲，这是该平台的优点之一，因为它可以用来种了许多不同类型的细胞和系统长时间。此外，成长能力细胞的多层的结构更加紧密EMUL阿泰的自然环境。 <img alt="图1" src="/files/ftp_upload/55452/55452fig1.jpg" /> 图1:描述LCE泡沫的步骤一步的制备和表征的通用程序。详情请参阅协议部分。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55452/55452fig2.jpg" /> 图2:3-臂SBC的交联方案。使用biscaprolactone或HDI如在用于泡沫状LCES的制备镍金属模板的存在交联剂的3臂SBC的交联方案被示出。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig2large.jpg" target="_blank">请点击此处查看该figu的放大版。</a>回覆。 <img alt="图3" src="/files/ftp_upload/55452/55452fig3.jpg" /> 图3:接着通过1 H NMR和FT-IR 1,2,3-三唑形成（成功的"点击"反应）方案。由叠氮基的卤素基团的位移由2100厘米的外观确认- 1个带使用衰减全反射（ATR）FT-IR。 1条带和单峰的出现，中的1 H NMR谱在7.30 ppm的观察-三唑环的形成是由2100厘米的消失证实。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55452/55452fig4.jpg" /> 图4:光学图像的交联之前的各种泡沫形状。金属泡沫被切割并成形为期望的形式，如图所示。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55452/55452fig5.jpg" /> 图5:前（A）和之后（B）的交联泡沫镍。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55452/55452fig6.jpg" /> 图6:LCE泡沫形态使用扫描电子显微镜（SEM）进行观察。基于SBC-（a和b）和基于LBC（C LCE泡沫的代表性SEM图像</使用Ni-860作为金属模板强&gt;以及d）弹性体被示出。箭头指示镂空支柱。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/55452/55452fig7.jpg" /> 图7:共聚焦显微照片显示附着于弹性体泡沫料播种后30天SH-SY5Y细胞的DAPI染色的核。 2D图像层叠在z -方向，以及在xz横截面的图像-中创建和yz -平面。这些图像表明，SH-SY5Y细胞（亮点）和附接在弹性体泡沫料的中空通道的壁内膨胀。将细胞在空间上扩展到多个层，并通过构建体延伸超过100μm（如图所示在xz -和yz -平面图像交叉秒条数）。 <a href="http://ecsource.jove.com/files/ftp_upload/55452/55452fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/55452/55452movie1.jpg" /> 电影1:一个LCE辊的变形和恢复的视频图像。 <a href="https://www.jove.com/files/ftp_upload/55452/Figure5_Movie.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures at JoVE.com

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

这项研究提出了一种方法来制备3D，基于生物相容的侧链液晶弹性体（LCES）可生物降解的，泡沫状细胞支架。共聚焦显微镜实验表明，泡沫状LCES允许细胞附着，增殖和C2C12s成肌细胞的自发排列。

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

synthesis-biocompatible-liquid-crystal-elastomer-foams-as-cell

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.4K 次观看.  Kent State University. 这项研究提出了一种方法来制备3D，基于生物相容的侧链液晶弹性体（LCES）可生物降解的，泡沫状细胞支架。共聚焦显微镜实验表明，泡沫状LCES允许细胞附着，增殖和C2C12s成肌细胞的自发排列。 

视频: Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

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 ...

科研

生物工程

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

单域液晶的弹性体和液晶橡胶纳米复合材料的制备

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...