Name: synthesis of biocompatible liquid crystal elastomer foams as cell scaffolds for 3d spatial cell cultures
Uploaded: 2017-04-11T15:00:00.000+00:00
Duration: 13 min 38 s
Description: Watch this Scientific Journal Video about Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures at JoVE.com

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>

<ol>
	<li>Khor, E., Lim, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantable+applications+of+chitin+and+chitosan.">Implantable applications of chitin and chitosan.</a> Biomaterials. 24 (13), 2339-2349 (2003).</li><li>Chung, H. J., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+engineered+and+drug+releasing+pre-fabricated+scaffolds+for+tissue+engineering.">Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering.</a> Adv. Drug Deliv. Rev. 59 (4-5), 249-262 (2007).</li><li>Yakacki, C. M., Gall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-Memory+Polymers+for+Biomedical+Applications.">Shape-Memory Polymers for Biomedical Applications.</a> Shape-Memory Polymers. 226, 147-175 (2010).</li><li>Agrawal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+liquid+crystal+elastomers+for+dynamic+cell+culture.">Stimuli-responsive liquid crystal elastomers for dynamic cell culture.</a> J. of Mat. Res. 30 (4), 453-462 (2015).</li><li>Agrawal, A., Yun, T. H., Pesek, S. L., Chapman, W. G., Verduzco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-responsive+liquid+crystal+elastomer+bilayers.">Shape-responsive liquid crystal elastomer bilayers.</a> Soft Matter. 10 (9), 1411-1415 (2014).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+and+Porous+Liquid+Crystal+Elastomer+Scaffolds+for+Spatial+Cell+Cultures.">Biodegradable and Porous Liquid Crystal Elastomer Scaffolds for Spatial Cell Cultures.</a> Macromol. Biosci. 15 (2), 200-214 (2015).</li><li>Yakacki, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailorable+and+programmable+liquid-crystalline+elastomers+using+a+two-stage+thiol-acrylate+reaction.">Tailorable and programmable liquid-crystalline elastomers using a two-stage thiol-acrylate reaction.</a> RSC Adv. 5 (25), 18997-19001 (2015).</li><li>deGennes, P. G., Hebert, M., Kant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+muscles+based+on+nematic+gels.">Artificial muscles based on nematic gels.</a> Macromolecular Symposia. 113, 39-49 (1997).</li><li>Fleischmann, E. -. K., Zentel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-Crystalline+Ordering+as+a+Concept+in+Materials+Science:+From+Semiconductors+to+Stimuli-Responsive+Devices.">Liquid-Crystalline Ordering as a Concept in Materials Science: From Semiconductors to Stimuli-Responsive Devices.</a> Angew. Chem. Int. Ed. 52 (34), 8810-8827 (2013).</li><li>Finkelmann, H., Kim, S. T., Munoz, A., Palffy-Muhoray, P., Taheri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+mirrorless+lasing+in+cholesteric+liquid+crystalline+elastomers.">Tunable mirrorless lasing in cholesteric liquid crystalline elastomers.</a> Adv. Mater. 13 (14), 1069-1072 (2001).</li><li>Artal, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHG+characterization+of+different+polar+materials+obtained+by+in+situ+photopolymerization.">SHG characterization of different polar materials obtained by in situ photopolymerization.</a> Macromolecules. 34 (12), 4244-4255 (2001).</li><li>Camacho-Lopez, M., Finkelmann, H., Palffy-Muhoray, P., Shelley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+liquid-crystal+elastomer+swims+into+the+dark.">Fast liquid-crystal elastomer swims into the dark.</a> Nat. Mater. 3 (5), 307-310 (2004).</li><li>Yamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomobile+polymer+materials:+Towards+light-driven+plastic+motors.">Photomobile polymer materials: Towards light-driven plastic motors.</a> Angew. Chem. Int. Ed. 47 (27), 4986-4988 (2008).</li><li>Ohm, C., Brehmer, M., Zentel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Crystalline+Elastomers+as+Actuators+and+Sensors.">Liquid Crystalline Elastomers as Actuators and Sensors.</a> Adv. Mater. 22 (31), 3366-3387 (2010).</li><li>Fleischmann, E. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-piece+micropumps+from+liquid+crystalline+core-shell+particles.">One-piece micropumps from liquid crystalline core-shell particles.</a> Nat. Commun. 3, (2012).</li><li>Herzer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printable+Optical+Sensors+Based+on+H-Bonded+Supramolecular+Cholesteric+Liquid+Crystal+Networks.">Printable Optical Sensors Based on H-Bonded Supramolecular Cholesteric Liquid Crystal Networks.</a> J. Am. Chem. Soc. 134 (18), 7608-7611 (2012).</li><li>Lockwood, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermotropic+liquid+crystals+as+substrates+for+imaging+the+reorganization+of+matrigel+by+human+embryonic+stem+cells.">Thermotropic liquid crystals as substrates for imaging the reorganization of matrigel by human embryonic stem cells.</a> Adv. Funct. Mater. 16 (5), 618-624 (2006).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+structural+variations+on+the+cellular+response+and+mechanical+properties+of+biocompatible,+biodegradable,+and+porous+smectic+liquid+crystal+elastomers.">Effects of structural variations on the cellular response and mechanical properties of biocompatible, biodegradable, and porous smectic liquid crystal elastomers.</a> Macromol. Biosci. , (2016).</li><li>Bera, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Crystal+Elastomer+Microspheres+as+Three-Dimensional+Cell+Scaffolds+Supporting+the+Attachment+and+Proliferation+of+Myoblasts.">Liquid Crystal Elastomer Microspheres as Three-Dimensional Cell Scaffolds Supporting the Attachment and Proliferation of Myoblasts.</a> ACS Appl. Mater. Interfaces. 7 (26), 14528-14535 (2015).</li><li>Bera, T., Malcuit, C., Clements, R. J., Hegmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Surfactant+during+Microemulsion+Photopolymerization+for+the+Creation+of+Three-Dimensional+Liquid+Crystal+Elastomer+Microsphere+Spatial+Cell+Scaffolds.">Role of Surfactant during Microemulsion Photopolymerization for the Creation of Three-Dimensional Liquid Crystal Elastomer Microsphere Spatial Cell Scaffolds.</a> Front. Mater. 3 (31), (2016).</li><li>McKee, C. T., Last, J. A., Russell, P., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indentation+Versus+Tensile+Measurements+of+Young's+Modulus+for+Soft+Biological+Tissues.">Indentation Versus Tensile Measurements of Young's Modulus for Soft Biological Tissues.</a> Tissue Eng. Part B Rev. 17 (3), 155-164 (2011).</li><li>Kung, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+three+dimensional+graphene+foam+supported+platinum-ruthenium+bimetallic+nanocatalysts+for+hydrogen+peroxide+based+electrochemical+biosensors.">Preparation and characterization of three dimensional graphene foam supported platinum-ruthenium bimetallic nanocatalysts for hydrogen peroxide based electrochemical biosensors.</a> Biosens. Bioelectron. 52, 1-7 (2014).</li><li>Amsden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curable,+biodegradable+elastomers:+emerging+biomaterials+for+drug+delivery+and+tissue+engineering.">Curable, biodegradable elastomers: emerging biomaterials for drug delivery and tissue engineering.</a> Soft Matter. 3 (11), 1335-1348 (2007).</li><li>Sinturel, C., Vayer, M., Morris, M., Hillmyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Vapor+Annealing+of+Block+Polymer+Thin+Films.">Solvent Vapor Annealing of Block Polymer Thin Films.</a> Macromolecules. 46 (14), 5399-5415 (2013).</li><li>Riboldi, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+myogenesis+on+highly+orientated+microfibrous+polyesterurethane+scaffolds.">Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds.</a> J. Biomed. Mater. Res. A. 84 (4), 1094-1101 (2008).</li><li>Chung, S., Moghe, A. K., Montero, G. A., Kim, S. H., King, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrous+scaffolds+electrospun+from+elastomeric+biodegradable+poly(L-lactide-co-epsilon-caprolactone)+copolymer.">Nanofibrous scaffolds electrospun from elastomeric biodegradable poly(L-lactide-co-epsilon-caprolactone) copolymer.</a> Biomed. Mater. 4 (1), 9 (2009).</li><li>Gao, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+3D+Liquid+Crystal+Elastomer+Cell+Scaffolds+and+Foams+with+Primary+and+Secondary+Porous+Architecture.">Biocompatible 3D Liquid Crystal Elastomer Cell Scaffolds and Foams with Primary and Secondary Porous Architecture.</a> ACS Macro Lett. 5 (1), 14-19 (2016).</li><li>Lenoir, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ring-opening+polymerization+of+alpha-chloro-is+an+element+of-caprolactone+and+chemical+modification+of+poly(alpha-chloro-is+an+element+of-caprolactone)+by+atom+transfer+radical+processes.">Ring-opening polymerization of alpha-chloro-is an element of-caprolactone and chemical modification of poly(alpha-chloro-is an element of-caprolactone) by atom transfer radical processes.</a> Macromolecules. 37 (11), 4055-4061 (2004).</li><li>Younes, H. M., Bravo-Grimaldo, E., Amsden, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization+and+in+vitro+degradation+of+a+biodegradable+elastomer.">Synthesis, characterization and in vitro degradation of a biodegradable elastomer.</a> Biomaterials. 25 (22), 5261-5269 (2004).</li><li>Donaldson, T., Henderson, P. A., Achard, M. F., Imrie, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+liquid+crystal+tetramers.">Chiral liquid crystal tetramers.</a> J. Mater. Chem. 21 (29), 10935-10941 (2011).</li><li>Palmgren, R., Karlsson, S., Albertsson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+degradable+crosslinked+polymers+based+on+1,5-dioxepan-2-one+and+crosslinker+of+bis-epsilon-caprolactone+type.">Synthesis of degradable crosslinked polymers based on 1,5-dioxepan-2-one and crosslinker of bis-epsilon-caprolactone type.</a> J. Pol. Sci. A Polym. Chem. 35 (9), 1635-1649 (1997).</li></ol>

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?...

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。

<table><tbody><tr><td>1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane</td><td>Alfa Aesar</td><td>L16606</td><td>Silanizing agent</td></tr><tr><td>2-bis(4-hydroxy-cyclohexyl)propane</td><td>TCI</td><td>&lt;a href=&quot;http://www.tcichemicals.com/eshop/en/us/commodity/B0928/&quot;&gt;B0928&lt;/a&gt;</td><td>Reagent</td></tr><tr><td>2-chlorohexanone&amp;nbsp;</td><td>Alfa Aesar</td><td>A18613</td><td>Reagent</td></tr><tr><td>2-heptanone&amp;nbsp;</td><td>Sigma Aldrich</td><td>W254401</td><td>Solvent</td></tr><tr><td>2-propanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>278475</td><td>Solvent</td></tr><tr><td>3-chloroperbenzoic acid, m-CPBA</td><td>Sigma Aldrich</td><td>273031</td><td>Reagent</td></tr><tr><td>4-dimethylaminopyridine</td><td>Alfa Aesar</td><td>A13016</td><td>Reagent</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole, DAPI&amp;nbsp;</td><td>Invitrogen</td><td>D1306</td><td>Nuclear Stain</td></tr><tr><td>5-hexynoic acid&amp;nbsp;</td><td>Alfa Aesar</td><td>B25132-06</td><td>Reagent</td></tr><tr><td>Acetic acid</td><td>VWR</td><td>36289</td><td>Solvent</td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>34850</td><td>Solvent</td></tr><tr><td>Alcohol 200 proof ACS Grade&amp;nbsp;</td><td>VWR</td><td>71001-866</td><td>Reagent</td></tr><tr><td>Benzene</td><td>Alfa Aesar</td><td>AA33290</td><td>Solvent</td></tr><tr><td>&amp;epsilon;-caprolactone&amp;nbsp;</td><td>Alfa Aesar</td><td>A10299-0E</td><td>Reagent</td></tr><tr><td>Chloroform</td><td>VWR</td><td>BDH1109</td><td>Solvent</td></tr><tr><td>Cholesterol</td><td>Sigma Aldrich</td><td>C8503</td><td>Reagent</td></tr><tr><td>Chromium(VI) oxide</td><td>Sigma Aldrich</td><td>232653</td><td>Reagent</td></tr><tr><td>Copper(I) iodide</td><td>Strem Chemicals</td><td>100211-060</td><td>Reagent</td></tr><tr><td>&lt;em&gt;D&lt;/em&gt;,&lt;em&gt;L&lt;/em&gt;-Lactide&amp;nbsp;</td><td>Alfa Aesar</td><td>L09026</td><td>Reagent</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>320269</td><td>Solvent</td></tr><tr><td>Diethyl ether&amp;nbsp;</td><td>Emd Millipore</td><td>EX0190</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium, DEME&amp;nbsp;</td><td>CORNING Cellgo</td><td>10-013</td><td>Cell Media</td></tr><tr><td>Ethanol</td><td>Alfa Aesar</td><td>33361</td><td>Solvent</td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>SIGMA Life Science</td><td>F8775</td><td>Fixative</td></tr><tr><td>Fetal bovine serum, FBS&amp;nbsp;</td><td>HyClone</td><td>SH30071.01</td><td>Media Component</td></tr><tr><td>Filter paper, Grade 415, qualitative, crepe</td><td>VWR</td><td>28320</td><td>Filtration</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td>Central node (3-arm)</td></tr><tr><td>Hexamethylene diisocyanate, HDI</td><td>Sigma Aldrich</td><td>52649</td><td>Crosslinker</td></tr><tr><td>Iron(III) chloride&amp;nbsp;</td><td>Alfa Aesar</td><td>12357</td><td>Etching agent</td></tr><tr><td>Isopropyl alcohol</td><td>VWR</td><td>BDH1133</td><td>Solvent</td></tr><tr><td>Methanol</td><td>Alfa Aesar</td><td>L13255</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&#039;&lt;/em&gt;-dicyclohexylcarbodiimide</td><td>Aldrich</td><td>D80002</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Nickel metal template</td><td>American Elements</td><td>Ni-860</td><td>Foam template</td></tr><tr><td>Neuroblastomas cells (SH-SY5Y)</td><td>ATCC</td><td>CRL-2266</td><td>Cell line</td></tr><tr><td>Penicillin streptomycin&amp;nbsp;</td><td>Thermo SCIENTIFIC</td><td>15140122</td><td>Antibiotics</td></tr><tr><td>Polyethylene glycol 2000, PEG</td><td>Alfa Aesar</td><td>B22181</td><td>Reagent</td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>VWR</td><td>97064-646</td><td>Reagent</td></tr><tr><td>Sodium bicarbonate</td><td>AMRESCO</td><td>865</td><td>Drying salt</td></tr><tr><td>Sodium chloride</td><td>BDH</td><td>BDH9286</td><td>Drying salt</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Scientific</td><td>S-374</td><td>Drying salt</td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma Aldrich</td><td>S9638</td><td>Drying salt</td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>239313</td><td>Drying salt</td></tr><tr><td>Tetrahydrofuran</td><td>Alfa Aesar</td><td>41819</td><td>Solvent</td></tr><tr><td>Thiosulfate de sodium</td><td>AMRESCO</td><td>393</td><td>Drying salt</td></tr><tr><td>Tin(II) 2-ethylhexanoate</td><td>Aldrich</td><td>S3252</td><td>Reagent</td></tr><tr><td>Toluene</td><td>Alfa Aesar</td><td>22903</td><td>Solvent</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td>Reagent</td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH30042.01</td><td>Cell Detachment</td></tr><tr><td>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?...

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

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

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 ...

科研

生物工程

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 ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

糖注射水凝胶的制备及其在3D 细胞培养中的应用

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

化学

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

用于细胞培养和皮肤应用的薄膜复合硅弹性体: 制造和表征

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

发育生物学

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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

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

三维肽支架中哺乳动物细胞的培养

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

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...