该项目是由美国国立卫生研究院（R21EB013717）和IUPUI OVCR（RSFG）。作者感谢汉施女士，她的技术援助。

A.大分子多肽合成<ol><li>综合四臂PEG -降冰片烯（PEG4NB）和光引发剂的锂arylphosphanate（LAP）使用已建立的协议。18,19 </li><li>合成双 - 半胱氨酸，含有凝乳蛋白酶敏感的肽CGGY↓C（箭头表示胰凝乳蛋白酶裂解位点），在微波肽合成（CEM查找SPS）使用标准的固相肽合成。 <ol class="lalpha"><li>计算树脂（酰胺结合Rink-MBHA树脂）所需的量的基础上的取代比率的树脂和合成规模。溶胀树脂的二甲基甲酰胺（DMF）中，在15分钟的反应容器（RV）。 </li><li>除去Fmoc保护基团的树脂，使用的脱保护溶液含有20％的哌啶和0.1M HOBt的DMF。使用在下面的表中列出的参数的微波辅助的芴甲氧羰基-脱保护（可用于所有Fmoc-氨基酸）: <table border="1"><tbody><tr><td> 合成规模 </STRONG&gt; </td><td> 0.1毫摩尔 </td><td> 0.2毫摩尔 </td></tr><tr><td>功率</td><td> 20 W </td><td> 50 W </td></tr><tr><td>温度</td><td> 75°C </td><td> 75°C </td></tr><tr><td>时间</td><td> 3分钟</td><td> 3分钟</td></tr></tbody></table></li><li>脱保护后，冲洗树脂用DMF，并进行茚三酮试验来确认的Fmoc去除（曝光的N-末端的胺）。 2分钟后在95℃树脂变成蓝色</li><li>成功后的脱保护，Fmoc-氨基酸的C-末端（合成运行从C-N-末端）的活化剂的碱溶液中（0.28中号）的DMF的二异丙基乙胺（DIEA），下表中列出的参数的第一耦合。 <table border="1"><tbody><tr><td> 合成规模 </td><td> 0.1毫摩尔 </td><td> 0.2毫摩尔 </td&gt; </tr><tr><td>功率</td><td> 20 W </td><td> 50 W </td></tr><tr><td>温度* </td><td> 75°C </td><td> 75°C </td></tr><tr><td>时间* </td><td> 5分钟</td><td> 5分钟</td></tr></tbody></table> *囊肿和他的50°C和10分钟，以减少外消旋化。 </li><li>冲洗树脂，并进行茚三酮试验来定性确认耦合的完成（N-末端胺消失）。树脂应明确5分钟后，在95°C。重复偶联步骤（A.2.d），茚三酮测试返回肯定结果（蓝色）。 </li><li>重复脱保护（步骤A.2.b节）和耦合（步骤A.2.d）的额外的氨基酸序列中的结束的最终脱保护步骤。 </li><li>漏的RV，冲洗树脂用25毫升的二氯甲烷（DCM）。此步骤是为了冲洗掉DMF中，这是一个基础，并会阻碍肽裂解。 </li><li>准备裂解鸡尾酒混合250米的克苯酚在4.75毫升三氟乙酸（TFA），0.125毫升三异丙基硅烷（TIS）和0.125毫升DDH 2 O </li><li>加入裂解液RV和切割为30分钟的微波多肽合成器（功率= 20 W，温度= 38°C）。 </li><li>收集的肽溶液在50毫升离心管中，使用真空歧管。 </li><li>沉淀物裂​​解的肽在40毫升乙醚中，涡管和离心机以3,000 rpm离心5分钟收集所述肽。 </li><li>排出上清液，重复步骤A.2.k两次。 </li><li>收集并干燥在真空中的肽。 </li><li>采用高效液相色谱法和净化的肽，它与质谱表征。 </li></ol></li></ol> B.材料的制备和灭菌<ol><li>准备大分子单体20％（重量）PEG4NB溶液在PBS，涡的混合物，直到完全溶解，并消毒的大分子单体溶液用注射器过滤器。存储的灭菌溶液在-20°C（准备等份长期储存）。 </li><li>准备和2％（重量）的光引发剂（LAP）的PBS溶液进行消毒。存储的无菌溶液在室温避光。 </li><li>在PBS中溶解的肽（CGGYC），旋涡的混合物和注射器过滤器进行灭菌的溶液。分装在-20°C肽溶液和存储之前，使用（防止冻融循环）。 </li><li>确定巯基（-SH）Ellman试剂的准备肽溶液的浓度（遵循制造商的协议）。此步骤将得到准确的肽浓度（ 即 ，[SH] / 2）]是所需的硫醇-烯照片点击反应中要使用的计算的肽量。 </li><li>准备凝胶模具切割顶端部分off1毫升无菌一次性注射器，用剃刀刀片（用于凝胶除去的开口尖端）。用高压灭菌器，消毒的注射器模具。 </li><li>基于所需的凝胶制剂（ 硫醇烯比= 1）和浓度的股票队友里亚尔，计算所需的体积的聚合物，肽的交联剂，光引发剂，和所需的缓冲溶液。 </li></ol> C.细胞制备<ol><li>平衡细胞培养介质（高葡萄糖DMEM中含有10％牛胎儿血清，100 U / ml青霉素，100微克/毫升链霉素，250 ng / ml的两性霉素B的抗生素抗真菌和50μM的β-巯基乙醇），Hank氏平衡盐溶液（ HBSS中，Ca 2 +的 ，镁2 +免费），和胰蛋白酶-EDTA至37℃。 </li><li>取出烧瓶含有MIN6β细胞从CO 2培养箱中，将烧瓶放置在层流罩。 </li><li>吸干从烧瓶的细胞培养基，并用HBSS冲洗细胞。 </li><li> Trypsinize使用3毫升2X（0.1％）胰蛋白酶-EDTA的细胞孵育4分钟的烧瓶在37℃和5％CO 2的 。 <ul><li>注意:使用2X胰蛋白酶的解决方案使MIN6β-细胞完全分离成单细胞悬液。 </li></ul> &lt;/ P&gt; <li>孵育后，点选烧瓶的罩的表面上，轻轻地完全分离细胞。确认使用显微镜的细胞的分离和分解。 </li><li>加入等体积的细胞培养液中的胰蛋白酶。混合溶液，以及通过轻轻移液，以1,000 rpm的5分钟，然后将混合物转移到一个规范的管和离心机。 </li><li>吸取上清，并轻轻重新悬浮的细胞在4 - 5毫升培养基。 </li><li>确定用50％的台盼蓝血细胞计数器的缓冲区中的细胞密度。 </li></ol> D.水凝胶的制备及电池封装<ol><li>一旦细胞准备就绪后，组合（1:1的巯基和烯比的基础上）需要大量的PEG4NB，CGGYC，LAP，细胞溶液，HBSS离心管中。 </li><li>混合的溶液，轻轻地用移液管，得到均匀的混合物中，并加入25微升所得溶液的注射器模具。公开的注射器UV灯（365纳米，5毫瓦/厘米2），持续2分钟。 </li><li>以下的光聚合，水垫到一个无菌的24 -孔板含有细胞的培养基中的凝胶，孵育的水凝胶，在37℃和5％CO 2的 。 </li><li>洗净细胞充满水凝胶的细胞培养液中30分钟，用清水冲洗干净松散贴壁细胞和未交联的大分子单体。将凝胶转移到新鲜培养基中。 </li><li>刷新细胞培养介质每2到3天。 </li></ol> E.细胞活性检测封装的细胞形态可使用光学显微镜观察（自合成的水凝胶是透明的）。细胞活力，可以可视化和LIVE / DEAD染色和定量使用的AlamarBlue试剂的定性测量。 E.1。 LIVE / DEAD染色<ol><li>在室温下解冻LIVE / DEAD染色试剂。 </li><li>在一个典型的管加PBS根据样本的数目（n）的被染色: <str王总成交量的PBS = N×500微升</li><li>移液器0.25μl的钙黄绿素AM和2微升的EthD-1每1毫升的PBS中含有PBS规范管。振荡溶液混合组件。 </li><li>在轨道摇床1小时，室温孵育细胞充满水凝胶LIVE / DEAD染色溶液（0.5毫升/样本）。 </li><li>用巴斯德吸管，去除的染料溶液，并用PBS冲洗样品两次。 </li><li>将盖玻片或载玻片上的样品。观察细胞，利用共聚焦显微镜图像。 </li></ol> E.2。的AlamarBlue含量<ol><li>准备10 / v％的在细胞培养基中的AlamarBlue溶液。 </li><li>将水凝胶删除从孵化器和吸出每口井没有任何接触水凝胶的媒体。 </li><li>孵育细胞充满水凝胶和一个空的阱（阴性对照），在500微升10％的AlamarBlue溶液16小时。 </li><li>经过孵化，移液管200微升在96孔板上，用酶标仪（激发波长:560 nm，发射:590nm处）和测量荧光的AlamarBlue溶液。 <ul><li>较高的细胞代谢活动降低了染料给予较高的荧光读数。 </li><li>一定要包括至少10％的AlamarBlue的解决方案为空白两口井。在分析数据时，减去平均空白荧光值的样品的荧光值。 </li></ul></li></ol> F.糜蛋白酶介导的凝胶侵蚀和球体恢复<ol><li>准备1毫克/毫升的胰蛋白酶-α-胰凝乳蛋白酶溶液中HBBS和无菌过滤的溶液。 </li><li>孵育在胰凝乳蛋白酶溶液（500微升每个凝胶）的水凝胶在室温轻轻摇动，直到实现完整的凝胶侵蚀。 <ul><li>对于25微升凝胶，完整的侵蚀的时间大约是5分钟。 </li></ul></li><li>板在冰上放置几分钟，以减少的活性糜蛋白酶。 </li><li>转移的细胞溶液在300rpm在1ml管和离心机，4℃下持续2分钟。 </li><li>小心地取出上清液使用牧场吸管，轻轻悬浮球体在HBSS。 </li><li>回收的旋转椭球体的溶液转移到24孔板上，并在冰上放置板允许球体安顿下来。 </li><li>大小分析，获得的解决球体，使用光学显微镜相衬图像。测量恢复软件，如尼康元软件ImageJ的球体直径。 </li></ol> G.回收的细胞球体的功能分析 G.1。从回收的细胞球体的葡萄糖刺激的胰岛素分泌<ol><li>在24孔板中，培养凝胶在500μl2mM的低血糖克布斯林格氏缓冲液（KRB）1小时。 </li><li>蚕食的凝胶和恢复细胞球体的步骤中一至中六。 </li><li>不作任何认真吸取上清液与细胞球体，悬浮在500微升2毫米的低血糖KRB球体。 </li><li> 100微升细胞球状体溶液转移至一个0.6毫升的试管中，并将其放置在冰上，细胞内ATP定量。 </li><li>剩余的细胞旋转椭球体的溶液在半（分成两个微量离心管）拆分离心2分钟，在300 rpm和4℃。 </li><li>吸液上清液（几乎至底部），重悬在1ml的25mM高糖KRB和第二个一半在1毫升2mM的低葡萄糖KRB细胞球体上半年。 </li><li>轻轻吸液管和24孔板的溶液中回收的细胞球体转移。孵育板1小时，在37℃和5％CO 2的 。 </li><li>孵育之后，将溶液转移到一个离心管中，离心2分钟，以1,000 rpm，4℃。 </li><li>从离心分离管500μl的上清液转移至另一微量离心管中并储存在4℃下对胰岛素酶联免疫吸附法（ELISA） </li><li>执行胰岛素ELISA下列制造商的协议。 </li></ol> G.2。 CellTiter格洛检测<ol><li> ATP标准:准备一个10μMATP解决方案的进行连续稀释，取得了一系列的8个标准的解决方案。 </li><li>到白色96孔板中，加入50微升旋转椭球体的细胞溶液（从步骤G.1.6）和标准。 </li><li>对于每个孔中含有的样品和标准品，添加50微升的CellTiter格洛试剂，在室温下孵育15分钟。 </li><li>培养后，用酶标仪和校准样品ATP浓度，使用所产生的ATP标准的线性回归曲线的样品和标准品的测量发光计数。 </li><li>确定总的ATP浓度样品和样品稀释倍数乘以一个细胞充满水凝胶的细胞内ATP浓度。 </li></ol> G.3。确定胰岛素分泌<ol><li>计算amount的胰岛素分泌从回收的球粒通过服用高葡萄糖培养基中分泌的低葡萄糖培养基中的胰岛素分泌量的胰岛素的量的比率。 </li><li>规范的量化胰岛素的细胞球体的内容与相应数量的细胞内ATP的恢复细胞球体。 </li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-click+chemistry:+a+multifaceted+toolbox+for+small+molecule+and+polymer+synthesis.">Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis.</a> Chemical Society reviews. 39, 1355-1387 (2010).</li><li>Fairbanks, B. D., 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=A+Versatile+Synthetic+Extracellular+Matrix+Mimic+via+Thiol-Norbornene+Photopolymerization.">A Versatile Synthetic Extracellular Matrix Mimic via Thiol-Norbornene Photopolymerization.</a> Adv. Mater. 21, 5005 (2009).</li><li>Fairbanks, B. D., Schwartz, M. P., Bowman, C. N., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinitiated+polymerization+of+PEG-diacrylate+with+lithium+phenyl-2,4,6-trimethylbenzoylphosphinate:+polymerization+rate+and+cytocompatibility.">Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility.</a> Biomaterials. 30, 6702-6707 (2009).</li><li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+release+from+hydrolytically+degradable+poly(ethylene+glycol)+hydrogels.">Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels.</a> Biotechnology and bioengineering. 108, 197-206 (2011).</li></ol>

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所描述的协议，详细介绍在逐步增长的光聚合形成的巯基烯水凝胶的细胞就容易封装。虽然硫醇官能团的降冰片烯的1:1的化学计量比使用在该协议中，其比例是可以根据实验调整。除了正确的配方，它是重要的是保持在预聚物溶液中的均匀性。特别是，使用温和的移液，以确保细胞以及分布在预聚物溶液中，以避免结块，在凝胶特性的细胞和变更。虽然用于凝胶交联聚合时间为2分钟，硫醇-烯的?...

水凝胶是亲水性的交联的聚合物，作为用于修复和再生组织的脚手架材料具有超常的潜力。1-3的高水含量的水凝胶的允许的氧容易扩散和交换的营养物质和细胞代谢产物，所有这些都是维持细胞活力的关键。此外，水凝胶是控制释放和细胞外，由于其高可调性的优良载体。2合成水凝胶，如制备聚乙二醇（PEG）越来越多地应用于组织工程的应用，主要是由于其细胞相容性，组织状弹性，和高的可调谐性材料的物理和机械性能。4-6 尽管通常使用的水凝胶的平台，研究表明，聚乙二醇二丙烯酸酯（PEGDA）凝胶由链增长photopolymerizations形成有一种倾向，损坏封装细胞杜里纳克网络交联和原位细胞封装。7细胞损伤主要是由于产生的自由基光引发剂的分子，它通过乙烯基PEGDA传播到水凝胶的交联聚合物链。不幸的是，这些自由基物种也可能导致在细胞封装应力和细胞损伤，尤其是对自由基敏感的细胞，胰腺β-细胞，如8-10为了获得更高的网目尺寸的更好的扩散，和细胞的存活，较高分子量PEGDA常常被用于电池封装。然而，这损害聚合动力学和导致次优凝胶生物物理特性。7,11,12在除了上述缺点外，它是非常困难的源自PEGDA水凝胶由于恢复细胞结构的异质性和非降解性质交联网络。虽然可以掺入蛋白酶敏感的肽到的PEG大分子的骨干，呈现否则惰性的PEGDA水凝胶敏感的酶裂解，结合经常使用昂贵的试剂，并导致网络还含有高度的异质性，由于链增长聚合的性质。13-15 最近，通过逐步增长硫醇-烯光聚合形成的PEG-肽水凝胶已被证明具有超过由链增长光聚合水凝胶形成的细胞封装优惠属性7的硫醇-烯水凝胶的凝胶动力学优于被归因于"点击"自然的反应，巯基和烯的功能。相比链增长聚合PEGDA，硫醇-烯反应是较少的氧抑制16,17硫醇-烯水凝胶也有较高的聚合效率和更好的凝胶相比，链增长PEGDA水凝胶，7的生物物理性质的凝胶速度更快的结果。 18 &lt;/ SUP&gt;，这将导致在有限的细胞造成损害的自由基物种在光聚合。 此前，硫醇-烯水凝胶形成由4臂PEG -降冰片烯（PEG4NB）大分子单体和双-含有半胱氨酸的肽的交联剂，如蛋白酶敏感的肽已被用于电池封装。7,18高可调性的PEG水凝胶网络提供灵活的和可控的三维微环境调查细胞的存活和活性，蛋白酶敏感的肽序列，而使用提供温和的方式进行恢复内自然形成的水凝胶的细胞结构。在这个协议中，我们利用逐步增长的光聚合的硫醇 - 烯水凝胶采用4 - 臂的PEG-降冰片烯（PEG4NB）和胰凝乳蛋白酶敏感的肽交联剂（CGGY↓C）用于封装MIN6β-细胞。此协议系统阐述研究MIN6存活，增殖和球体形成的技术在硫醇 - 烯水凝胶的β-细胞。我们还提供β-细胞的的球体恢复和回收的球体的生物学特性的方法。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>四臂PEG（20kDa） </td><td>美国键凯科技</td><td> 4ARM-PEG-20K </td><td></td></tr><tr><td> Fmoc-氨基酸</td><td> Anaspec </td><td></td><td></td></tr><tr><td> LIVE / DEAD细胞活性试剂盒</td><td> Invitrogen公司</td><td> L3224 </td><td>包括钙黄绿素AM和乙锭同型二聚体-1 </td></tr><tr><td>的AlamarBlue试剂</td><td> ABD Serotec公司</td><td> BUF012 </td><td></td></tr><tr><td> CellTiter Glo试剂</td><td> Promega公司</td><td> G7570 </td><td></td></tr><tr><td> DPBS </td><td>龙沙</td><td> 17-512F </td><td>不含Ca +2和Mg +2 </td></tr><tr><td> HBSS </td><td>龙沙</td><td> 10547F </td><td>没有钙离子和Mg +2 </td></tr><tr><td>高糖DMEM </td><td> Hyclone公司</td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gibco公司</td><td> 16000-044 </td><td></td></tr><tr><td>抗生素，抗真菌</td><td> Invitrogen公司</td><td> 15240-062 </td><td></td></tr><tr><td> β-巯基乙醇</td><td> Sigma-Aldrich公司</td><td> M7522-100ML </td><td></td></tr><tr><td>胰蛋白酶-EDTA </td><td> Invitrogen公司</td><td> 15400-054 </td><td></td></tr><tr><td>胰蛋白酶 - 自由α-胰凝乳蛋白酶</td><td>沃辛顿生化公司</td><td> LS001432 </td><td></td></tr><tr><td>鼠标Inusin ELISA试剂盒</td><td> Mercodia </td><td> 10-1247-01 </td><td></td></tr><tr><td> 1毫升一次性注射器</td><td> BD生物科学</td><td></td><td></td></tr></tbody></table>

generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels

水凝胶是亲水性的交联的聚合物提供了一个三维的组织类似用于培养的治疗相关的细胞或组织的弹性和高渗透性的微环境。从聚（乙二醇）（PEG）衍生物制备的水凝胶被越来越多地用于各种组织工程应用，部分由于其可调和细胞相容性属性。在这个协议中，我们使用硫醇 - 烯逐步增长photopolymerizations的的制造PEG-肽水凝胶封装胰腺MIN6 B细胞。将凝胶形成了由4 - 臂的PEG - 降冰片烯（PEG4NB）大分子和胰凝乳蛋白酶敏感的肽的交联剂（CGGYC）。的亲水性和非污损性质PEG提供在3D中的细胞存活和增殖的细胞相容性的微环境，而使用糜蛋白酶敏感的肽序列（ÇGGY↓C，箭头表示酶切割位点，而终端囊肿加入EINE残留物硫醇 - 烯交联），可以迅速恢复细胞内形成水凝胶的结构。以下协议阐述技术:（1）在硫醇 - 烯水凝胶封装MIN6β细胞，（2）的定性和定量的细胞生存力分析，以确定细胞存活和增殖;（3）回收细胞球体使用胰凝乳蛋白酶介导的凝胶侵蚀;和（4）的结构和功能分析的回收球体。

<html> 图1-4示出了具有代表性的结果，封装，生存，增殖，形成球体，和硫醇-烯水凝胶球体复苏。 图1示出的反应步骤（1）增长的硫醇-烯光聚合使用PEG4NB和CGGYC示意图，和（ 2）胰凝乳蛋白酶介导的凝胶表面侵蚀机理侵蚀，如下图2和图3展示可行性利用LIVE / DEAD染色的AlamarBlue检测结果。我们观察到在PEG4NB-CGGYC水凝胶即使在低堆积密度的细胞的细胞?...

Watch this Scientific Journal Video about Generation and Recovery of Î²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

下面的协议提供了用于封装在逐步增长的PEG-肽水凝胶形成的硫醇 - 烯照片点击反应胰腺β-细胞的技术。这种材料平台不仅提供了细胞相容性的微环境，细胞封装，但也允许用户控制的快速恢复细胞结构内形成的水凝胶。

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

generation-recovery-cell-spheroids-from-step-growth-peg-peptide

Research

JoVE Journal

Bioengineering

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

14.1K 次观看.  Indiana University - Purdue University at Indianapolis. 下面的协议提供了用于封装在逐步增长的PEG-肽水凝胶形成的硫醇 - 烯照片点击反应胰腺β-细胞的技术。这种材料平台不仅提供了细胞相容性的微环境，细胞封装，但也允许用户控制的快速恢复细胞结构内形成的水凝胶。

视频: Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

发育生物学

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

在 PEG 水凝胶中制备肿瘤球状体后进行 3D 封装

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

作者聚焦:胶质母细胞瘤微环境研究的体外水凝胶模型

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

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通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用