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>

没有利益冲突的声明。

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 &amp;#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 &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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 ...

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 ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

破碎散装水凝胶并加工成颗粒状水凝胶，用于生物医学应用

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

用于肝病研究的外周血人肝球

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...