DOZ的日内瓦基金会颁发的赠款支持。支持博士后奖学金资助从匹兹堡组织工程​​倡议的SN。

1。分离脂肪干细胞（ASC） 注:所有的程序，在室温下进行，除非另有说明。 <ol><li>分离大鼠肾周和附睾脂肪用无菌汉克的缓冲盐溶液，含1％胎牛血清（FBS），如先前所述6（HBSS中）。 </li><li>百果组织和转移到25毫升管离心8分钟在室温和500克50毫升含1％胎牛血清的HBSS 1-2克。 </li><li>收集的自由浮动的脂肪层，并转移到125毫升三角瓶和治疗II型胶原酶（200 U / ml的）25毫升在45分钟的HBSS 37°，轨道摇床（125转） </li><li>小心地取出液体部分（油和脂肪层以下），并依次通过100μm和70μm的尼龙网过滤器过​​滤。离心滤液在500克，在室温下10分钟，吸出supernatant和25毫升的HBSS洗两次沉淀。 </li><li>悬浮细胞沉淀在50毫升的培养基中补充MesenPRO RS生长添加物，抗生素，抗霉菌（100单位/毫升100微克/毫升硫酸链霉素，青霉素G，和0.25微克/毫升两性霉素B）（MesenPRO RS基础培养基） ，2毫米到2的T75瓶（25毫升/瓶）L-谷氨酰胺和吸管细胞。 </li><li>文化ASC在37°C（使用2-4升序所有实验）在5％CO 2培养箱孵化器。 </li></ol> 2。制备壳聚糖微球（CSM） 注:所有的程序，在室温下进行，除非另有说明。 <ol><li> CSM的准备由水油乳化过程中，随着离子的凝聚技术，用我们以前的协议6。乳化在油相混合的豆浆100毫升的壳聚糖水溶液（6毫升3％W / V壳聚糖在0.5 M醋酸）油，正辛醇（1:2 V / V）和5％山梨醇单油酸酯（跨度80）乳化剂，使用开销（1700转）和磁力搅拌（1000转）同时在相反的方向。这种混合确保早期形成之前，在交联胶束可以留在溶液中，并没有解决的底部双方法。此外，磁力搅拌棒的艾滋病在聚合过程中胶束的形成和刚化壳聚糖。 </li><li>不断搅拌约1小时，直到获得一个稳定的中水乳化油的混合物。离子交联开始增加1.5毫升1％W / V正辛醇15分钟，每4小时（共24毫升）的氢氧化钾。 </li><li>交联反应完成后，慢慢地倾倒出油的混合物含有的CSM阶段，马上加入100 mL丙酮球。用丙酮清洗球，直到完全除去油相。 </li><li>干燥在真空干燥器和肛门恢复领域YZE没有进一步的处理。使用粒度分析仪测定平均CSM粒径，表面积每毫克和单位立方体体积。 </li><li>对于随后的实验中，用无菌水的CSM三次，以除去残留的盐和无水酒精消毒。 </li></ol> 3。在CSM数确定的游离氨基酸组 注:所有的程序，在室温下进行，除非另有说明。 <ol><li>免费使用的硝基苯磺酸（TNBS）的Bubins和Ofner 7的酸法目前CSM的离子交联后的氨基酸组确定的数量。用1 mL 0.5％三硝基苯磺酸溶液50毫升的玻璃试管中4小时在40°C孵育5毫克的微球，并在60°C 3毫升6N盐酸水解时间为2h。 </li><li>样品冷却至室温，加入5毫升的去离子水和10毫升乙酸乙酯提取免费硝基苯磺酸乙醚。 </li><li> 5毫升的水相等分预热至40°C的水浴蒸发冷却至室温，任何残余的乙醚，15分钟和15毫升水稀释。 </li><li>与壳聚糖不使用三硝基苯磺酸溶液作为空白和壳聚糖为CSM准备用于确定氨基酸组总数分光光度计在345 nm处测量吸光度。估计CSM的相对壳聚糖的游离氨基酸组的数量。 </li></ol> 4。在CSM加载升序 注:所有的程序，在室温下进行，除非另有说明。 <ol><li>平衡在一夜之间无菌的HBSS 2.5节从5毫克和消毒的CSM添加到8微米孔径的膜培养板插入（24孔板）。 </li><li>后落户到膜的CSM，小心吸的HBSS中，添加300μL培养基中生长介质的插入和700微升内的THE境外插入。 </li><li>升序重悬在适当的浓度（1×10 4 4×10 4）200μL生长介质和种子在培养板内插入的CSM。 ，播种后，内插入的文化语言的最终体积为500μL。 </li><li> CSM升序种子孵育24小时在5％CO 2培养箱孵化器在37°C。 </li></ol> 5。在CSM确定ASC的装载和细胞活力的百分比 注:所有的程序，在室温下进行，除非另有说明。 <ol><li>孵化后，收集，而不会干扰细胞已迁移到插入膜ASC装在无菌的1.5 ml离心管的CSM。 </li><li>去除残留的培养基，并添加250μL新鲜生长培养基管。 </li><li>每管加入25微升MTT法[3 - （4,5 - dimethylthiozole -2 - 基）-2,5 - 二苯基溴化]解决方案（5毫克/毫升）和5％CO 2培养箱孵化器孵化为4小时，在37°C。 </li><li>孵化后，取出介质，添加250μL二甲基亚砜，涡2-5分钟，以溶解甲臜的复杂混合物。 </li><li>在2700 xĞCSM的离心5分钟，并确定在570作为参考使用630纳米的纳米测量上清液的吸光度。 </li><li>确定细胞数量与CSM的相对吸光度值获得从可行升序知名的关联。 </li></ol> 6。表征的ASC-CSM-嵌入式胶原蛋白凝胶 注:所有的程序，在室温下进行，除非另有说明。 <ol><li>混合ASC装CSM（含5毫克≈2×10 4细胞）1型从鼠尾腱胶原蛋白的提取，使用2列印氢氧化钠调节pH值至6.8后，根据伯恩斯坦8和fibrillate方法（7.5毫克/毫升）。 </l我&gt; <li>纤胶原蛋白ASC-CSM的混合物加入12孔板，孵育30分钟在5％CO 2培养箱孵化器在37°C。 </li><li>完成后房颤，在5％CO 2培养箱孵化至14天孵化，在37°C胶原蛋白ASC-CSM的凝胶</li><li>使用标准的显微镜技术观察从CSM的细胞到凝胶的释放和迁移。 </li></ol> 7。代表结果在本研究中，我们已经开发提供成胶原蛋白凝胶支架壳聚糖微球（CSM）的干细胞在体外战略。大小均匀（直径在175-225微米）和组成的多孔CSM的准备，并作为细胞载体（ 图2）。孵化与CSM的ASC，细胞附着在一个2×10 4 CSM的cells/5mg的浓度。这些细胞能在微蔓延，同时延长丝状伪足微多孔缝隙（ 图3）。一旦细胞加载的CSM与胶原凝胶混合，细胞立刻开始迁移到凝胶（ 图4）。 <img alt="表1" src="/files/ftp_upload/3624/3624table1.jpg" /> 表1。在干细胞输送系统使用壳聚糖和胶原蛋白的生物优势。 <img alt="图1" src="/files/ftp_upload/3624/3624fig1.jpg" /> 图1。示意图描绘两用的ASC-CSM装胶原支架的整体战略。 图2，3，4内的示意图，以帮助解释图像注释。 <img alt="图2" src="/files/ftp_upload/3624/3624fig2.jpg" /> 图2示意图描绘的干细胞接种于壳聚糖微球的过程。在proc的ESS涉及共培养与CSM在8升序 - 微米孔径的膜培养板插入。 24小时后，球从插入删除，并嵌入到生物材料的基体的准备。 <img alt="图3" src="/files/ftp_upload/3624/3624fig3.jpg" /> 图3。CSM的加载与ASC的形态特征。 A组描绘了一个ASC装的CSM光显微镜，而B组显示了同一领域的共聚焦荧光显微镜获得的图像叠加的观点。升序预装了钙黄绿素AM（绿色）。 C组描绘了一个卸载微球的SEM图像的图像，而面板D显示加载到微（星号）的细胞。 TEM图像显示在面板E卸载微球的截面。众多的孔隙和裂缝遍布微球。 F显示面板的连接并延伸到综合招聘考试的丝状伪足与细胞的横截面微球（箭头）恶习。原放大倍率:A和B = 70X，C = 500X，丁= 2000×，E及F = 2,500 X。 <img alt="图4" src="/files/ftp_upload/3624/3624fig4.jpg" /> 图4。迁移到三维胶原支架的CSM的ASC。板A和B细胞迁移从微球和胶原基质进入第3天（一个箭头）描绘的CSM。后12天，B组显示一个类似的文化。透射电子显微镜（TEM）影像描绘在C，D和E在C和D星号显示，一直与细胞迁移从微球（箭头）的横截面的微球。描述了更高的放大倍率，面板D面板E，并显示出细胞伪足的胶原纤维（插图）。原放大倍率:＆，B = 100X，C＆D = 6000 X;Ë= 20000，插图= 150,000 X。 <img alt="图5" src="/files/ftp_upload/3624/3624fig5.jpg" /> 图5。示意图描绘的CSM在再生医学和药物输送广阔的用途。

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没有竞争的金融利益存在。 免责声明所载的意见或主张是作者的私人意见，是不被理解为官方或反映国防部或美国政府部门的意见。作者是美国政府的雇员，以及作为其执行公务的一部分准备工作。所有的工作是由美国陆军医学研究和装备司令部的支持。这项研究是根据美国陆军医学研究和装备司令部机构审查委员会，并按照批准的协议，审查和批准了一项协议。

干细胞为基础的治疗的一个主要障碍是细胞运送到指定维修地区发展的有效方法。由于病人到病人的变异，组织类型，损伤的大小和深度;提供干细胞的方法，必须逐案的基础上确定。虽然嵌入矩阵内的干细胞，并提供他们的伤口，似乎是组织工程的下一个合乎逻辑的做法，一些技术上的障碍仍然存在。这包括嵌入细胞的能力附加到基体，并提供周围的生物相容性，可以附加在细胞增殖和失巢凋亡的9-10前，重新建立一个合适的微环境。这个过程需要细胞上调基质重塑机械，这个过程可能需要数天前完成所需的干细胞的增殖，迁移和分化的细胞过程。为了规避这些问题，我们Ĥ大道到壳聚糖微球的的预紧干细胞嵌入到一个合适的基质，修复受损组织之前开发出一种方法。利用我们独特的离子交联的方法来制备微球，该协议是能够准备&gt; 90％的微球，直径在175-225微米，从而产生良好的微型细胞的载体。更重要的是，交联后，微球保持正电，这是非常有利于细胞附着。这些微球的特性，使干细胞附着到CSM迅速。一旦连接，这些干细胞微球是稳定的，能够实现空间内支架的安排，如胶原蛋白凝胶，模仿受伤的组织。总之，这个协议提供了一种新的方法来加载ASC上的CSM和说明，对微球的细胞可以迁移，同时保留自己的干细胞表型。多孔CSM的描述提供了一个优秀的微升序附件，仍然允许细胞迁移到周围基质。 摘要: 关键步骤<ul><li>使用统一大小的微球直径的变化直接影响微球为每毫克细胞附着的表面积。 </li><li>均匀的微球床内插入的文化准备以最佳的细胞均匀附着在CSM。 </li><li>确保媒体的体积增加外以及细胞培养插入是高于内以及半月板。通常情况下，内，外体积比:1:2是合适的，因为这有利于细胞附着的微球。 </li><li>加载内支架微球均匀分布的细胞。如果ASC - CSM的分布过于密集，细胞不会从他们的微迁移。 </li><li> CSM和分发所需的数量bution密度取决于应用程序和受伤的组织面积。 </li></ul>限制<ul><li>此方法仅限于微球总面积。如果需要较高的细胞数，需要更多的球，以增加表面积。 </li><li>过程使细胞接种于预制微，不拟对细胞封装的目的。 </li><li>在这个过程中开发的壳聚糖微球可以支持锚地依赖性细胞，但没有其他的细胞类型。 </li><li>由于壳聚糖微球的研制使用0.5M的醋酸溶液，乳化油的混合物（豆油:正辛醇）和恢复使用有机溶剂，不积极治疗基易受上述条件。 </li><li> CSM的主要目的，以提供细胞/持续时间短（5-10天）的药物，由于其降解速度更快率，不同于那些旨在持续时间长的可用性。 </li></ul>可能的修改和多功能性（ 图5） <ul><li>微球的大小可能会修改，以改变总面积。例如，微球的大小可能会减少，增加的表面积/毫克氯磺化聚乙烯，这反过来又会增加总表面积和可以连接到微球的细胞数，每增加细胞微球负载。 </li><li>壳聚糖微球可作为载体，加载其他类型的细胞（内皮细胞，周细胞，成纤维细胞等）。 </li><li>细胞装领域（无论是单一或多种细胞类型）可以嵌入在特定地点的支架。这一战略将有助于在多细胞组织再生的组织工程支架的建设。 </li><li>除了提供细胞，球也可以被用来提供治疗药物（也就是说，生长因子，分化诱导，和/或抗生素）。这些药物微球内支架也可用于增强愈合和再生过程中结合细胞微球。 </li></ul>故障排除<ul><li> CSM的准备，如果发生在规模较大的变化，那么一些项目可以调整，以产生所需的CSM大小11。 <ul type="circle"><li>壳聚糖溶液的粘度。新鲜配制的股票是首选的解决方案，从批次，批次准备下保持恒定粘度</li><li>调整量的表面活性剂（跨度80）用于准备乳液。 </li><li>确保架空搅拌器的速度在整个过程中保持不变（速度可能会随电源波动</li><li>在搅拌传授大气可能造成动荡和大小的变化（这可以通过柯避免EP的液体石激起千层浪，在刚过的开销搅拌器叶片搅拌形成。 </li></ul></li><li>微球，以确保最佳细胞负荷，量热MTT法可以执行。这将给每毫克微球装入一个可行的细胞数目估计。 </li><li>防止悬浮在细胞培养基体积小如胶原凝胶内支架前混合细胞微球聚集。 </li></ul>

<table border="1" ><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>汉克斯BalancedSalt溶液（HBSS中） </td><td> Gibco公司</td><td> 14175 </td><td>耗材</td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH30071.03 </td><td>耗材</td></tr><tr><td>二型胶原酶</td><td> Sigma-Aldrich公司</td><td> C6685 </td><td>耗材</td></tr><tr><td> 70微米尼龙网过滤器</td><td> BD公司</td><td> 352350 </td><td>耗材</td></tr><tr><td> 100μm的尼龙网过滤器</td><td> BD公司</td><td> 352360 </td><td>耗材</td></tr><tr><td> MesenPRO生长介质系统</td><td> Invitrogen公司</td><td> 12746-012 </td><td>耗材</td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td>耗材</td></tr><tr><td>的T75组织培养瓶</td><td> BD公司</td><td> 137787 </td><td>耗材</td></tr><tr><td>壳聚糖</td><td> Sigma-Aldrich公司</td><td> 448869 </td><td>耗材</td></tr><tr><td>醋酸</td><td> Sigma-Aldrich公司</td><td> 320099 </td><td>耗材</td></tr><tr><td>正辛醇</td><td> Acros Organics公司</td><td> 150630025 </td><td>耗材</td></tr><tr><td>山梨醇单声道，油酸</td><td> Sigma-Aldrich公司</td><td> S6760 </td><td>耗材</td></tr><tr><td>氢氧化钾</td><td> Sigma-Aldrich公司</td><td> P1767 </td><td>耗材</td></tr><tr><td>丙酮</td><td> Fisher Scientific则</td><td> L - 4859 </td><td>耗材</td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司</td><td> 270741 </td><td>耗材</td></tr><tr><td>硝基苯酸</td><td> Sigma-Aldrich公司</td><td> P2297 </td><td>耗材</td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 320331 </td><td>耗材</td></tr><tr><td>乙醚</td><td> Sigma-Aldrich公司</td><td> 472-484 </td><td>耗材</td></tr><tr><td> 8微米的细胞培养板插入</td><td> BD公司</td><td> 353097 </td><td>耗材</td></tr><tr><td> 1.5毫升离心管</td><td>费舍尔</td><td> 05-408-129 </td><td>耗材</td></tr><tr><td> MTT法试剂</td><td> Invitrogen公司</td><td> M6494 </td><td>耗材</td></tr><tr><td>二甲基亚砜</td><td> Sigma-Aldrich公司</td><td> D8779 </td><td>耗材</td></tr><tr><td> qtracker细胞标记试剂盒（问跟踪655） </td><td>分子探针</td><td> Q2502PMP </td><td>耗材</td></tr><tr><td> 1型胶原</td><td> travigen </td><td> 3447-020-01 </td><td>耗材</td></tr><tr><td>氢氧化钠</td><td> Sigma-Aldrich公司</td><td> S8045 </td><td>耗材</td></tr><tr><td> 12孔细胞培养板</td><td> BD公司</td><td> 353043 </td><td>耗材</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5417R </td><td>设备</td></tr><tr><td>轨道摇床</td><td>新不伦瑞克省Scienctific </td><td> C24的</td><td>设备</tD&gt; </tr><tr><td>湿润的孵化器与空气，5％的CO 2 </td><td> Thermo Scientific的</td><td> 370型</td><td>设备</td></tr><tr><td>架空搅拌器</td><td>伊嘉</td><td> visc6000 </td><td>设备</td></tr><tr><td>磁力搅拌器</td><td>康宁</td><td> PC-210 </td><td>设备</td></tr><tr><td>真空干燥器</td><td> - </td><td> - </td><td>设备</td></tr><tr><td>粒度分析仪</td><td>马尔文</td><td> STP2000 Spraytec </td><td>设备</td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> isotemp210 </td><td>设备</TD&gt; </tr><tr><td>分光光度计</td><td>贝克曼</td><td>贝克曼DU800UV/Visible分光光度计</td><td>设备</td></tr><tr><td>涡旋</td><td> diagger </td><td> 3030A </td><td>设备</td></tr><tr><td>酶标仪</td><td>分子器件</td><td> SpectraMax M2的</td><td>设备</td></tr><tr><td>光/荧光显微镜</td><td>奥林巴斯</td><td> IX71 </td><td>设备</td></tr><tr><td>共聚焦显微镜</td><td>奥林巴斯</td><td> FV-500激光共聚焦显微镜</td><td>设备</td></tr><tr><td>扫描电子显微镜</td><td>卡尔·蔡司麦克风roImaging </td><td>狮子座435副总裁</td><td>设备</td></tr><tr><td>透射电子显微镜</td><td> JEOL </td><td> JEOL 1230 </td><td>设备</td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

多能干细胞已被证明是非常有用的在再生医学领域的1-3。然而，为了有效使用这些细胞的组织再生，变量的数目必须考虑。这些变量包括:植入网站的总体积和表面面积，该组织的力学性能和组织的微环境，其中包括血管的数量和细胞外基质的组成部分。因此，被用来传递这些细胞的材料必须与定义的化学成分的生物相容性，同时保持了机械强度，模仿宿主组织。这些材料还必须渗透到细胞附着和增殖提供了有利的微环境的氧气和营养物质。壳聚糖阳离子多糖，具有优良的生物相容性，可以很容易地化学改性，并具有较高的亲和力体内 MAC绑定romolecules 4-5。壳聚糖模仿外基质糖胺部分，使其能够正常细胞黏附，迁移和增殖的基质。在这项研究中，我们利用壳聚糖微球的形式提供基于胶原蛋白的三维支架6脂肪干细胞（ASC）。一个理想的细胞微球比确定培养时间和细胞密度达到最大数量的细胞可以加载。一旦升序接种到壳聚糖微球（CSM），它们被嵌入在胶原支架和可长时间保持文化。总之，这项研究提供了一种方法，准确地提供三维生物材料支架内的干细胞。

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

在目前的干细胞疗法的一个主要障碍是确定最有效的方法来提供这些宿主组织细胞。在这里，我们描述了一个基于壳聚糖的交付方法，该方法是有效的方法简单，同时让脂肪干细胞维持其多能。

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Nanyang Technological University

Research

JoVE Journal

Bioengineering

16.7K 次观看.  United States Army Institute of Surgical Research. 在目前的干细胞疗法的一个主要障碍是确定最有效的方法来提供这些宿主组织细胞。在这里，我们描述了一个基于壳聚糖的交付方法，该方法是有效的方法简单，同时让脂肪干细胞维持其多能。

视频: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...