我们感谢鼠资源研究中心，美国密苏里大学，哥伦比亚大学，密苏里州，提供GFP鼠; Neuralstem公司提供人类神经干细胞。这项工作是由退伍军人管理局，美国国立卫生研究院（NS09881），加拿大脊髓研究组织，该克雷格H.尼尔森基金会，伯纳德和安妮·斯皮策慈善基金的支持。

所有动物的协议是由弗吉尼亚州圣地亚哥机构动物护理和使用委员会（IACUC）批准。实验动物护理和安全指引，美国国立卫生研究院严格遵循。动物可以免费获得食物和水在整个研究和充分治疗减少疼痛和不适。 1，含生长因子鸡尾酒纤维蛋白组分的制备<ol><li>溶解25毫克大鼠纤维蛋白原在0.5ml PBS中，以得到50毫克/毫升2×原液（见材料表）。纤维蛋白原是难于溶解，并应置于37℃培养箱或水浴和动摇每隔5-10分钟1-2小时。 </li><li>溶解100 UN大鼠凝血酶在2ml的10mM无菌的CaCl 2，得到50 U / ml的2×储液。 </li><li>溶解在PBS中的生长因子以下列浓度，得到100微升储备溶液:1毫克/毫升:BDNF; NT-3（高浓度股票鞘内输注体内 <sup&gt; 19）; 200微克/毫升:GDNF;胰岛素样生长因子; bFGF的;表皮生长因子;血小板衍生生长因子;的aFGF;肝细胞生长因子（除用于细胞培养大约1000倍以上）。 </li><li>溶解25毫克MDL28170（蛋白酶抑制剂）的130毫升DMSO中，得到50mM的DMSO储备溶液，然后稀释50倍的PBS中，得到1mM储备溶液。 </li><li>混合100μl的各生长因子组分和100μlMDL28170（1mM储备溶液），以产生1,000微升生长因子鸡尾酒溶液（ 图1）。 </li><li>混合500μl的血纤维蛋白原2×原液用500μl生长因子鸡尾酒，得到25毫克/毫升纤维蛋白原工作溶液含有生长因子和分装成10或20微升贮存于-70℃。在-70℃下的溶液是稳定的，最多至12个月</li><li>同样，混合500微升的凝血酶倍原液用500μl生长因子鸡尾酒，得到25单位/毫升凝血酶工作液含有生长因子和分装成类似10或20微升卷进行存储一个吨-70℃长达12个月。 </li><li>上接枝的日子，将纤维蛋白原和含凝血酶生长因子鸡尾酒在冰上，直到与细胞混合。 </li></ol> 2，T3脊髓横断<ol><li>用可接受的方法麻醉成年雌性菲舍尔344大鼠或无胸腺裸鼠体内。主题深度麻醉，当反应到尾巴和爪子掐是完全不存在。注:我们通常用150-200克的大鼠菲舍尔或180-220克无胸腺大鼠和氯胺酮麻醉的鸡尾酒（2毫升/千克）（25毫克/毫升），甲苯噻嗪（1.3毫克/毫升）和乙酰丙嗪（0.25毫克/毫升）。 </li><li>适用眼膏，以防止干燥或眼睛的伤害，而动物在麻醉状态下。 </li><li>刮胡子的上胸面积和清洁与优碘皮肤。 </li><li>切使用＃15刀片在皮肤和肌肉，用外科手术牵开器暴露T3椎体，并在T3椎骨执行椎板使用咬骨钳，露出的T3 / 4脊髓。 </li><li>做一个LONgitudinal正中切口在使用＃11刀片大约2毫米长的硬脑膜。 </li><li>将虹膜切除剪硬脑膜下切开右外侧整个脊髓。使在同一侧的另一个切1.5毫米更多cadually。 </li><li>两切段用钝器23号针头连接到中等强度之间的真空吸脊髓组织。使用可视化验证，以确保完全横断腹和横向。这将完成一个侧面半切。 </li><li>为了延长病变为全宽脊髓横断，将镜切口进入左hemicord。试图离开硬脑膜完整，比先切等，嫁接时，两个星期后，保留该公司移植物/纤维蛋白基质中的病变部位。 </li><li>缝合肌肉，应用抗生素粉，主食皮肤。 </li><li>注入乳酸林格氏（3毫升）中，Bannamine（2.5-5毫克/千克）和氨苄青霉素（80-100毫克/千克）手术和脉后，立即（见材料表）ntain大鼠在一个温暖的孵化器，直到体温重新建立。 </li><li>排空膀胱，并每天两次约两个星期注射林格氏和氨苄西林溶液直至建立反射性膀胱排空（ 图1B）。 </li></ol> 3。的新鲜分离胚胎一天准备14脊髓神经干细胞<ol><li>获得近交转基因绿色荧光蛋白F344大鼠（F344-的Tg（UBC-EGFP）F455Rrrc）从大鼠资源研究中心，美国密苏里大学。 </li><li>注射40微克脱-甘氨酸10，[D-丙氨酸6]-促黄体生成激素释放激素乙基酰胺（LHRH）2后，在PBS中稀释，在200的浓度微克/毫升腹膜内（IP）的每成熟雌性大鼠（8周或更老） -3小时光照诱导的胚胎收集的同步。 </li><li>交配成熟的绿色荧光蛋白纯合或杂雄性大鼠过夜注射后4天。 </li><li>收集胚胎在几天13.5-14.5交媾（PC）在冷的HBSS缓冲液，检查下一个"NightSea"手电筒和过滤眼镜（BLS1 - 蓝星手电筒和型号VG1屏障过滤器）GFP表达或荧光显微镜。 </li><li>从每个GFP阳性胎儿解剖脊髓出无菌，取出覆脑膜及附加脊神经节与虹膜切除术剪刀和细珠宝商镊子。 </li><li>收集解剖脊髓中，加入2ml冷的HBSS缓冲液在15毫升cornical管并保持在冰上。 </li><li>按照参考第20到分离解剖脊髓: <ol><li>简单地说，加2毫升0.25％胰蛋白酶-EDTA含有解剖脊髓管（线数可以一起消化，1线可以产生足够的细胞为一个主题的移植物）和消化，在37℃下10-12分钟。 </li><li>停止胰蛋白酶反应通过加入含有10％FBS的管和离心组织于2500 rpm离心2分钟的10ml DMEM。 </li><li>悬浮组织在2毫升Neurobasal培养基合作ntaining 2％B27，以及使用越来越小的火抛光的巴斯德吸管磨碎的脊髓组织。 </li><li>离心将细胞以2,500 rpm离心2分钟，再悬浮于2ml的Neurobasal培养基中含有B27，并用40μm的细胞过滤器过滤器过滤细胞。 </li></ol></li><li>计数细胞，用血细胞计数和分裂细胞同样分为两个Eppendorf管中。 </li><li>离心机的细胞，彻底消除上清液（1-2分钟都必须撤出所有上清液用低真空小费），所以按剩余上清细胞再悬浮后，生长因子鸡尾酒将不会被摊薄。 </li><li>重悬细胞的每一半中含有的生长因子，分别以250,000细胞/微升的浓度（细胞沉淀计数大约⅓最终体积）的纤维蛋白原或凝血酶的工作溶液，并放置在冰上事先移植（ 图1A）。请注意，当与细胞混合纤维蛋白原可能自发地胶前s的凝血酶在病变/移植部位相结合;避免过早胶凝，立刻体内细胞移植前混合细胞中纤维蛋白原。 </li></ol> 4，移植<ol><li>两个星期后脊髓横断Reexpose的损毁T3 / 4脊髓。 </li><li> 5微升纤维蛋白原细胞和5微升凝血酶的细胞可以直接注射到九分，通过密封的疤痕组织和硬脑膜完整病灶部位，而无需重新打开病灶部位。 </li><li>使用27号针头以创建9个孔的疤痕组织和完整硬脑膜1-2周后的病变的表面上:在中心3（一个在中间点和两个支管，并间隔0.5毫米分开），并在这两个延髓3和在尾部接口（大约0.5mm或延髓尾端向病灶中心）3。 </li><li>然后纤维蛋白原细胞溶液体积的一半注入病灶部位和四分之一体积的三个核心点到3点吻端接口和AQ的uarter量为3分尾接口。 </li><li>然后，立即注入throbmbin细胞进入相同的斑点。血纤维蛋白原的细胞和病灶部位内凝血酶细胞的混合物会形成纤维蛋白凝胶，以保持细胞到损伤位点和生长因子鸡尾酒将支持细胞的生存。细胞使用的是连接到一个PicoSpritzer（通用阀门公司）拉玻璃移液管注入。 </li><li>移植培养的人神经干细胞21在与上述相同的程序，利用从人的试剂制成的纤维蛋白和生长因子鸡尾酒。 </li></ol>

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我们什么都没有透露。

一个用于NSC移植在脊髓损伤的主要障碍是生存于病灶中心不佳。在病灶部位的任何间隙或腔体有可能减少或削弱棘上轴突和下方的伤害分离的脊髓节段之间的功能神经元中继的形成。另外，移植的神经干细胞存活不佳可能影响其与宿主组织整合，并因此减少移植神经元与神经元的主机的连通性。可能的机制来解释细胞的损失包括急性脊髓损伤产生的细胞毒性环境中，主机脊髓继发变性，受伤的区?...

脊髓损伤（SCI）往往损害不仅白质束携带的信号和从脑，而且中央灰质，引起的interneurons和运动神经元的节段性损失。脊髓损伤的后果是两个马达和病变以下感觉功能的丧失。不幸的是，成人中枢神经系统（CNS）不自发地再生，从而产生永久功能缺损1。因此，重建受伤的成年脊髓和改进运动，感觉和植物神经功能是脊髓损伤研究的一个重要目标。神经干细胞（NS​​Cs），无论是从胚胎或成年中枢神经系统中直接分离，是引人注目的候选细胞，以取代失去的神经元和神经胶质细胞。此外，这些细胞具有形成新的功能性继电器跨越病变部位2,3恢复轴突传导的潜力。 迄今为止，还没有解剖的详细阐释，electrophysiological和神经元中继形成了神经干细胞移植的重度脊髓损伤后行为的影响。有几个原因:第一，移植的神经干细胞或胎儿中枢神经系统组织中存活不佳时，嫁接到大病灶腔。以往的研究表明大量的早期细胞脱落，留下大空囊状病灶腔4,5。在一些研究中移植的细胞随后会分裂，并填写病灶腔4,5，但几天到几周的延迟之后这可能会发生，以及随后病灶部位填充可能不完整或一致的。其次，高效的跟踪系统，可提供对细胞存活，分化/成熟，并移植神经干细胞生长的声音数据缺乏。大多数早期的研究采用顺行和逆行标记来跟踪从移植2,3轴突的预测。然而，这些技术只是部分，往往从移植细胞所产生的标记不清轴突预测及示踪方法都是主观吨引起的染料泄漏以外植入的细胞的工件。其他组的人使用特定的神经元标记物来标记人类胎儿神经干细胞到受伤的啮齿类动物脊髓5,6移 ​​植后轴突的预测。然而，在这些研究中， 移植并没有一贯良好的生存。最近，病毒递送的GFP报告基因的用于标记培养的神经干细胞7,8。但是，GFP的表达常常不一致，并且可以下调7。最近，利用转基因供体小鼠或大鼠稳定表达报告基因，绿色荧光蛋白，或人胎盘碱性磷酸酶，已显着提高体内 9,11移植的神经干细胞/祖细胞的跟踪。第三，一些研究表明，无论从胚胎或成年中枢神经系统导出体外培养的大鼠神经干细胞完全分化成神经胶质谱系当移植到完整的或受伤的成年脊髓心病的环境ð7,12,13，尽管事实是这些神经干细胞能够分化成神经元和神经胶质细胞在体外的，这表明局部环境可能要求干细胞的命运。可选地，培养的神经干细胞，尤其是那些从成年CNS衍生的，可具有固有defaulty属性分化为体内 13胶质谱系。 因为上面所讨论的限制，我们小组最近开发出一种新的协议，以提高胚胎NSC跟踪，生存，并在严重受伤的成年脊髓分化/成熟。简单地说，我们开始与表达绿色荧光蛋白报告基因在体内移植后14支撑GFP表达一个稳定的转基因菲舍尔344大鼠自交系。接下来，我们使用新鲜分离的神经干细胞从胚胎第14天菲舍尔344脊髓，发展阶段，其保存以产生神经元和神经胶质细胞的潜力。最后，我们的嵌入式新鲜分离的神经干细胞成含增长纤维蛋白基质因素15-17保留了细胞和均匀的大病灶腔内散发，旨在支持移植细胞的存活，分化和整合。移植物置入的T3完全横断，脊髓损伤后两星期网站。这些移植细胞始终充满完全横断网站和分化成神经元丰富的扩展大量的轴突到宿主脊髓长距离18。类似的结果用培养的人神经干细胞移植到免疫缺陷大鼠18获得的。

<table><tbody><tr><td>Fibrinogen (rat)</td><td>Sigma</td><td>F6755-25MG</td><td>2 hr at 37 &amp;deg;C to dissovle&lt;br/&gt; Stock Concentration: 50 mg/ml&lt;br/&gt; Final Concentration: 25 mg/ml</td></tr><tr><td>Thrombin (rat)</td><td>Sigma</td><td>T5772-100UN</td><td>Dissovle in 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; Stock Concentration: 50 U/ml&lt;br/&gt; Final Concentration: 25 mg/ml</td></tr><tr><td>bFGF (human)</td><td>Sigma</td><td>F0291 (25 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>EGF (murine)</td><td>Sigma</td><td>E1257 (0.1 mg)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>BDNF (human)</td><td>Peprotech</td><td>452-02 (1 mg)</td><td>Stock Concentration: 1,000 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 50 ng/&amp;mu;l</td></tr><tr><td>NT-3 (human)</td><td>Peprotech</td><td>452-03 (1 mg)</td><td>Stock Concentration: 1,000 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 50 ng/&amp;mu;l</td></tr><tr><td>GDNF (rat)</td><td>Sigma</td><td>G1401 (10 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>IGF-1 (mouse)</td><td>Sigma</td><td>I8779 (50 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>aFGF (human)</td><td>Sigma</td><td>F5542 (25 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l </td></tr><tr><td>PDGF-AA (human)</td><td>Sigma</td><td>P3076 (10 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>HGF (human)</td><td>Sigma</td><td>H9661 (5 &amp;#956;g)</td><td>Stock Concentration: 200 ng/&amp;mu;l&lt;br/&gt; Final Concentration: 10 ng/&amp;mu;l</td></tr><tr><td>MDL28170</td><td>Sigma</td><td>M6690 (25 mg)</td><td>Stock in DMSO&lt;br/&gt; Stock Concentration: 1 mM&lt;br/&gt; Final Concentration: 50 &amp;mu;M</td></tr><tr><td>PBS</td><td>Millipore</td><td>BSS-1005-B</td><td>Stock Concentration: 1x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Ketamine</td><td>Putney</td><td>26637-411-01</td><td>40-80 mg/kg&lt;br/&gt; Stock Concentration: 100 mg/ml&lt;br/&gt; Final Concentration: 25 mg/ml</td></tr><tr><td>Xylazine</td><td>Lloyd</td><td>0410,</td><td>2.5-8 mg/ml&lt;br/&gt; Stock Concentration: 100 mg/ml&lt;br/&gt; Final Concentration: 5.8 mg/ml</td></tr><tr><td>Acepromazine</td><td>Butler</td><td>003845</td><td>0.5-4 mg/ml&lt;br/&gt; Stock Concentration: 10 mg/ml&lt;br/&gt; Final Concentration: 0.25 mg/ml</td></tr><tr><td>Betadine</td><td>Healthpets</td><td>BET16OZ</td><td></td></tr><tr><td>Ringers</td><td>Abbott</td><td>04860-04-10</td><td>2-3 ml/inj</td></tr><tr><td>Banamine</td><td>Schering-Plough</td><td>0061-0851-03</td><td>2.5-5 mg/kg&lt;br/&gt; Stock Concentration: 50 mg/ml&lt;br/&gt; Final Concentration: 0.5 mg/ml</td></tr><tr><td>Ampicillin</td><td>Sandoz</td><td>0781-3404-85</td><td>80-100 mg/kg&lt;br/&gt;Final Concentration: 50 mg/ml</td></tr><tr><td>LH-RH</td><td>Sigma</td><td>L4513</td><td>200 &amp;mu;g/kg&lt;br/&gt; Final Concentration: 200 &amp;mu;g/ml</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-096</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200056</td><td>Stock Concentration: 0.25 %&lt;br/&gt; Final Concentration: 0.125 %</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995073</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000044</td><td>Stock Concentration: 100 %&lt;br/&gt; Final Concentration: 10 %</td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td>Stock Concentration: 100x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td colspan="4">Note, use human reagents for grafts of human NSCs</td></tr></tbody></table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

神经干细胞（NS​​Cs）能自我更新和分化成神经元和神经胶质细胞。移植的神经干细胞可以脊髓损伤（SCI）后替代丢失的神经元和神经胶质细胞，并能形成功能性继电器重新连接的上方和下方的病变脊髓节段。以前的研究接枝神经干细胞已经被不完全移植物存活的脊髓损伤腔内的限制。另外，接枝细胞存活，分化和扩展过程跟踪没有被优化。最后，在以往的研究，培养的大鼠神经干细胞通常报告分化成神经胶质细胞在移植到损伤脊髓，而不是神经元，除非命运被驱赶到一个特定的细胞类型。为了解决这些问题，我们开发了新的方法，以提高神经干细胞的存活，整合和分化，甚至严重的脊髓损伤部位。神经干细胞是新鲜从表达绿色佛罗里达州一个稳定的转基因菲舍尔344大鼠行胚胎第14天脊髓（E14）隔离uorescent蛋白（GFP），并嵌入到含有生长因子的纤维蛋白基质;该制剂的目的是保留移植细胞在病灶腔和支持细胞的生存。神经干细胞在纤维蛋白/生长因子鸡尾酒植入两周后胸3级（T3）完全性脊髓横断，从而避免炎症的高峰期。导致移植物完全充满病灶腔和分化为两个神经元，轴突延伸到宿主脊髓过相当长的距离，和神经胶质细胞。培养的人神经干细胞表达绿色荧光蛋白移植物导致类似的结果。因此，方法被定义为提高神经干细胞移植，存活和体内研究结果的分析。

的GFP免疫组织化学标记表明接枝的大鼠神经干细胞重新悬浮于磷酸盐缓冲盐水（PBS）（缺乏纤维蛋白基质和生长因子），在T3切断现场不良存活，只有附着在病灶/主机余量，留下大部分病灶部位的空的无移植的细胞（ 图2A）。神经干细胞的存活改善时共接枝与单独的纤维蛋白基质，但接枝并没有完全填满一个大腔病变（数据未显示）。因此，我们嵌入到神经干细胞含有生长因子鸡尾?...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

含有生长因子的纤维蛋白基质被用来留住嫁接神经干细胞分化为完全脊髓横断网站。移植细胞完全充满病灶腔和分化为多种神经细胞类型，包括神经元轴突延伸到宿主脊髓长距离。

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K 次观看.  Veterans Administration Medical Center, San Diego. 含有生长因子的纤维蛋白基质被用来留住嫁接神经干细胞分化为完全脊髓横断网站。移植细胞完全充满病灶腔和分化为多种神经细胞类型，包括神经元轴突延伸到宿主脊髓长距离。 

视频: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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

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

制备纤维蛋白三维支架干细胞培养应用

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

科研

生物工程

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

医学

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

实验策略急，慢性损伤后桥的损伤脊髓组织大差距

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

神经科学

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

导电支架调节和传递干细胞

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

氟 TrFE 导管内许旺细胞移植桥断大鼠脊髓残肢促进轴突再生跨越间隙

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...