我们要感谢美国国立卫生研究院和美国国家神经疾病和中风研究所的资助（R01NS079691）。

实验是根据杜克大学和加州大学洛杉矶分校的IUCAC进行的。本研究使用8至12周龄雄性C57Bl / 6J小鼠。这些动物被安置在受控温度（22±2°C）下，具有12小时的光暗循环期，并随意获得颗粒状食物和水。镇痛和镇静方案被描述为由IUCAC批准，但可能与其他实验室使用的方案不同。
如果动物经历过度不安，发声（表明无法控制的疼痛），自我创伤，行动能力下降或受损，皮肤感染的身体体征，食欲不振和/或体重减轻超过10%-15%，作为生存手术的结果，可能会过早安乐死。注射凝胶没有预期的不良影响，因为它是由大脑中天然存在的聚合物组成的。应每天监测动物，包括周末和节假日，并每隔一天重新称重一次。我们实验室和其他人的研究发现，在这些小的皮质或皮质下中风后，小鼠在梳理，体重减轻或戒断或慢性压力的体征/症状方面没有缺陷。如果动物表现出任何局部头部伤口感染的迹象，则应对它们实施安乐死。还应监测动物梳理不良、伤口状况和动物之间打架的证据（背部伤口）。如果有争斗或伤口裂开的证据，动物在接触兽医后应首先接受治疗。这通常涉及水中的抗生素和/或局部局部抗生素的应用。如果这些措施无法治愈，动物应该被安乐死。
1. 准备材料和仪器
<ol><li>在手术前准备光血栓染料。称出15毫克孟加拉玫瑰到2毫升微量离心管中。 注意:孟加拉玫瑰会造成严重的眼睛损伤/刺激。<ol>
<li>将孟加拉玫瑰溶解在1.5毫升无菌过滤的1x PBS中，使工作浓度达到10毫克/毫升。</li>
		<li>涡旋管子，直到看不到团块，表明染料已完全溶解，然后覆盖在箔片中直至进一步使用。 注意:所需的染料量取决于小鼠的数量。将染料以10μL / g的浓度在腹膜内IP内注射，例如，20g小鼠为200μL。</li>
	</ol>
</li>
	<li>打开加热垫，在麻醉（37°C）期间保持小鼠体温。</li>
	<li>准备高压灭菌剪刀，镊子，棉花，兽医眼药膏，手术胶水或缝合材料，蓝色或黑色手术标志物，无菌酒精和β碘湿巾。准备带有无菌钻头的骨钻头。</li>
	<li>将微球灭菌打开至160°C，以允许在小鼠手术之间对器械进行灭菌。留出一个50 mL锥形管无菌盐水或1x PBS溶液，供以后用于维持水合的操作区域。</li>
	<li>将激光连接到立体定位设备。戴上激光安全护目镜，测量激光的功率（mW）。 注意:在手术过程中，只要打开激光，就使用激光安全护目镜。<ol>
<li>按照激光制造说明调整激光控制台上的电流，直到激光功率输出读数为10 mW，并将电流设置为主屏幕控制台上的预设。然后再次调整电流以建立另一个预设，其中激光输出功率读数为40 mW。现在关闭激光，直到进一步使用。 注:10 mW将在使用前用于定向激光器。</li>
	</ol>
</li>
	<li>手术后为小鼠准备一个干净的笼子。然后，将小鼠放入异氟醚浓度为4%的诱导室中麻醉它，直到自发运动停止，其呼吸达到缓慢，稳定的速率。</li>
	<li>一旦睡着，称量鼠标以确定注射多少玫瑰孟加拉染料。然后，将鼠标转移到立体束装置中，维持异氟醚浓度为1.5%。 注意:增加异氟醚浓度可以扩张血管并防止小鼠之间的足够闭塞或大小一致的中风。</li>
	<li>将兽医眼药膏涂抹在双眼上。 注意:在整个过程中注意呼吸和体温，并偶尔捏住脚趾，以确保鼠标感觉不到任何东西。</li>
</ol>2. 光血栓性卒中模型9
<ol><li>剃掉老鼠头上的皮毛，然后用酒精擦拭布和β-碘擦拭物无菌地准备该区域。重复三次。 注意:如有必要，最后使用额外的酒精擦拭巾来清除所有β碘，因为它会引起皮肤刺激，导致小鼠在手术后头骨瘙痒。</li>
	<li>使用小手术剪刀，在耳朵到眼睛之间做一个横向切口，约1-2厘米。为此，用镊子向上拉皮肤以形成帐篷，向下切割一次，然后将剪刀插入开口并形成连续的中线切口。</li>
	<li>分开皮肤，用镊子轻轻按压顶骨以定位胸骨。使用手术标记用点标记胸膜。 注意:骨碎片运动突出显示顶骨的额边缘。胸膜是额骨和顶骨碎片相遇的中心点（图1A）。</li>
	<li>戴上激光安全护目镜，将激光移到鼠标的头骨上，将立体定位装置固定在90°角。选择10 mW预设并打开激光器。<ol>
<li>调整激光的 x 轴和 y 轴，直到它直接超过边角。重置数字显示控制台上的 x 和 y，然后将激光器移动到边距左侧 1.8 mm 处。现在，将激光尽可能靠近颅骨，不要让它接触颅骨。</li>
		<li>将激光移动到边角左侧 2.2 mm 处，以确保它可以在没有任何障碍物的情况下移动。关闭激光。</li>
	</ol></li>
	<li>使用一次性29 G针头腹腔注射适量的Rose Bengal染料（IP）。立即启动定时器7分钟，让染料全身循环。选择预设的 40 mW，无需打开激光器。 注意:一旦熟悉该过程，可以在小鼠无菌制备之前注射染料，并在7分钟结束之前完成以节省时间。</li>
	<li>当7分钟计时器关闭时，戴上激光安全护目镜，打开激光（40 mW），并设置10分钟计时器。<ol>
<li>10分钟后，将激光移动到边角左侧2.2毫米处，不将其关闭，并设置另一个10分钟计时器。</li>
		<li>第二个10分钟计时器关闭后，将激光器切换回10 mW，并将激光器移动到边角左侧2.0 mm处。用手术标记标记标记此位置。然后关闭激光。</li>
		<li>提起激光并从90°角度解锁，以便将其移离鼠标。此时，如果手术区域干燥，请用棉涂抹无菌盐水或PBS。</li>
	</ol></li>
	<li>在垂直于颅骨表面的2.0毫米标记处进行小爆发钻孔。 注意:小心慢慢钻孔，以避免钻孔太深并击中大脑。一旦钻头穿过头骨，阻力就会略有下降/变化，钻孔可能会导致出血。<ol>
<li>使用棉花吸收多余的血液。钻孔后，通常会从颅骨的切口动脉中看到一些血液 - 过多的血液意味着钻头会击中大脑。如果发生这种情况，请记录鼠标标识符，然后继续。</li>
	</ol></li>
	<li>在鼠标旁边放一个小擦拭布。使用镊子，将皮肤拉近以准备粘合。 注意:缝合可以在这里完成。缝合通常只需要3次缝合。<ol>
<li>使用镊子，抓住皮肤的两侧，将它们拉在一起并向上拉。在涂抹到鼠标之前，在擦拭巾的尖端末端轻拍任何大滴的手术胶水。</li>
		<li>小心翼翼地涂抹手术胶水，并用镊子将皮肤固定在一起10秒。继续，直到没有可见的开口。 注意:胶水很快出来 - 不要挤压瓶子。避免将皮肤粘在颅骨上或将胶水粘在眼睛里。</li>
	</ol></li>
	<li>粘合或缝合后，将小鼠放入新笼子中以从麻醉中恢复。动物需要5-10分钟才能恢复，并且有助于将笼子的一半放在加热垫上。</li>
</ol>3. 假中风手术
<ol><li>执行与上述第2节中描述的操作相同的所有程序，除了注射1x PBS或盐水而不是Rose Bengal染料。</li>
</ol>4. 注射水凝胶或其他生物材料
<ol><li>在用户定义的时间注射生物材料。在该实验中，注射在中风后5至7天之间进行。注射2 - 6μL水凝胶（用户定义）。<ol>
<li>打开加热垫，在麻醉（37°C）期间保持小鼠体温。</li>
		<li>准备高压灭菌剪刀，镊子，棉花，兽医眼膏，手术胶水或缝合材料，以及无菌酒精和β碘湿巾。准备带有无菌钻头的骨钻头和带有金属30 G针头的25μL玻璃汉密尔顿注射器。</li>
		<li>将磁珠灭菌打开至160°C，以允许对小鼠之间的器械进行灭菌。</li>
		<li>将喷射泵连接到立体定位装置。将流速设置为 1 μL/min，将体积设置为 4 μL。</li>
		<li>手术后为小鼠准备一个干净的笼子。</li>
	</ol></li>
	<li>将小鼠放入异氟醚浓度为4%的诱导室中麻醉它，直到自发运动停止，其呼吸达到缓慢，稳定的速率。<ol>
<li>一旦睡着，将小鼠转移到立体定位装置中，维持异氟醚浓度为1.5%。</li>
		<li>将兽医眼药膏涂抹在双眼上。</li>
		<li>剃掉任何可能已经重新长出小鼠头部的皮毛，并使用酒精擦拭布然后使用β-碘擦拭物无菌地准备该区域。重复三次。 注意:如有必要，最后使用额外的酒精湿巾来清除所有的β碘，因为它会引起皮肤刺激并导致小鼠在手术后头骨瘙痒。</li>
	</ol></li>
	<li>使用小剪刀和/或镊子，重新打开大脑上方的皮肤。使用含无菌盐水或PBS的棉花清洁颅骨上的任何碎屑。一个白黄色的圆圈（笔程， 图1B）和钻头上的毛刺孔将可见。如果死组织不可见，则可能没有发生中风。如果毛刺孔未在冲程上方居中，请重新钻孔以使其居中。</li>
	<li>将注射泵定向到鼠标上方，并锁定在90°。在回装材料之前，用无菌盐水或PBS溶液清洁注射器。<ol>
<li>清洁后，拆卸玻璃注射器，使没有针头。使用25μL正置换移液器将注射器与10μL水凝胶（或其他生物材料，有或没有细胞）回加载注射器。</li>
		<li>使用注射器柱塞，将凝胶一直推到注射器的前面。然后，重新组装注射器并继续推动，直到凝胶从针头中明显渗出。</li>
	</ol></li>
	<li>将注射器放到泵上。可能需要调整泵的背面，以便注射器适合。为此，请将流速调整为30μL/min，根据需要移动方向选择 "撤液 "或" 注射"， 然后按 "开始"。当泵的背面位于正确的位置时，点击 停止 。<ol>
<li>拧紧泵的背面，使注射器牢固。如果之前进行了调整，请确保将流速设置回1μL / min并设置为注射。</li>
	</ol></li>
	<li>沿 x 和 y 方向移动泵，使其在孔上定向。沿 z 方向向下移动泵，直到注射器的针头接触到孔的顶部。</li>
	<li>重置数字显示控制台上的 z。然后，将注射器沿z方向向下移动0.750 mm。如果注射器弯曲或有阻力，则颅骨愈合或未完全钻孔，需要重新钻孔。</li>
	<li>按泵控制台上的 "开始" 键，以 1 μL/min 的速率注入 4 - 6 μL 水凝胶。</li>
	<li>当泵停止进样时，设置一个5分钟的计时器，以使凝胶开始交联。</li>
	<li>5分钟后，通过转动z nob慢慢拉起注射器。观察是否有任何材料意外拉出或从孔中泄漏。解锁泵，当针头离头骨足够远时，将其移离鼠标。</li>
	<li>使用镊子，手术胶水或缝合线，关闭头部上方的皮肤。将鼠标放入干净的笼子中，观察其恢复情况。小鼠从麻醉中恢复大约需要5-10分钟。</li>
</ol>5. 灌注和样本采集
<ol><li>使用异氟醚麻醉小鼠。</li>
	<li>将动物固定在仰卧位，并用正中切口打开腹腔。（可选）夹住降腹主动脉以使用较少的固定剂和PBS。</li>
	<li>切开隔膜，向上移动肋骨的两侧以打开胸腔。将灌注插管插入左心室，并在右心房切开一个开口。开始缓慢灌注 10-20 mL PBS 或直到液体半透明。</li>
	<li>清除后，换用 4% PFA，再加 10 – 20 mL。松开双臂的销钉允许它们在PFA注入时收缩。一旦他们停止移动，再等30秒。</li>
	<li>斩首动物并拉回皮肤以暴露头骨。使用钝而细的镊子，小心地取出头骨的碎片以暴露大脑。移除卒中部位上方的颅骨时需要特别小心。这里可以使用小型手术剪刀来帮助切除颅骨。最大的挑战是凝胶粘在头骨上，并在头骨被移除时从大脑中出来。</li>
	<li>一旦大脑脱离，放入10-15毫升4%PFA过夜（不超过24小时）。</li>
	<li>第二天将大脑从PFA移动到30%蔗糖。一旦大脑沉入底部（约2-3天），大脑就准备好进行冷冻切除。此时也可以将其保留用于存储。</li>
</ol>6. 冷冻切除和染色
<ol><li>将大脑从蔗糖中取出，放在载玻片上。用剃须刀片切掉大脑后部的一小部分，形成一个平坦的部分，可以安装在卡盘上。<ol>
<li>将一小块最佳切割温度化合物（OCT）放在卡盘上，然后将大脑平放一面朝下，放在OCT中的卡盘上。将此标本放在干冰上，直到冷冻或冷冻块上的低温恒温器中。 注意:可以添加OCT以完全包围大脑，以帮助防止材料在切片过程中与大脑分离。</li>
	</ol>
</li>
	<li>在-20°C的低温恒温器上，在10个明胶包被的载玻片上以30μm厚的部分连续切割大脑。切片时要小心，确保不会丢失水凝胶。如果这部分很困难，请在凝胶上方的大脑顶部放置一些OCT（图3）。储存在-80°C直至进一步使用。</li>
	<li>将载玻片从-80°C中取出，放在染色盘上以使其干燥。尚未冷冻但刚刚切片的载玻片也可以立即染色。</li>
	<li>使用疏水笔，在幻灯片上的脑切片周围画一个圆圈。干燥后，用1x过滤的PBS重新水润脑切片，并在室温下置于摇摇杯上15分钟。每张载玻片需要约1 mL缓冲液，具有9-12个脑切片。</li>
	<li>在室温下用1x过滤的PBS洗涤载玻片5分钟，重复三次。</li>
	<li>在室温下用10%驴血清在0.01%PBS-Triton X中在肚皮舞者身上阻断载玻片30分钟至一小时。每张载玻片大约需要200μL。</li>
	<li>将一抗与0.01%PBS-Triton X在4°C的摇摇杯上孵育10%驴血清中过夜。首先测试一抗以确定正确的浓度。这里使用了GFAP（1:400），Iba1（1:250），Glut1（1:500）（图4）。</li>
	<li>第二天用1x过滤的PBS洗涤载玻片，在室温下洗涤5分钟，重复三次。</li>
	<li>在室温下，将二抗与10%驴血清在0.01%PBS-Triton X和DAPI中孵育2小时。1:1，000 用于所有辅助数据库和 DAPI。</li>
	<li>用1x过滤的PBS洗涤载玻片5分钟。</li>
	<li>让切片在室温下在黑暗中干燥。这可能需要20分钟到4小时;将载玻片放入干燥器中可以加快该过程。</li>
	<li>脱水载玻片在升压浓度的酒精（每个溶液1分钟）为50%，70%，95%，100%和100%。</li>
	<li>在第一次二甲苯浴中脱脂1分钟，然后在第二次二甲苯浴中脱脂5分钟。</li>
	<li>快速将载玻片逐个取出，并在大脑切片被二甲苯润湿时加入安装介质。在顶部快速添加玻璃盖玻片。使用分别用于成像的载玻片和显微镜所需的正确长度和厚度的盖玻片。</li>
	<li>通过用移液器吸头从载玻片中心向外运动轻轻按压盖板下的任何气泡，摆脱盖玻片下的任何气泡。让DPX在室温下干燥4小时，在黑暗中过夜。</li>
	<li>使用剃须刀片刮掉溅到载玻片上的任何干燥安装介质。用镜头清洁剂擦拭幻灯片。载玻片现在可以用荧光显微镜成像。 注意:最好将载玻片存放在黑暗中或4°C下，直到成像完成。</li>
</ol>

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作者宣称TS是Tempo Therapeutics的创始人，该公司旨在将MAP水凝胶商业化。

在这里，我们展示了一个易于重复的，微创的，永久性的中风模型，并描述了如何在中风后五天将生物材料注射到梗死中。使用光血栓染料Rose Bengal和连接到立体束装置的520nm准直激光器使我们能够以更高的精度将行程定位在小鼠的运动皮层。卒中五天后，在照射中心用眼睛可以看到梗死的位置，距胸侧2.0mm。然后使用注射泵将水凝胶准确地注射到行程核心中。该模型中手术期间和中风后的死亡率?...

中风是美国残疾的主要原因和第五大死因1。大约87%的卒中是缺血性的，而其余13%中的大多数是出血性2。缺血性卒中被定义为动脉中流向周围组织的血流阻塞。这种闭塞导致缺氧和随后的坏死，这通常导致幸存患者的永久性残疾。虽然中风3的死亡率有所下降，但预计到2030年其患病率将增加到340万人4。残疾幸存者的增加以及随之而来的经济负担导致了对中风研究的推动，该研究的重点是神经修复机制。中风后有一个炎症期，导致疤痕的形成，阻止坏死区域扩张。坏死核心周围的区域被称为"围死期梗死"，并且有强有力的证据表明，该区域的可塑性（包括增加的血管生成，神经发生和轴突发芽）与动物模型和人类观察到的恢复直接相关5。由于没有体外模型可以正确复制中风后的复杂相互作用，因此动物模型对于中风研究至关重要。
有几种体内模型可用于产生缺血性中风。小鼠最常用的模型之一是通过远端或近端（通过动脉内丝）闭塞的脑中动脉闭塞或MCAo。近端模型，也称为细丝MCAo（fMCAo），通常会导致大的缺血性卒中，包括大脑半球的5%至50%，这取决于因素的数量6。在这些模型中，缝合线或细丝从颈内动脉推进到大脑中动脉（MCA）的底部，并保持在适当的位置一段时间。这种闭塞方法可以是暂时的或永久性的，产生以纹状体为中心的梗死，并且可能涉及也可能不涉及覆盖的皮层6。由此产生的笔画大小变化很大，并且需要成像技术（例如激光多普勒）来确认每个小鼠中该程序的有效性。持续超过30分钟的动脉内或腔内灯丝闭塞在尺寸范围的较大末端产生中风。一些研究人员专注于较短的灯丝闭塞时间，这需要大量的实验重点和实验室验证7。小鼠中的细丝MCAo模型遵循与人类中风病例相似的细胞死亡，缺血进展和围死区形成的阶段;然而，较大的中风更接近于恶性脑梗死的疾病状态，这是不太常见，不太可治疗的人类中风6。同时，远端 MCA 闭塞需要更复杂的手术和颅骨切除术。在这个模型中，沿着大脑表面运行的MCA的远端部分被缝合带或烧灼直接闭塞。在该技术的某些变体中，颈动脉是单侧或短暂双侧闭塞的。远端 MCAo 的一个好处是，它会产生基于皮质的卒中，其大小比灯丝模型变化较小。然而，由于颈外动脉（ECA）的横断，远端模型产生较差的行为输出，这也是fMCAo6的一个问题。
另一种已知侵入性较小的卒中模型是光血栓形成（PT）模型。PT模型导致缺血的明确定义位置，并且与高生存率相关8。该技术依赖于腹腔内注射的光敏染料，只需用光或激光照射所需组织9，即可实现血管内光氧化。在激发时，形成氧自由基，引起内皮损伤，从而激活受照区域中的血小板聚集和凝块形成8，9。对行程大小和位置的严格控制，以及PT模型的高再现性，使其成为研究生物材料的理想选择。虽然使用激光和立体自聚焦坐标可以实现精度，但存在一些缺点，可能会使该模型在少数研究中不那么理想。与 fMCAo 模型不同，PT 行程模型不能重新熔化。因此，用于研究针对再灌注后损伤的神经保护剂或再灌注后机制的材料在这里没有用8。此外，由于PT模型的微血管损伤，可见相对较小的缺血性半影。相反，局部血管源性水肿发生，这是人类卒中的特征，使得该模型不适合专注于围梗死区域的临床前药物研究6，8。
中风中生物材料策略的总体目标是提供生物活性剂或作为脑组织生长的替代细胞外基质。我们将使用我们的方法探索的一种策略是将水凝胶直接输送到中风核心，而不是在梗死周围组织，其中许多当前的细胞疗法都提供10。这种方法的基本原理是，递送到核心中发现的坏死组织将避免破坏周围的健康或恢复组织。我们假设生物材料中包含的任何活性剂的扩散将能够从核心到达围陷圈，特别是因为我们发现水凝胶生物材料的输送减少了神经胶质疤痕的厚度11。这一点很重要，因为围陷区域已被证明在卒中后表现出神经可塑性，使其成为一个有吸引力的靶标。此外，将替代基质递送到卒中核心可以加载血管生成12 或神经源性13 因子，以指导新组织的形成，以及用于递送的细胞14。通过使用基质可以大大增强细胞递送，因为它可以保护细胞免受递送过程中存在的苛刻注射力和局部环境的影响，并鼓励分化和植入15。
这些可注射的治疗性生物材料在中风应用中具有临床相关性，因为目前还没有药物疗法可以刺激中风后的神经元恢复。参与恢复的底层神经回路位于与中风核心16相邻的脑组织中，而中风核心本身缺乏活的神经组织。我们预计，将生物材料递送到坏死性卒中核心具有通过前面提到的许多机制刺激相邻组织走向再生过程的潜力，包括生长因子13的库释放，刺激生长中的组织并促进恢复的脑组织发育11，12，免疫反应的改变17，以及干细胞衍生疗法的递送14， 18.然而，为了有效地研究这些应用的可能性，需要一种一致且可重复的方法来诱导中风和注射生物材料。PT 行程模型使用的技术可精确控制行程的方向和位置。连接到立体定位装置的激光引导方向，连接到立体定位装置的泵控制材料的注入速率，而无需其他形式的成像。因此，我们选择描述在小鼠运动皮层中执行PT中风以及将生物材料注射到中风核心的方法。在这里，我们使用微孔退火颗粒（MAP）水凝胶作为注射生物材料，不添加细胞或生长因子。此外，我们解释了如何使用完整的生物材料成功检索大脑，并讨论了用于分析中风结果的免疫化学测定，无论是否注射生物材料。

<table><tbody><tr><td>10% Normal Goat Serum</td><td>VWR</td><td>100504-028</td><td>For blocking buffer</td></tr><tr><td>2-ply alcohol pre pad, Sterile, Medium</td><td>Medline</td><td>MDS090735</td><td></td></tr><tr><td>25uL Hamilton Syringe 702RN, no needle</td><td>Fishcer Scientific</td><td>14824663</td><td>Syringes used to inject biomaterials</td></tr><tr><td>25uL Positive displacement pipette</td><td>Gilson</td><td>M-25</td><td></td></tr><tr><td>2x Beam Expander, 400-650nm</td><td>Thorlabs</td><td>GBE02-A</td><td>Laser beam expander</td></tr><tr><td>Adjustable Stage Platform</td><td>Kopf Instruments</td><td>901</td><td></td></tr><tr><td>Anti-Glucose Transporter GLUT1 antibody, rabbit</td><td>Abcam</td><td>ab113435</td><td></td></tr><tr><td>Anti-Iba1 Antibody, goat</td><td>abcam</td><td>ab5076</td><td></td></tr><tr><td>BD Vacutainer Safety-Lok Blood Collection Sets. 25G, 12&quot;</td><td>Medsupply</td><td>367294</td><td>For perfusions</td></tr><tr><td>BKF12- Matte Black Aluminum Foil</td><td>Thorlabs</td><td>BKF12</td><td>To cover anything that is reflective when using laser.</td></tr><tr><td>Bone Iris Mini Scissors - 3-1/2&quot;</td><td>Sklar surgical instruments</td><td>64-2035</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Jackson Laboratory</td><td>000664</td><td>8-12 weeks of age</td></tr><tr><td>Cage Assembly Rob</td><td>Thorlabs</td><td>ER3-P4</td><td>3&quot; Long, diameter 6mm, 4 pack - for attaching laser to sterotax</td></tr><tr><td>Carbon Steel Burrs -0.5mm Diameter</td><td>Fine Science Tools</td><td>19007-05</td><td>For creating burr hole</td></tr><tr><td>Chromium(III) potassium sulfate dodecahydrate</td><td>VWR</td><td>EM1.01036.0250</td><td></td></tr><tr><td>Compact Controller for pigtailed lasers</td><td>Thorlabs</td><td>CLD1010LP</td><td></td></tr><tr><td>Cotton Swabs</td><td>VWR</td><td>89031-288</td><td></td></tr><tr><td>CP 25 pipette tips</td><td>Gilson</td><td>F148012</td><td></td></tr><tr><td>Donkey anti-goat IgG H&amp;amp;L (488)</td><td>abcam</td><td>ab150129</td><td></td></tr><tr><td>Donkey Anti-rabbit IgG H&amp;amp;L (647)</td><td>abcam</td><td>ab150075</td><td></td></tr><tr><td>Donkey Anti-rat IgG H&amp;amp;L (555)</td><td>abcam</td><td>ab150154</td><td></td></tr><tr><td>EMS DPX Mountant</td><td>Elecron Microscopy Sciences</td><td>13512</td><td>Mounting solution for slides</td></tr><tr><td>EMS Gelatin Powder Type A 300 Bloom</td><td>Electron Microscopy Sciences</td><td>16564</td><td>For gelatin coating slides</td></tr><tr><td>EMS Paraformaldehyde, Granular</td><td>VWR</td><td>100504-162</td><td>For making 45 PFA</td></tr><tr><td>ESD Worstation kit</td><td>Elmstat</td><td>WSKK5324SB</td><td>Need for setting up the laser</td></tr><tr><td>Fiber Bench Wall Plate, unthreaded</td><td>Thorlabs</td><td>HCA3</td><td>Need for connecting laser to Kopf shaft</td></tr><tr><td>FiberPort</td><td>Thorlabs</td><td>PAF-X-5-A</td><td>FC/APC&amp;amp; APC, f=4.6mm, 350-700nm, diameter 0.75mm</td></tr><tr><td>Fine Scissors - straight/sharp-blunt/10cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>GFAP Antibody, rat</td><td>Thermo Fisher Scientific</td><td>13-0300</td><td></td></tr><tr><td>Heating Plate</td><td>Kopf Instruments</td><td>HP-4M</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td>H-4000</td><td>For staining slides</td></tr><tr><td>IMPAC 6-Integrated Multi Patient Anesthesia Center</td><td>VetEquip</td><td>901808</td><td></td></tr><tr><td>Iodine Prep Pads</td><td>Medx Supple</td><td>MED MDS093917H</td><td></td></tr><tr><td>Jewlers Forceps #5</td><td>GFS chemicals</td><td>46085</td><td></td></tr><tr><td>Laser Safety Glasses</td><td>Thorlabs</td><td>LG10B</td><td>Amber Lenses, 35% Visible light (googles versions available too)</td></tr><tr><td>M27-1084 Powerful LED Dual Goose-neck</td><td>United Scope</td><td>LED-11C</td><td></td></tr><tr><td>Medical USP Grade Oxygen</td><td>Airgas</td><td>OX USP250</td><td></td></tr><tr><td>Miltex Adson Dressing Forceps, Disecting-grade</td><td>Intefra Miltex</td><td>V96-118</td><td></td></tr><tr><td>Mini Cord/Cordless Small Animal Trimmer</td><td>Harvard aparatus</td><td>72-6110</td><td></td></tr><tr><td>Mini-pump variable flow</td><td>Thomas Scientific</td><td>70730-064</td><td>Pump for perfusions</td></tr><tr><td>Mouse Brain Matrices, Coronal Slices, 1mm</td><td>Kent Scientific</td><td>RBMA-200c</td><td>For TTC slices</td></tr><tr><td>Mouse Gas Anethesia Head Holder</td><td>Kopf Instruments</td><td>923-B</td><td></td></tr><tr><td>Nanojet Control Box</td><td>Chemyx</td><td>10050</td><td></td></tr><tr><td>Nanojet pump header</td><td>Chemxy</td><td>10051</td><td>Attach to stereotaxic device for injecting biomaterial</td></tr><tr><td>Needle RN 30G PT STY 3, 0.5 inch</td><td>Fishcer Scientific</td><td>NC9459562</td><td></td></tr><tr><td>Non-rupture ear bars 60&amp;ordm;</td><td>Kopf Instruments</td><td>922</td><td></td></tr><tr><td>PBS buffer pH 7.4</td><td>VWR</td><td>97062 338</td><td></td></tr><tr><td>Pigtaled laser 520 nm, 100mW, 5G Pin</td><td>Thorlabs</td><td>LP520-MF100</td><td></td></tr><tr><td>Positive charge glass slides</td><td>Hareta</td><td>AHS90-WH</td><td></td></tr><tr><td>Power engergy meter</td><td>Thorlabs</td><td>PM100D</td><td>Used to measure your mW laser output</td></tr><tr><td>Puralube Vet Ointment</td><td>Dr. Foster Smith</td><td>9N-76855</td><td></td></tr><tr><td>Rectal Probe Mouse</td><td>kopf Instruments</td><td>Ret-3-ISO</td><td></td></tr><tr><td>Rose Bengal Dye 95%</td><td>Sigma-Aldrich</td><td>330000-5G</td><td></td></tr><tr><td>Shaft Modified 8-32 threaded hole 1/2&quot; depth</td><td>Kopf Instruments</td><td>1770-02</td><td>For connecting laser to sterotaxic device</td></tr><tr><td>Slim photodiode power sensor</td><td>Thorlabs</td><td>S130VC</td><td>Used with power energy meter</td></tr><tr><td>SM1-Threaed 30 mm Cage Plate 0.35&quot; thick 2 Retaining</td><td>Thorlabs</td><td>CP02</td><td>For connecting laser expander</td></tr><tr><td>Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL</td><td>VWR</td><td>10002-726</td><td>To inject rose bengal</td></tr><tr><td>StainTray Slide Staining System</td><td>Simport Scientific</td><td>M920-2</td><td>For staining slides</td></tr><tr><td>Sterotaxic device</td><td>Kopf Instruments</td><td>940</td><td>Small Animal Stereotaxic Instrument</td></tr><tr><td>Student Adson Forceps -1x2 teeth</td><td>Fine Science Tools</td><td>91127-12</td><td></td></tr><tr><td>Student Fine Forceps - straight/broad Shanks</td><td>Fine Science Tools</td><td>91113-10</td><td></td></tr><tr><td>Temperature Controller</td><td>Kopf Instruments</td><td>TCAT-2LV</td><td></td></tr><tr><td>Tissue-Tek OCT compound</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>Triton X-100</td><td>VWR</td><td>97063-864</td><td></td></tr><tr><td>Upper Bracket Clamp</td><td>Kopf Instruments</td><td>1770-c</td><td>For connecting laser to sterotaxic device</td></tr><tr><td>Vetbond Tissue Adhessive 3mL</td><td>Santa Cruz Biotechnology</td><td>sc-361931</td><td></td></tr><tr><td>Vogue Professional My Manicurist</td><td>Bargin Source</td><td>6400</td><td>For Burrs</td></tr><tr><td>VWR Bead Sterilizers</td><td>VWR</td><td>75999-328</td><td></td></tr><tr><td>Tissue Tek OCT compound</td><td>Sakura</td><td>4583</td><td>For tissue embeding</td></tr></tbody></table>

injection of hydrogel biomaterial scaffolds to the brain after stroke

中风是美国残疾的主要原因和第五大死因。大约87%的中风是缺血性中风，被定义为向大脑供血的血管突然阻塞。在阻塞的几分钟内，细胞开始死亡并导致不可挽回的组织损伤。目前的治疗重点是血栓去除或裂解，以允许再灌注并防止更严重的脑损伤。虽然短暂的脑可塑性可能会随着时间的推移挽救一些受损组织，但很大一部分患者留下了永远无法解决的神经功能障碍。缺乏治疗中风引起的神经功能障碍的治疗选择，强调需要制定新的策略来治疗这一不断增长的患者群体。可注射的生物材料目前正在设计中，通过递送活性剂或干细胞来增强大脑可塑性并改善内源性修复。测试这些方法的一种方法是利用啮齿动物中风模型，将生物材料注射到中风核心中，并评估修复情况。了解卒中核心的精确位置对于卒中后的准确治疗至关重要，因此，最好使用导致可预测的卒中位置的卒中模型，以避免在注射前需要成像。以下方案将涵盖如何诱导光血栓性中风，如何以受控和精确的方式注射水凝胶，以及如何提取和冷冻切除大脑，同时保持生物材料完整。此外，我们将重点介绍如何将这些相同的水凝胶材料用于干细胞的共递送。该协议可以推广到使用其他可注射的生物材料进入中风核心。

这种方法的目的是展示如何在中风后将生物材料注射到大脑中。使用带有玫瑰孟加拉和520nm激光的光血栓形成模型来控制卒中病变的大小和位置的方向。卒中五天后，梗死可以在手术过程中（图1B）和TTC和成像IHC染色载玻片（图2）可视化。使用2倍透镜增加激光直径会导致行程病变的视觉增加，其范围超过2.5 mm，而仅使用激光的单个1 mm部分（<strong class="...

Watch this Scientific Journal Video about Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke at JoVE.com

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

中风是一个全球性问题，治疗选择最少，目前没有用于再生丢失的脑组织的临床治疗方法。在这里，我们描述了在啮齿动物的运动皮层中产生精确光血栓性中风的方法，并随后注射水凝胶生物材料，以研究它们对中风后组织再生的影响。

中风后向大脑注射水凝胶生物材料支架

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

injection-of-hydrogel-biomaterial-scaffolds-to-the-brain-after-stroke

Research

JoVE Journal

Biology

5.0K 次观看.  Duke University. 中风是一个全球性问题，治疗选择最少，目前没有用于再生丢失的脑组织的临床治疗方法。在这里，我们描述了在啮齿动物的运动皮层中产生精确光血栓性中风的方法，并随后注射水凝胶生物材料，以研究它们对中风后组织再生的影响。

视频: Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

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

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

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 ...

科研

神经科学

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

用于治疗性水凝胶研究的胶质母细胞瘤复发术后切除模型

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective ...

癌症研究

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 ...

生物工程

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

医学

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

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 ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

用于外周神经工程的石墨烯基3D生物杂化水凝胶生物墨水的制备与表征

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

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.

使用干细胞递送开发脊髓损伤的组合疗法

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