作者感谢 NSERC、CFI、AIHS、艾伯塔省卫生服务机构以及戴维大脑研究基金会的资助。

1天的大鼠幼崽的脑组织提取协议, 被斩首的安乐死, 得到了阿尔伯塔大学动物护理和使用委员会的批准。
1. 小胶质细胞和星形胶质细胞隔离67,68
注: 所有用于隔离和细胞培养的介质在水浴中预热至37° c。汉克的平衡盐溶液 (HBSS) 有1% 青霉素-链霉素 (PS)。所有 Dulbecco 的改良鹰的媒体与火腿的 F12 营养混合物 (DMEM/F12) 补充10% 胎牛血清 (FBS) 和1% 青霉素-链霉素 (PS)。只有与胰蛋白酶的混合物缺乏 FBS。本协议中的所有材料均为无菌 (过滤器、酒精、购买和蒸压), 步骤编号1.8 后面的每一步都在一个无菌技术的生物安全柜中执行。
<ol><li>每脑, 预热15毫升的 HBSS 和12.5 毫升 DMEM/F12 到37° c, 在50毫升圆锥离心管, 约2毫升0.25% 胰蛋白酶与1毫米乙酸酸 (EDTA) 在15毫升圆锥离心管。加热额外的25毫升的 DMEM/F12。热胰蛋白酶约10分钟前使用, 以防止失去酶活性。</li>
	<li>倒入足够的温热 HBSS 成无菌, 6 和10厘米的培养皿覆盖表面。准备一个10厘米的菜, 每两个大脑加上一个额外的6厘米的菜。</li>
	<li>在每个 10 cm2盘中放置多达2个大脑 (图 2A)。</li>
	<li>在解剖显微镜下, 用弯曲和直钳将皮质沿中线和小脑与脑干分开。这会产生每个脑部的三部分组织 (图 2B)。</li>
	<li>剥离薄的, 半透明的, 层 (脑膜) 的组织包含血管从每个部分的组织和丢弃, 转移剩余的组织成一个单独的培养皿。抓住薄层暴露的组织削减, 用镊子, 并仔细拉, 直到主要部分是 ' 挂断 ' 的脑组织。最后通过轻轻地扯拽来去除这部分。有关此步骤之前、期间和之后的代表性图像, 请参见图 2C-2E 。</li>
	<li>在生物安全柜中, 从培养皿中取出剩余的 HBSS, 小心地保持组织。用手术刀将组织浸渍, 并将组织转移到预热的15毫升管与胰蛋白酶。</li>
	<li>在37° c 浴中孵育此管25分钟以消化组织酶。</li>
	<li>离心管在 500 x g, 在室温下2分钟, 倒出上清液。</li>
	<li>使用10毫升血清吸管, 添加10毫升的温暖 DMEM/F12, 使胰蛋白酶失效, 并研制溶液的5倍, 以打破组织。</li>
	<li>离心管在 500 x g, 在室温下2分钟, 倒出上清液。</li>
	<li>使用10毫升血清吸管, 重新悬浮颗粒成10毫升的温暖 DMEM/F12, 并转移到一个50毫升圆锥离心管的解决方案。</li>
	<li>使用10毫升注射器与18口径针, 研制的解决方案3次。</li>
	<li>将此解决方案分配给每个大脑12.5 毫升温 DMEM/F12 的增量。</li>
	<li>吸管1毫升的 triturated 溶液的增量到一个12井板 (1 每个脑子)。</li>
	<li>孵育在37° c 和 5% CO2在一个湿的细胞培养孵化器。三天后更换介质, 每隔三天刷新一次介质。2周后, 可以采集细胞进行实验。</li>
</ol>2. 单体合成69
<ol><li>在50毫升锥形离心管中重375毫克透明质酸 (HA) 盐, 加入37.5 毫升蒸馏水。</li>
	<li>用 vortexer 冲洗溶液1分钟, 几种混合物, 直到它看起来均匀并失去凝胶状粘度。这个过程可能需要6小时。</li>
	<li>将溶液转移到一个100毫升的烧杯中, 用磁力搅拌棒 (38 mm), 并在室温下用力搅拌。</li>
	<li>仔细滴定5.0 米氢氧化钠到混合 HA 和测量与 ph 值纸直到 ph 值稳定在8和12之间。</li>
	<li>收集3毫升的新鲜甲基丙烯酸酐 (MA, 94%), 在贮存过程中被氮气覆盖。 注: MA 被暴露在大气中很长时间 (天), 它将失去它的反应性。 注意: 所有使用的 MA 都应该在通风橱里做。</li>
	<li>在混合 HA 溶液中加入200µL 的 MA。反应降低溶液的 pH 值低于8。</li>
	<li>重复步骤2.4 到 2.6, 直到将 3 mL MA 添加到解决方案中。这个过程可能需要1-2 小时。</li>
	<li>允许反应在4° c 的夜间继续, 不混合。</li>
	<li>400毫升的冷乙醇 (乙醇) 在一个500毫升的烧杯, 并允许沉淀 24-72 小时在4° c。56
</li>
	<li>小心地将溶液的主要部分醒酒到一个单独的烧杯中处理, 然后在50毫升锥形离心管中收集沉淀。如有必要, 可在2-4 根管子中分布。</li>
	<li>在离心机中离心 200 x g 的管子, 在室温下2分钟, 处理上清液。</li>
	<li>顶部的管与冷乙醇, 混合的解决方案与 vortexer 为1分钟, 并重复步骤 2.10-2. 11。</li>
	<li>加入25毫升无菌去离子水, 混合溶液与 vortexer 为1分钟, 几种 (> 20 赫) 为1小时, 并孵化在4° c 过夜。</li>
	<li>将溶液放在-80 ° c 的冷冻柜中15分钟, 或直至冷冻或在液氮中冻结。 注意: 使用液氮时, 使用防护手套、面罩和围裙。</li>
	<li>冷冻干燥的管 (低于0.1 毫巴和-30 ° c) 为 24-48 小时, 直到一个类似雪的粉末产生。</li>
	<li>在这一点上, 通过1H 核磁共振测试样品的纯度, 其中甲基丙烯酸甲酯的质子 (峰值在6.1 和 5.6 ppm), 相对于甲基质子 (1.9 ppm), 可以确认 HA 骨干。95% 甲基丙烯酸甲酯与甲基质子的最低比率是可以接受的。69
</li>
</ol>3. 凝胶形成和3D 封装65
<ol><li>
片和模具准备
	<ol>
<li>在50毫升锥形离心管中制作一个50毫升蒸馏水溶液, 2% 3-(基) 丙基甲基丙烯酸酯。</li>
		<li>放置18毫米玻璃片在溶液和岩石1小时室温。可同时添加多达50片。</li>
		<li>在100毫升去离子水的3烧杯中依次浸洗片。</li>
		<li>用干燥和烤箱在40° c 的真空干燥片。</li>
		<li>快速浸泡在70% 乙醇与镊子的个人片。</li>
		<li>没有干燥, 把每个片到一个12井板井。</li>
		<li>每井用不育的去离子水 (每井1毫升) 洗涤和抽吸。</li>
		<li>在每个井中加入1毫升无菌2µg/毫升的聚 l-赖氨酸 (PLL), 孵育 > 2 小时。</li>
		<li>吸气锁相环溶液, 使片空气干燥。</li>
		<li>沉浸 polydimethyl 硅氧烷 (见图 1) 在70% 乙醇, 并放置在每片的中心, 并允许空气干燥, 并创建一个密封的模具和玻璃。<ol>
<li>在一个平底的聚苯乙烯盘中, 用10:1 的比例铸造聚苯氧烷预混剂来制备硅橡胶模具。所制备的硅橡胶的数量选择了约1毫米厚的板材。要在该公司中形成油井, 请用直径为10的圆冲切薄板。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
细胞制备 注意: 协议中的所有步骤都是在生物安全柜中用无菌技术进行的。磷酸缓冲盐水 (PBS) 溶液被调整到 pH 值7.4 的所有步骤。<ol>
<li>24小时前在哈马的细胞封装, 刷新2周的主要文化 (从步骤 1.15) 在12井板培养基。每井添加1毫升的 DMEM/F12 (10% FBS 和 1% PS) 培养基。</li>
		<li>为每12个细胞板准备12.5 毫升稀释胰蛋白酶 (0.25% 胰蛋白酶-EDTA 稀释30% 与 DMEM/F12 媒介) 和25毫升 DMEM/F12 (10% FBS 和 1% PS) 和温暖在37° c 在水浴。</li>
		<li>使用10毫升血清学吸管 (> 0.5 毫升) 从板中收集条件培养基, 彻底抽出剩余液体, 用1毫升的稀释胰蛋白酶 (0.075% 胰蛋白酶-EDTA) 在孵化箱中20-30 分钟 (37 ° c, 5% CO2) 中更换, 直到汇合的细胞层与板块分离。</li>
		<li>用1毫升吸管回收悬浮的细胞材料 (显示为一个单一的漂浮件) 和收集在一个15毫升锥形离心管。用等量的 DMEM/F12 (10% FBS 和 1% PS) 稀释, 在室温下离心 200 x g, 以2分钟, 丢弃上清液。</li>
		<li>重在10毫升温暖 DMEM/F12 (10% FBS 和 1% PS) 的细胞颗粒, 研制5次与10毫升吸管, 和离心在 200 x g 为2分钟。</li>
		<li>醒酒上清液, 重新悬浮在5毫升温暖 DMEM/F12 细胞, 并转移到一个50毫升圆锥离心管的细胞。</li>
		<li>使用10毫升注射器与18口径针, 研制的细胞溶液3次, 并过滤器的悬浮通过40µm 细胞筛到一个新的锥形离心管。</li>
		<li>收集10µL 的细胞悬液, 稀释1:100 在温暖的 DMEM/F12, 并计数细胞与自动细胞计数器</li>
		<li>在37° c 的水浴中孵育直到封装步骤。</li>
	</ol>
</li>
	<li>
单元封装 注意: 协议中的所有步骤都是在生物安全柜中用无菌技术进行的。<ol>
<li>每12个3D 的水凝胶暖12.5 毫升的 DMEM/F12 和12.5 毫升的条件 DMEM/F12 培养基收集在细胞准备。</li>
		<li>称最后浓度为0.5% 重/vol. 的 methacrylated 透明质酸 (哈马) 的数量需要在无菌过滤 PBS 中溶解, 浓度为 (2% 度/卷);浓度比最终浓度高4倍, 用于容纳细胞和其他试剂的加入。一个12井板的水凝胶需要〜350µL PBS 与7毫克哈马。</li>
		<li>几种 (> 20 赫) 解决方案直到哈马完全溶解60分钟。</li>
		<li>准备10% 个三乙醇胺 (茶叶) 和 1-乙烯基 2-咯 (NVP) 的单独的解决方案, 并在1毫升等分的无菌 PBS pH 7.4 中, 1 毫米的曙红 Y 的溶液。</li>
		<li>对于一个12井板, 做最后1.4 毫升混合物 1 x 107细胞, 0.5% 重量/卷哈马 (350 µL 2% 重量/卷), 0.1% 茶 (14 µL 10%), 0.1% NVP (14 µL 10%), 和0.01 毫米 (14 µL 1 毫米), 20% 基板混合物 (280 µL)。剩余的音量是 PBS。</li>
		<li>轻轻地混合解决方案和吸管100µL 到每个硅橡胶模具与1毫升尖端。</li>
		<li>将样品暴露在一个高强度的绿色 LED 灯 (〜 520 nm, 60 兆瓦, 在一个封闭的 20 cm x 20 cm x 20 cm 箱) 为5分钟的室温。</li>
		<li>加1毫升的温暖条件 DMEM/F12 到12井板。</li>
		<li>抓住每一个拇指和食指之间的片, 慢慢剥离的硅橡胶模具与弯曲的镊子小心不取代凝胶, 并把它放入一个良好的条件介质。</li>
		<li>在每个井中加入额外的1毫升温暖 DMEM/F12。</li>
		<li>孵育板在37° c 与 5% CO2为2星期, 刷新细胞媒介每星期由移1毫升媒介从每口井和替换以1毫升 DMEM/F12。</li>
	</ol>
</li>
</ol>4. 显微镜
<ol><li>
免疫 注: 采用倒置共焦显微镜对这些样品进行图像处理, 并利用 ImageJ 对图像进行加工和显示。在哈马培养3周后, 小胶质细胞、星形胶质、突和细胞核将被标记。<ol>
<li>在37° c 浴中预热 10% PBS 缓冲福尔马林 (pH 7.0)。 注意: 虽然福尔马林可以在油烟机外使用, 但这种材料是与组织反应的, 并且应始终小心和手套处理。应单独处理。</li>
		<li>小心地从盘子里吸出介质, 并将1毫升的10% 福尔马林抽到每一个井。</li>
		<li>孵育板在37° c 在 5% CO2为 20 min。</li>
		<li>从每个板吸液, 吸管2毫升 PBS (pH 7.4) 到每井, 孵化15分钟。</li>
		<li>重复步骤4.1.4 两次。</li>
		<li>吸液从板块, 增加1毫升每井 PBS 与10% 正常马血清 (NHS) 和0.5% 海卫 X-100, 并轻轻晃动最低速度) 为1小时。</li>
		<li>重复步骤4.1.4 三次。</li>
		<li>吸液并在 PBS 中加入1毫升的以下混合物: 兔电离钙结合适配器分子 (Iba1) 1:1, 000, 鸡胶质纤维蛋白 (胶质) 1:5, 000, 小鼠 2 ', 3 '-循环-nucelotide 3 '-phophodiesterase (CNPase) 1:1, 000, 1% NHS, 0.1% 细胞穿孔表面活性剂。在4° c 的夜间孵育, 尽可能轻柔地摇晃盘子。</li>
		<li>重复步骤4.1.4 三次, 与 1 h 孵化之间。</li>
		<li>吸液和添加1毫升每井以下混合物在 PBS: 647 nm 兴奋驴抗兔抗体 1:200, 546 nm 兴奋山羊抗鸡抗体 1:200, 488 nm 兴奋驴抗鼠抗体 1:200, 1% NHS, 1:1, 000 蓝色核染色。在室温下孵育1小时, 轻轻摇晃盘子。</li>
		<li>重复步骤4三次, 与 1 h 孵化之间。</li>
		<li>保存水凝胶, 抽吸缓冲液, 浸泡在0.5 毫升的细胞安装介质中, 1 分钟. 吸管取出安装介质, 轻轻地将片放在凝胶上, 在玻璃之间形成一层。添加少量的安装介质到片的未被发现的一侧, 以防止水凝胶的干燥。 注: 为了排除故障, 凝胶可以在步骤4.1.11 上成像。在进一步的加工之前看到正确的标签。</li>
	</ol>
</li>
	<li>
扫描电子显微镜 (SEM) 注: 为了形象化的水凝胶, 用扫描电子显微镜来图像这些样品。<ol>
<li>在37° c 水浴中预先加热2.5% 戊二醛和2% 甲醛在 PBS (0.1 M) 中的定影混合物。 注意: 这些试剂与组织具有高度的反应性, 应小心和手套处理。它们应尽可能地处理在油烟罩中, 并单独处置。</li>
		<li>从细胞培养皿中吸取培养基, 加入1毫升的预加热定影液。</li>
		<li>孵育板在37° c 在 5% CO2为 20 min。</li>
		<li>将盘子放在4° c (冰箱/冷却器) 过夜。</li>
		<li>从油井中抽出固定液, 加入1毫升的 PBS。</li>
		<li>从每个板块吸出液体, 在每个井中加入2毫升的 PBS, 孵育15分钟。</li>
		<li>重复步骤4.2.6 两次。</li>
		<li>吸入 pbs 和添加1毫升1% 锇氧化缓冲在 pbs 每井。 注意: 直到样品干燥, 这一步和所有后续步骤都在通风柜中执行。</li>
		<li>允许固定发生在30分钟的锇蒸气从液体可以固定在同一板块的其他样品;因此, 在此步骤之前, 请相应地对示例进行分区。 注意: 锇氧化与组织和挥发性高度反应。这是危险的, 应该处理在油烟罩与手套。它和任何立即清洗应另行处置。</li>
		<li>重复步骤4.2.6 三次。</li>
		<li>在下面的列表中吸入液体并加入脱水混合物, 按顺序, 并孵育 (室温) 为指定时间。开始逐渐加入乙醇 (乙醇), 随后烷 (HMDS)。请参见每个增量的表 2 。 注: 当添加 HMDS 时, 片能强力附着在塑料上, 变得非常难以去除。在第一次使用 HMDS 后, 可以在玻璃滑块下放置一个小塑料平台来防止这种情况。在第二次乙醇孵化后, 样品可以在液氮中取出并淬火几秒钟以冻结试样。</li>
		<li>小心地除去样品, 用双面导电胶带将样品贴在扫描电子显微镜存根上。</li>
		<li>溅射涂层的样品与黄金的最低金额。将样品放在支架上, 在15毫安的地方操作机器2分钟。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

为了建立一个3D 的培养系统来塑造神经胶质 bioreactivity 和胶质瘢痕的过程, 我们开发了一个能支持原代培养的小胶质细胞、星形胶质细胞和突的系统, 并能使细胞膜的强健特性形态学和细胞间的相互作用。从显微显示, 每个细胞类型的形态学是明显不同的 2D, 3 d-哈马, 和3D 哈马基板混合平台。在2D 系统中, 形态学在表面的平面上明显偏倚, 但与3D 的哈马相比, 小胶质细胞和星状体在母体内普遍较小, 但胶质簇除外。在扫描电子显微中, 细胞通常在哈马形成圆形。这可能很大程度上是由于细胞被封装, 有有限的能力, 以修改矩阵的运动和自由移动和增长的过程。在哈马的细胞生长通常是径向的星形胶质细胞和突。虽然这些细胞扩展过程, 他们的组织建议, 他们的运动在整个矩阵和能力与其他细胞的互动是有限的。此外, 以前的工作已经表明, 冻裂的哈马是多孔没有互联的网络, 这可能解释这种缺乏的副产物65,70。这可能是有可能的, 其中一些细胞没有生存的聚合, 保留在矩阵中, 但生存能力系统评估在我们以前的工作, 并保持 80% (0.5% 瓦特/五) 在一周的过程中, 在一个星期65。
为了改善3D 培养基质中的细胞相互作用, 我们引入了基板混合物作为一个生物相容性和可修改的矩阵分量。细胞与基质的整合被发现明显改善, 所有细胞类型显示广泛的过程相比, 2D 文化和3D 哈马。基底叶片混合物由多种基底板组成, 主要用于支持神经干细胞前体的胶质分化71。这是不意外的胶质细胞反应的基底板混合物, 随后周围的3D 矩阵, 但融合的程度, 建议一个健康的组织样的环境。比较哈马与哈马-基底层的混合, 水凝胶的形态没有明显的不同, 因此细胞可能更积极地重塑它。考虑到添加这种生物活性成分的简单性, 使用不同的叶片和/或蛋白质提示来支持神经元的培养条件并不是不可想象的。在未来的潜在方向, 这个系统可以用来区分神经元的神经祖细胞与混合胶质细胞的各种有影响力的鞘或神经炎症损伤模型。
在中枢神经系统损伤或生物材料排斥发生严重和破坏性的情况下, 居民组织发起炎症反应, 使神经和鞘恶化。这通常导致形成一个神经胶质瘢痕来划分损伤部位, 从而防止再生。本研究对 methacrylated 光聚合透明质酸基3D 支架的方法进行了详细的分析, 目的是模拟胶质瘢痕形成后的初始细胞反应;胶质反应性, 星形胶质细胞的招募和肥大, 胶质损伤和撤退。3D 细胞支架的设计, 以纳入所有三胶质细胞类型, 并进一步修改与多组分基底叶片混合物。通过共焦和扫描电镜成像, 确定了该细胞在哈马的包封, 并在引入基底叶片混合物时能更好地进行整合。这些结果可能导致一些新的应用。仅哈马可能会创造共同文化系统, 其目的是限制细胞与细胞相互作用, 或研究用凝胶作为药物负载扩散有限矩阵的蒸馏药物的传递。水凝胶将基板混合物加入到哈马基质中, 可以形成更一致的组织样环境, 能够模拟急性炎症、胶质或胶质瘢痕, 并能进一步提高胶质细胞的高吞吐量特性。bioreactivity 用于植入物、药物和器械的开发, 以及其他减少和恢复炎症损伤的策略。

中枢神经系统 (cns) 的炎症一直被认为是急性 (如: 如, 缺血性中风, 外伤性脑和脊髓损伤) 和慢性 (如: 老年痴呆, 帕金森氏症, 亨廷顿氏病) 中枢神经损伤的标志,但越来越被认为是神经退行性疾病和精神障碍的原因。持续或不适当的炎症可能导致神经损伤和鞘 (如多发性硬化), 并对大脑发育产生负面影响 (如:如、精神分裂症、自闭症) 和心境状态 (如、抑郁症、焦虑, 躁郁症)。此外, 使用植入设备的新的治疗策略 (e. g., 脑-计算机-接口1,2,3, 深脑刺激4,5, 椎管内microstimulation6,7,8,9,10在设备和 CNS 之间的接口上生成可预知的炎症反应, 从而导致保护组织的反应, 可能导致在植入物的生命周期的失效或设备故障11。中枢神经系统的炎症通常是由小胶质细胞发起的, 它作为中枢神经系统的常驻免疫单元, 负责组织监测和安装异物反应 (已被审查的12)。根据侮辱的严重性, 小胶质细胞信号和招募额外的单元类型到受伤地点。具体来说, 小胶质细胞激活星形细胞, 这反过来充当继发性炎症单元和形成一个密集的保护屏障, 以包含伤害网站13,14。小胶质细胞也可以在外周免疫系统中启动一个活动级联, 这可能导致 BBB 的分解以允许免疫浸润 (在参考文献15中进行了回顾)。
对于植入中枢神经系统的设备, 由于设备插入而导致的组织损伤以及异物的持续存在, 可能会引发一种称为胶质瘢痕的过程。在此过程中, 小胶质细胞迁移到损伤部位并增殖。他们还开始释放炎症因子, 以抵消潜在的威胁, 并招募更多的胶质细胞。随后, 激活的星形胶质细胞变得肥大, 开始封装植入的设备形成连续的纤维屏障16。炎症信号还有助于促进从种植体附近撤出神经元过程, 并最终招募成纤维细胞, 以加强发育中的神经胶质瘢痕17。突, 负责鞘内的神经元, 以提高电导率, 不生存这个过程和遥远的细胞被分割从植入物的疤痕18。胶质瘢痕极大地降低了植入装置的功能和寿命, 特别是用于记录电极, 并最终限制了神经接口19的功能。
在 CNS20、21、22、23中, 已利用多种方法提高植入设备的生物相容性和界面活性。对这些神经接口的生物相容性设计24进行了广泛的回顾。最突出的策略包括围绕电极与相容涂层, 如 polyelthyleneglycol (PEG), 聚乳酸-乙醇酸 (PLGA) 的25, 或增强电极与导电聚合物, 如聚乙烯 (乙烯dioxythiophene) (PEDOT) 和聚吡咯 (吡)26,27,28,29,30,31。生物活性涂层也被用于提供神经组织生长的线索, 使用来自细胞外基质的配体, 包括胶原蛋白、fibronectins 和透明质酸32,33,34 ,35,36,37。这些涂层的生物活性已进一步探索与生长因子释放系统模拟自然细胞分泌物30,38,39,40,41,42,43,44,45,46,47,48,49,50. 同时, 一些研究小组选择了重塑电极的几何形状、柔韧性和成分, 以减少设备和组织之间的机械不匹配51,52,53 ,54,55,56,57。总之, 这些策略已经导致了下一代神经界面设备的许多有希望的改进, 但是长期的兼容性是一个持续的问题, 而且在复杂和耗时的体内模型中可能会阻碍进展。.
以动物模型为基础的方法可以限制实验的吞吐量, 增加测试电极生物相容性的成本。体外使用常规细胞培养技术的方法提供了一个更经济高效的替代方案, 但无法重述设备和组织之间的交互的复杂程度58。特别是, 使用2D 细胞培养的表面涂层的测试限制了电极几何的建模以及机械不匹配和微思想对生成导致设备故障的主机响应的影响59,60。
为了克服与2D 细胞培养有关的问题, 水凝胶培养的神经细胞已经开发了广泛的各种应用, 药理学研究61, 以指导神经细胞分化62, 以了解疾病路径63,64, 或在与其他单元格类型的 co 区域性中进行分层, 以模拟单元格迁移、神经保护或模型组织环境61。水凝胶易于形成不同的大小和几何可以包含多种类型的主要或永生细胞文化, 并高度适合分析的常用技术, 如共用荧光显微镜。为了建立一个模型的胶质瘢痕的过程, 我们最近开发和特征的透明质酸基3D 水凝胶系统的高通量测试的胶质反应植入电极 (图 1)65。该系统有几个明显的优点: 1) 原发胶质细胞 (小胶质、星形胶质和突) 被封装在一个由透明质酸聚合物组成的3D 基质中, 它是一种内源性细胞外基质成分;2) 矩阵刚度可进行 "调谐", 以再现脑或脊髓组织的力学特性;和 3) 细胞可以封装在矩阵的快速台阶顶部的方法使用聚合与绿色光, 限制毒性在封装。该系统可实现体内生物相容性的关键功能: 设备以与组织相类似的方式插入到水凝胶中, 对植入设备的细胞反应进行监测, 以实现范围广泛的参数65。这些包括设备与各种结构的水凝胶涂层和电刺激脉冲之间的机械不匹配。该系统还包括胶质和相关的前体, 它们经常存在并在胶质瘢痕中被吸收。小胶质细胞的损伤、死亡和吞噬作用是炎症性损伤的高度表现, 作为一种模型可以减少疤痕或恢复, 他们有能力证明神经元重新鞘66。
本文介绍了一种合成和形成混合透明质酸水凝胶的方法, 并结合商用基底膜配方, 以改善细胞合并。此外, 我们将展示的主要培养的神经胶质细胞 (小胶质, 星形胶质, 和突) 和分析的文化生长, 使用细胞化学和共聚焦显微镜。

<table><tbody><tr><td>&lt;strong&gt;1. Materials for HAMA synthesis and photopolymerization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hyaluronic acid (HA)</td><td>Sigma-Aldrich</td><td>53747-10G</td><td>Streptococcus equi, MW: 1.5 - 1.8 X 10^6</td></tr><tr><td>Methacrylic anhydride (MA)</td><td>Sigma-Aldrich</td><td>275585-100ML</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>221465-25G</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Commerical Alcohols Inc.</td><td></td><td>Anhydrous</td></tr><tr><td>Phosphate buffered saline (pH 7.4) tablets</td><td>Fisher Scientific</td><td>18912014</td><td></td></tr><tr><td>Triethanolamine (TEA)</td><td>Sigma-Aldrich</td><td>90279-100ML</td><td></td></tr><tr><td>1-Vinyl-2pyrrolidinone (NVP)</td><td>Sigma-Aldrich</td><td>V3409-5G</td><td></td></tr><tr><td>EosinY (EY)</td><td>Sigma-Aldrich</td><td>E6003-25G</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Sigma-Aldrich</td><td>440159-100ML</td><td></td></tr><tr><td>Beaker (100 mL)</td><td>Corning</td><td>1000-100</td><td></td></tr><tr><td>Beaker (500 mL)</td><td>Corning</td><td>1000-600</td><td></td></tr><tr><td>pH paper (Labstick)</td><td>Sigma-Aldrich</td><td>9580</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Materials for glial cell isolation and cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>P1-2 Sprague Dawley rat pups</td><td>Charles River</td><td>CD Sprague Dawley rat strain code 001</td><td></td></tr><tr><td>Dissector scissors - slim blades (small)</td><td>Fine Science Tools</td><td>14081-09</td><td></td></tr><tr><td>Surgical scissors - Toughcut (large)</td><td>Fine Science Tools</td><td>14130-17</td><td></td></tr><tr><td>Fine forceps (Dumont #5)</td><td>Fine Science Tools</td><td>11521-10</td><td></td></tr><tr><td>Curved fine forceps (Dumont #7)</td><td>Fine Science Tools</td><td>11271-30</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution (HBSS)</td><td>Gibco</td><td>14170-112</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium and Ham&#039;s nutrient mixture F-12 (DMEM/F12)</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>Penicillin-streptomycin (PS)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>12483-020</td><td></td></tr><tr><td>0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA)</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P-6282</td><td></td></tr><tr><td>50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>05-539-13</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>05-539-5</td><td></td></tr><tr><td>12 well Tissue culture treated plates (Cellstar)</td><td>Greiner Bio-One</td><td>665 108</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Fisher Scientific</td><td>13-676-10F</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Fisher Scientific</td><td>12-676-10K</td><td></td></tr><tr><td>Petri dish (60 mm X 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Petri dish (100 mm X 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Microscope Coverslip (18 mm)</td><td>Fisher Scientific</td><td>12-545-100 18CIR</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Materials for microscopy (confocal and scanning electron microscopy)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse monoclonal anti-CNPase</td><td>abcam</td><td>ab6319</td><td></td></tr><tr><td>Rabbit anti-Iba1</td><td>Wako Laboratory Chemicals</td><td>019-17741</td><td></td></tr><tr><td>Chicken anti-GFAP</td><td>abcam</td><td>ab4674</td><td></td></tr><tr><td>Hoechst 33342</td><td>Fisher Scientific</td><td>62249</td><td></td></tr><tr><td>Fluoromount-G</td><td>Fisher Scientific</td><td>00-4958-02</td><td></td></tr><tr><td>Formalin</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td>Buffered (10%)</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electon Microscopy Sciences</td><td>157-8</td><td>Buffered (8%)</td></tr><tr><td>Guteraldehyde</td><td>Electon Microscopy Sciences</td><td>16019</td><td>Buffered (8%)</td></tr><tr><td>Osmium tetraoxide</td><td>Electon Microscopy Sciences</td><td>19152</td><td>Buffered (2%)</td></tr><tr><td>Hexamethyldilazane (HMDS)</td><td>Electon Microscopy Sciences</td><td>16700</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Electon Microscopy Sciences</td><td>15055</td><td>Anhydrous</td></tr><tr><td>Microscope Slide (25 X 75 X 1 mm)</td><td>VWR International</td><td>48311-703</td><td></td></tr></tbody></table>

improved 3d hydrogel cultures of primary glial cells for in vitro modelling of neuroinflammation

在中枢神经系统, 许多急性损伤和神经退行性疾病, 以及植入设备或生物材料的设计, 以提高功能的结果相同的结果: 过量炎症导致胶质, 细胞毒性, 和/或形成一个共同加剧伤害或防止健康恢复的神经胶质瘢痕。为了建立一个系统来塑造神经胶质瘢痕形成和研究炎症过程, 我们已经产生了一个3D 细胞支架, 能够容纳原代培养的胶质细胞: 小胶质组织, 调节异物反应, 并启动炎症事件, 星形胶质细胞的反应形成一个纤维状的疤痕, 和突, 通常易受炎症损伤。本工作提供了一个详细的分步方法, 对透明质酸基3D 水凝胶支架与大鼠脑源性胶质细胞的制备、培养和显微表征。此外, 通过共焦免疫荧光和扫描电子显微镜对细胞包封和水凝胶支架的特性进行了研究, 并展示了用生物活性基质修饰支架的能力, 以及将商业基板混合物加入到改进的细胞整合中。

为了在高吞吐量下对神经组织宿主反应和胶质瘢痕进行建模,体外系统需要一个具有生物相容的基质材料的3D 细胞支架, 在原位形成过程中不会发生细胞毒事件, 并且可以修改用生物活性成分指导仁慈的反应。为此, 我们创建了一个基于透明质酸的3D 细胞支架系统, 并封装了一个主要的混合胶质细胞群体, 以研究细胞间的相互作用和胶质 bioreactivity。对细胞和支架进行了简单的视觉研究。细胞亚群的形态学, 包括突 (CNPase 在绿色), 小胶质 (Iba1 在红色) 和星形胶质细胞 (黄胶质), 通过共聚焦荧光显微镜 (图 3Ai第二 Ci ii) 显示。扫描电镜 (图 3Aiii-iv-Ciii-iv) 也显示了组合支架和细胞形态学。在这些图像中, 显示了低 (i 和 iii) 和高 (ii 和 iv) 分辨率代表图像。将 2D PLL 片涂层 (图 3A) 与 3D 0.5% w/v 型哈马支架 (图 3B) 进行比较, 并将20% 伏/v 基板混合物混合在 0.5% w/v 型哈马 (图 3C) 中。这样做是为了证明在2D 和3D 细胞培养的形态学差异, 以及在支架上引入生物层的效果。在补充信息中也提供了3D 重建。
随着3D 平台的引入, 每个细胞类型的细胞形态发生了变化。在2D 粘附培养 (PLL 涂层玻璃片), 突出现典型的圆形和形成的小网络通常连接与轻度标记的胶质蛋白的过程。小胶质细胞比其他单元小, 并有较小的分支过程, 并没有延伸到远离他们的细胞体 (< 20 µm)。星形胶质细胞是更多样化的形状, 有些具有径向形态学与较小的细胞体和广泛的薄, 分枝过程, 而另一些是纤维状的扁平的外观和很少的广泛的过程, 大衣的表面。在3D 的哈马文化中, 突出现在星团中, 分支成小球体。小胶质细胞是圆的以少量过程, 并且星形胶质是被环绕或分支的辐状地入网络从一个单独群。一般而言, 这些细胞似乎已经从单点或停留在哈马地区, 而不与其他细胞, 这是不同的2D 文化, 并暗示通过矩阵的流动性有限。当20% 伏/v 的基板混合物添加到光聚合混合物, 所有的胶质亚型与3D 平台集成, 并形成了更广泛的, 交互式分支结构。突扩展的球状突起与非基底层的混合形态相似, 但另外出现的过程是沿整个支架中的星形胶质细胞过程分支的薄互连网络延伸。小胶质细胞被分散在这些网络中, 随着薄的过程与组织中的形态学更加一致。总的来说, 这种形态表明, 所有三种胶质细胞都可以自由地整合到哈马-基底板混合物中, 可能通过增加对混合基质的粘附, 或者通过增加对脚手架的改造能力。
通过扫描电镜观察, 发现细胞形态学证实了共焦显微。哈马矩阵出现在表面光滑的10-50 µm 孔表面。细胞附着在哈马的表面, 在下面的层中也依稀可见。这些胶质细胞没有形成可比较的形态, 那些在2D 文化中指出, 然而, 随着基板混合物的添加, 广泛的网络观察。在哈马-基底板混合面上观察到较细的星形胶质细胞纤维, 并以无缝的方式延伸到基体中。这可以依稀可见的矩阵折叠周围的细胞, 建议细胞可能会改变脚手架, 形成了他们想要的构象。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56615/56615fig1.jpg"> 图 1: 基于 methacrylated 透明质酸 (哈马) 的3D 细胞培养和聚合协议的示意图.胶质细胞 (2 周大鼠脑培养) 与哈马和 pipetted 混合在一个玻璃片上的烷 (硅橡胶) 霉菌。这种混合物是暴露在一个高强度的绿色 LED 光聚合水凝胶5分钟。模具被移除, 凝胶在培养基中孵化。有代表性的细胞包括小胶质 (红)、星状胶质细胞 (黄色) 和突 (绿色)。<a href="//cloudflare.jove.com/files/ftp_upload/56615/56615fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56615/56615fig2.jpg"> 图 2: 1 天大鼠脑的解剖.整个大脑的图像 (A), 解剖皮质和小脑 (B), 一个皮质与脑膜 (C), 剥离的脑膜与钳子 (D), 和无 meninge 皮质 (E)。<a href="//cloudflare.jove.com/files/ftp_upload/56615/56615fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56615/56615fig3.jpg"> 图 3:荧光共焦 (i. ii) 和扫描电子 (iii-iv) 显微的混合胶质细胞的人口在 2D PLL 涂层 (A), 哈马 (B), 和哈马-基础叶片混合物 (C)。显示了较低的 (i、iii) 和更高 (ii、iv) 放大图像。标签包括 Iba1 在红色的小胶质细胞, 黄胶中的星形胶质, CNPase 在绿色的突, 和赫斯特核染色。用于扫描电子显微的共焦图像和 50 (iii) 和20µm (iv) 的比例条 = 25 µm。水凝胶为0.5% 伏/v, 基底层混合物体积为20%。<a href="//cloudflare.jove.com/files/ftp_upload/56615/56615fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 3D 荧光显微与混合胶质细胞群的哈马-基底叶片混合物的重建.标签包括 Iba1 在红色的小胶质细胞, 黄胶中的星形胶质, CNPase 在绿色的突, 和赫斯特核染色。水凝胶是0.5% 瓦特/v, Getrex 是20% 的体积。<a href="http://cloudflare.jove.com/files/ftp_upload/56615/Figure_1a_Final.avi">请单击此处下载此文件.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>哈马</td>
			<td>基底叶片</td>
			<td>nvp</td>
			<td>茶</td>
			<td>他们</td>
		</tr>
<tr>
<td>工作浓度</td>
			<td>0.5% 瓦特/伏</td>
			<td>20% 伏/五</td>
			<td>0.10%</td>
			<td>0.10%</td>
			<td>. 01 毫米</td>
		</tr>
<tr>
<td>12-井板</td>
			<td>2% 瓦特/伏</td>
			<td>100% 瓦特/伏</td>
			<td>10% 伏/五</td>
			<td>10% 伏/五</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>1.4 毫升共计</td>
			<td>350µL</td>
			<td>280µL</td>
			<td>14µL</td>
			<td>14µL</td>
			<td>14µL</td>
		</tr>
</tbody></table>表 1: 单元封装的示例。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>内容</td>
			<td>孵化时间</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>30%</td>
			<td>30分钟</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>50%</td>
			<td>30分钟</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>70%</td>
			<td>30分钟</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>90%</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>100%</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>乙醇</td>
			<td>100%</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>乙醇: HMDS</td>
			<td>75:25</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>乙醇: HMDS</td>
			<td>50:50:00</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>乙醇: HMDS</td>
			<td>25:75</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>HMDS</td>
			<td>100%</td>
			<td>20分钟</td>
		</tr>
<tr>
<td>HMDS</td>
			<td>100%</td>
			<td>一</td>
		</tr>
</tbody></table>表 2: 脱水时乙醇和 HMDS 的渐进递增。

Watch this Scientific Journal Video about Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation at JoVE.com

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

在此, 我们为大鼠脑源性胶质细胞的3D 培养提供了一项协议, 包括星状细胞、小胶质和突。我们展示了原细胞培养, methacrylated 透明质酸 (哈马) 水凝胶合成, HAMAphoto 聚合和细胞封装, 和样品处理共聚焦和扫描电子显微成像。

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 ...

improved-3d-hydrogel-cultures-primary-glial-cells-for-vitro-modelling

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

14.6K 次观看.  University of Alberta. 在此, 我们为大鼠脑源性胶质细胞的3D 培养提供了一项协议, 包括星状细胞、小胶质和突。我们展示了原细胞培养, methacrylated 透明质酸 (哈马) 水凝胶合成, HAMAphoto 聚合和细胞封装, 和样品处理共聚焦和扫描电子显微成像。

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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

科研

生物工程

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

神经科学

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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 ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

胚胎小鼠脑混合细胞培养物研究感染和先天免疫

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

作者聚焦:从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

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

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从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫