Name: ultrathin porated elastic hydrogels as a biomimetic basement membrane for dual cell culture
Uploaded: 2017-12-26T13:00:00.000+00:00
Duration: 11 min 34 s
Description: Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

作者希望感谢 Prof. 的 Prof. Chinedum Osuji 的谈话和材料科学的专业知识。这项工作的经费由 Dubinsky 新倡议奖和国家卫生研究院 NIBIB BRPR01 EB16629-01A1 提供。

在使用之前请阅读所有材料的材料安全数据表 (MSDS), 并在任何时候使用安全预防措施。
1. 氧化锌针的合成
<ol>
	<li>准备250毫升的0.04 米锌 (没有3)2* 6H2O 解决方案, 加入2.9749 克的硝酸锌到250毫升的水。</li>
	<li>将6克氢氧化钠加到150毫升的水中, 制备150毫升的1米氢氧化钠。</li>
	<li>在带有搅拌器的热板上设置矿物油浴, 并在室温下将500毫升圆底烧瓶浸入油浴中。</li>
	<li>添加250毫升锌 (无3)2* 6H2O 到烧瓶中, 并开始搅拌反应。</li>
	<li>添加150毫升氢氧化钠溶液 (白色沉淀将形成短暂, 然后消失, 因为这两个解决方案继续混合)。搅拌2小时发现。</li>
	<li>将烧瓶加热至 55-60 ° c, 继续搅拌24小时。</li>
	<li>关闭热量, 让溶液在搅拌时冷却到室温。</li>
	<li>过滤解决方案的 chner 漏斗与11µm 孔径大小, 并允许干燥过夜, 破获。当过滤纸不再湿从浇溶液和白色粉末形成了在漏斗孔, 微粒完全地烘干。</li>
	<li>收集 ZnO 针, 并准备扫描电子显微镜 (SEM), 以确认针状形态。简单地, 将碳带贴在大头针上, 用金属刮刀将 ZnO 针涂抹在碳带上。溅射涂层与8毫米铱在密度 22.4 g/cm3和获取图象在10伏。</li>
</ol>
2. 在盐酸诱导的 PEG 释放的显微镜下添加牺牲锌层
<ol>
	<li>在200毫升甲醇中溶解1.756 克醋酸锌, 制备醋酸锌溶液。</li>
	<li>清洁 3 "x 1" 纯显微镜幻灯片与70% 乙醇和一次性擦拭。允许空气干燥10分钟。</li>
	<li>将一个150毫米的玻璃培养皿放到热板上, 预热至 150-160 ° c。</li>
	<li>使用吸管灯泡的玻璃吸管, 通过应用5滴醋酸锌, 在幻灯片上形成一层薄薄的薄片, 用镊子和涂层幻灯片握住玻璃幻灯片。允许过剩的解决方案滴回库存解决方案。</li>
	<li>将滑块放在预热培养皿 (150-160 ° c) 中, 将醋酸锌涂层侧面朝上。在板上留15分钟。</li>
	<li>用镊子将滑轨移开, 使其冷却至室温。该幻灯片将出现涂上白色条纹。</li>
	<li>使用一次性擦拭去除剩余的多余的锌, 去除突出的白色条纹。表面应该是均匀的。</li>
	<li>将准备好的幻灯片暴露在生物安全罩中的 UV 光中至少1小时, 以确保不育。 注意: 紫外线对眼睛和暴露的皮肤有害。避免直接接触眼睛或皮肤, 在不使用时关闭电源。</li>
</ol>
3. 硅胶隔离器的制备
<ol>
	<li>切割硅胶板到正方形小于 1 "x 1" (隔离器必须能够适应6井道)。</li>
	<li>8-12 毫米孔在正方形的中心与活检拳打。</li>
	<li>高压釜硅胶隔离器20分钟在121.0 ° c 和1.12 公斤/厘米。</li>
</ol>
4. peg 溶液的制备及 peg 凝胶的合成
注: PEG 的功能化和聚合已被我们的实验室和其他人广泛地探索和详述。8,10,11,13,14,15,16,17
<ol>
	<li>结合 RGD 细胞黏附肽与丙烯-PEG-NHS 在50毫米碳酸氢钠 (pH 8.5) 的1:1 摩尔比, 使功能化的胺末端的肽与丙烯酸酯基团, 一个生物化学的反应, 这是以前其特点是产量大于85% 的效率。17</li>
	<li>将 22-甲氧基2苯基与 1-乙烯-2-吡咯烷酮混合, 在300毫克/毫升的浓度下制备光引发剂。</li>
	<li>在单独的1.5 毫升管, 权衡以下:20 毫克的 ZnO 针, 12.5 毫克的 peg DA, 和2.5 毫克的 peg-RGD。</li>
	<li>在270µL 10% FBS/PBS 溶液中悬浮20毫克氧化锌针;漩涡混合 (大约十五年代)。</li>
	<li>简要地 (和 #60; 1 s) 在一个台式微型离心机旋转解决方案, 使任何 ZnO 骨料到该管的底部。</li>
	<li>收集250µL 从顶部的 ZnO 溶液, 以确保骨料保持在底部的管。结合 peg 和 peg-RGD, 创建一个约2.5 毫米 RGD 的 peg 解决方案。漩涡混合 (大约十五年代)。</li>
	<li>添加2µL 苯乙酮/n-乙烯基吡咯烷酮照片引发剂和涡流简要混合 (约5秒)。</li>
	<li>添加20µL 的聚合物溶液沿中心的 ZnO 涂层的幻灯片。</li>
	<li>在上面慢慢降低第二张幻灯片, 与 zno 涂层面向下 (PEG 解决方案应在两个 zno 涂层层)。尽量防止气泡的形成。沿主轴横向移动幻灯片, 以允许任何气泡逃逸, 并将解决方案传播到一个薄薄的层。用聚合物溶液覆盖大部分的滑动。使用顶部幻灯片创建轻微的悬垂, 以确保在以后的步骤中可以将幻灯片拆分。</li>
	<li>在 365 nm 紫外线灯 (大约10兆瓦/cm2) 下的幻灯片上进行交联, 15 分钟。</li>
</ol>
5. 从玻璃玻片中释放 PEG 凝胶
<ol>
	<li>将压力施加到悬垂滑块上, 并以缓慢的速度手动拉出两张幻灯片, 以避免使幻灯片破裂。允许凝胶干燥至少5分钟, 或过夜。</li>
	<li>将幻灯片放在无菌的 25 mm 玻璃培养皿中, 并将1米 HCl 溶液轻轻地倒入幻灯片上, 只用足够的时间覆盖幻灯片 (大约 10-20 毫升)。轻轻摇动培养皿;当 HCl 溶解牺牲的锌涂层和针头时, 该凝胶应该开始从滑梯上脱落。一旦凝胶从滑梯上释放出来, 将 HCl 倒入股票溶液中。</li>
	<li>将大约25毫升的 1x PBS 轻轻地倒入培养皿中冲洗, 直到滑块和凝胶被淹没。</li></ol>小心地将 PBS 倒入垃圾箱。
	<li>轻轻地将大约25毫升的 1x PBS 倒入培养皿中, 直到凝胶漂浮在溶液中。</li>
	<li>使用镊子, 小心地滑动硅胶下的有机硅隔离器, 并提起凝胶上。</li>
	<li>将隔离器和凝胶转移到6个装满无菌 1x PBS 的盘子里。</li>
	<li>重复这个过程, 直到所有的凝胶已经从幻灯片中删除, 并浸泡在 PBS。将准备好的凝胶暴露在生物安全罩中至少1小时, 以确保不育。</li>

6. 种子细胞双层
<ol>
	<li>准备 A549 细胞进行播种。简要地, 冲洗75厘米2烧瓶与5毫升的 1x PBS, 添加3毫升0.25% 胰蛋白酶-EDTA, 让坐在 2-3 分钟在37° c。<ol>
		<li>机械搅拌烧瓶, 淬火反应与3毫升 Dulbecco 氏改良鹰培养基与10% 胎牛血清和1% 青霉素-链霉素, 并收集到一个15毫升圆锥烧瓶。冲洗与额外4毫升的完整 Dulbecco 的修改鹰的媒体和收集到同一个圆锥。旋转细胞在 475 x g 为6分钟。</li>
	</ol>
	</li>
	<li>当细胞旋转下来, 添加一小滴完整的 Dulbecco 的改良鹰介质到每一个6井板的井。使用镊子转移硅胶隔离器与凝胶进入新的6井板, 这样, 凝胶是休息的媒体下降。现在, 凝胶被扩散到一个薄薄的层, 检查, 以确保没有大孔可见的眼睛。这应在细胞播种前立即完成, 以避免凝胶的干燥和开裂。</li>
	<li>Dulbecco 氏改良鹰培养基中的重细胞, 10% 胎牛血清和1% 青霉素-链霉素, 浓度为 6 x 105细胞/毫升。添加细胞悬浮滴明智的凝胶的中心, 试图保持一个半月板的穿孔出面积的硅膜。允许细胞坚持4小时。</li>
	<li>4小时后, 加入0.5 毫升的 Dulbecco 的改良鹰完全培养基到井中, 并在夜间孵育在37° c, 以允许完全粘合。</li>
	<li>第二天, 准备内皮细胞播种。<ol>
		<li>冲洗75厘米2烧瓶与5毫升的 1X PBS, 添加3毫升0.25% 胰蛋白酶-EDTA, 让坐在 2-3 分钟在37° c。</li>
		<li>机械地搅动烧瓶, 淬火反应与3毫升 M199 培养基与20% 胎牛血清, 1% 青霉素-链霉素, 和1% 生长补充剂, 并收集到一个15毫升圆锥。</li>
		<li>冲洗4毫升的 M199 完整的媒体和收集到同一个圆锥。旋转细胞在 475 x g 为6分钟。</li>
	</ol>
	</li>
	<li>当细胞向下旋转时, 在一个新的6井板中, 在每个井中添加一小滴完整的培养基199。在硅树脂的中心放置一个新的有机硅隔离器在井与媒介下落。</li>
	<li>使用镊子, 小心地翻转硅胶隔离器支持 PEG 凝胶与 A549 细胞到隔离在新的6井板。</li>
	<li>重内皮在浓度为 6 x 105细胞/毫升, 并添加细胞悬浮到中心的翻转凝胶滴明智, 试图保持一个半月板上的凝胶在穿孔出面积的硅膜。允许细胞坚持2小时。</li>
	<li>2小时后, 轻轻地将2毫升的 M199 完全介质添加到每个井中。</li>
</ol>
7. 免疫荧光
<ol>
	<li>小心地从凝胶中取出介质, 用手动吸管, 并将大约500µL 4% 甲醛 (粉煤灰) 添加到凝胶的中心, 其中细胞被播种。让我们在室温下坐30分钟。 警告: 粉煤灰是一种有毒化学物质;穿戴防护和保证处理在生物安全内阁做。</li>
	<li>小心地取出粉煤灰, 并在 PBS 中添加大约500µL 2% BSA 的每一个凝胶。让我们在室温下坐1小时。</li>
	<li>小心删除 BSA。在 PBS 中以稀释1:100 的 2% BSA 制备初级抗体, 并在每个凝胶中加入500µL。让我们在室温下坐1小时。用500µL 1x PBS 轻轻冲洗。<ol>
		<li>重复相同的过程与二次抗体。使用以下抗体: 1) 人 CD144 (VE 钙黏附素) 克隆 16B1;2) 人 CD324 (e-钙黏蛋白) 克隆 6714;3) 人 CD31 (PECAM-1) 克隆 C-20;4) anti-A549;5) anti-mouse FITC;6) 山羊 Alexa 氟647。为了形象化 F 肌动蛋白, 添加500µL 的笔 (10 µg/毫升在 PBS) 20 分钟。</li>
	</ol>
	</li>
	<li>小心地去除二次抗体或笔并且增加500µL DAPI (0.1 µg/毫升) 为 20 min. 小心地删除 DAPI 溶液, 并轻轻地将1.5 毫升的 1x PBS 添加到每个井中, 以使凝胶处于悬浮中。<ol>
		<li>使用镊子和硅胶隔离器, 将一个胶凝体转移到玻璃玻片上。在成像前立即对每个凝胶进行处理, 并在成像后将凝胶替换为 PBS 溶液。</li>
		<li>或者, 使用 DAPI 安装介质安装凝胶。用指甲油密封好样品, 在2天内获得图像, 防止凝胶的干燥和开裂。有关疑难解答, 请参阅表1。</li>
	</ol>
	</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+normal+and+fibrotic+human+lung+matrices+as+a+culture+system+for+in+vitro+investigation.">Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.</a> Am J Respir Crit Care Med. 186, 866-876 (2012).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes:+structure,+assembly+and+role+in+tumour+angiogenesis.">Basement membranes: structure, assembly and role in tumour angiogenesis.</a> Nat Rev Cancer. 3, 422-433 (2003).</li><li>Roudsari, L. C., Keffs, S. E., Witt, A. S., Gill, B. J., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Poly(ethylene+glycol)-based+Tumor+Angiogenesis+Model+to+Study+the+Influence+of+Vascular+Cells+on+Lung+Tumor+Cell+Behavior.">A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior.</a> Scientific Reports. 6, 1-15 (2016).</li><li>Bermudez, L. E., Sangari, F. J., Kolonoski, P., Petrofsky, M., Goodman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+the+translocation+of+Mycobacterium+tuberculosis+across+a+bilayer+of+epithelial+and+endothelial+cells+as+a+model+of+the+alveolar+wall+is+a+consequence+of+transport+within+mononuclear+phagocytes+and+invasion+of+alveolar+epithelial+cells.">The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells.</a> Infect Immun. 70, 140-146 (2002).</li></ol>

作者没有什么可透露的。

这里详述的协议允许我们创建一个可调谐的 PEG 水凝胶作为仿生 BM 支架。具体而言, 通过改变 PEG 分子量、肽共轭策略和氧化锌微晶结构或浓度, 可以分别修改水凝胶的弹性模量、生物化学性质和多孔结构。超薄的 PEG 支架具有更高的孔隙密度和更小的孔径, 更模拟了在体内基底膜中发现的特征, 与标准的渗透性支持模型 (表 2) 相比。此外, 通过这种方法, 我们实现了一个平均厚度小于20µm 的超薄结构。虽然聚碳酸酯的渗透性支持模型已经实现了一个超薄层只有10µm, 我们的知识, 这是第一个自由浮动和可调谐的水凝胶系统, 以达到这个厚度。最后, PEG 水凝胶有大约100帕的弹性模量, 这是比传统的渗透性支撑、组织培养塑料和玻璃的数量级不那么僵硬, 这些都在 GPa 范围内都具有弹性模量, 因此更可与低 (和 #60; 10 帕) 的弹性模量在体内细胞外基质 (表 2)。
实现低弹性模量是本文所提出的工作的主要目标之一, 也是对传统聚碳酸酯渗透性支撑模型使用的一个限制。众所周知, 基底刚度可以决定细胞的行为, 工作表明, 基底刚度可以指导细胞的命运分化细胞。12硬基板也通常用于模拟疾病状态, 包括纤维化组织或癌变肿瘤, 因此不能准确地代表健康组织。18,19,20,21为了研究静止的细胞表型和功能, 关键是要使用模拟弹性模量的基底, 这是相关的体内。该系统的主要优点之一是能够调节凝胶的力学, 这可以通过使用不同分子量的 PEG 来实现。由于许多疾病与 ECM 硬化有关, 该系统提供了一个仿生平台来研究健康与患病 BM 对细胞功能的影响。该系统的进一步修改也可以通过改变包括细胞黏附的肽片段来实现。为了本研究的目的, RGD 被使用由于它无处不在的表达在许多 BM 蛋白质和它的能力促进牢固的细胞附件。除了 RGD, 层粘连蛋白片段也可用于促进层粘连蛋白-上皮相互作用, 引导心尖基细胞极化。1正如 BM 的硬度在疾病中改变一样, 基底膜的组成在病理条件下被重塑。采用胺-反应型 peg-NHS 前驱体, 可以将多种肽和蛋白质共轭到 PEG 聚合物基。11,14,15这特别有利于研究整合素参与对不同整合器受体的影响。例如, 用纤维连接蛋白和胶原蛋白表达的 RGD 序列具有多种整合, 但对整合素αvβ3具有较高的特异性。为了特别模仿纤维连接蛋白丰富的基质, 可加入亮氨酸-天门冬氨酸-缬氨酸 (LDV) 序列, 以参与α4β1, 或模仿胶原膜, 天冬氨酸-甘氨酸-谷氨酸-丙酸 (DGEA), 可以增加参与整合素α2β18
内皮上皮双层只是一个细胞双层的例子, 可以创建与模型在这里提出。其他双层存在自然在体内, 仅由一个薄 BM 或 ECM, 包括内皮-细胞和内皮平滑肌细胞双层。因此, 该方法的应用广泛, 从肺医学到血管生物学。此外, 由于这些水凝胶的独特孔隙度, 它们提供了进行功能研究的能力, 以前的凝胶模型无法支持。通过在我们的超薄水凝胶系统中加入毛孔, 我们已经为渗透性支持模型创建了一个增强的仿生替代品, 使调查细菌浸润, 一个常见的发生在肺部结核的上皮内皮屏障。3,6,14,22同样, 通过测量凝胶的 RGD 功能化细胞黏附表面上的细胞捕获率, 可以对炎症细胞在血管细胞培养中的轮回或趋化性进行研究, 如前所示。16这是一个明显的优势优于以往的水凝胶的方法, 已经达到共培养条件, 通过封装。这种系统允许两个细胞类型之间的接近和细胞间的接触, 并已成功地测量功能, 如血管生成和血管, 但缺乏 two-dimensional 系统提供的无障碍, 这提供了一个很更简单的平台, 用于测量泄漏, 渗透, 或在共细胞屏障的活动迁移。17
因此, 此处演示的方法为跨多个领域的各种研究提供了一个平台, 并为从体外研究中生成更多相关数据提供了一个垫脚石, 以进一步了解体内病理和机制。

细胞外基质 (ECM) 构成了支持细胞附着的蛋白质支架, 在不同细胞类型之间充当屏障, 是复杂组织和器官的重要组成部分。与间质结缔组织不同, 基底膜 (BM) 是一种特殊类型的 ECM, 它是一种屏障, 可以将组织间隔分开。BMs 大约有100µm 厚, 因此可以在两边的细胞之间进行直接和间接的交流。两个常见的例子的 bms 是血管 bms, 发现在微血管壁之间的周和内皮细胞, 和气道 bms, 发现之间的内皮细胞和上皮组织。BMs 在调节细胞功能, 如细胞极性和迁移, 在健康和疾病中起着重要的作用。1 BMs 的组成、刚度、结构和孔隙度在不同的器官系统间变化, 以促进不同的生理功能。例如, BM 毛孔对于维持细胞间的通讯、可溶性分子的扩散, 以及在感染期间炎症或细菌迁移的免疫细胞是至关重要的。在气道中, 孔隙横跨 BM 的全厚度, 直径范围从0.75 到3.86 µm.2
BM 的稀薄的本质保证, 虽然细胞类型是物理上分开的, 通过分泌-和接触介导的信令的胞间通信被保留。因此, 研究人类疾病在体外,研究人员依靠多孔渗透支持插入到培养细胞双层。3这些模型对于了解在健康和疾病中发挥作用的蜂窝通信非常重要。3,4,5,6,7渗透性支持插入满足了了解细胞信号如何调节生理过程的基本要求, 如白细胞的招募和细菌浸润;然而, 这些插入物有很大的局限性, 无法模仿人类的 BM. 渗透性支持插入缺乏机械和生化调谐, 并且简单化的多孔结构不模仿造成不规则的毛孔的纤维结构典型的 BMs。因此, 越来越需要可调谐的系统, 可以重现影响细胞过程的自然 BM 属性。
聚合物基板是发展仿生 BMs 研究细胞双层的理想候选对象, 它在更接近于体内环境的背景下进行。8,9,10,11,12聚合物是机械可调谐的, 可以进行化学修饰, 以加入仿生肽片段.11,12,13 bioinert 聚合物聚乙二醇 (peg) 可用于构建仿生 BMs, 最近的工作详细介绍了可用于支持细胞生长的多孔网络的机械可调谐 PEG 精氨酸-甘氨酸 (RGD) 凝胶的合成和炎症细胞趋化。14虽然发布的 PEG-based 基板提供了比可渗透支持更逼真的人类 ECM 模型, 但这些模型中的许多都非常厚, 有大约775µm 的深度, 从而限制了用细胞细胞创建双层培养物的能力。联系.14
在这里, 我们提出了一个协议, 建立一个 PEG 聚合物 BM 模仿, 克服了目前的细胞双层培养技术的许多限制。我们已经开发了一种模板化方法, 它将氧化锌作为一种用于制造微晶生产的广泛使用的材料, 在合成和交联过程中加入到聚合物中, 随后被选择性地从产生的散装聚合物。这个过程产生一个随机的多孔网络, 模仿人类 BMs 的曲折和互联的孔隙网络。此外, 通过改变针生产过程中的反应化学计量, 可以通过更改氧化锌微的大小和形状来改变孔隙率。在这里开发的技术创建了一个超薄的水凝胶, 模仿人类 bm 的厚度. 最后, 这些 bm 结构的力学、孔隙率和生物化学组成很容易被改变, 产生微环境, 这是最类似于在体内看到的。

<table><tbody><tr><td>1M Hydrogel Chloride (HCl)</td><td>EMD</td><td>HX0603-75 2.5L</td><td>Sterile. Use in fume hood with eye protection and gloves.</td></tr><tr><td>1X PBS</td><td>Gibco</td><td>14040-133 500 mL</td><td>None</td></tr><tr><td>Zinc Nitrate Hexahydrate (Zn(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>228737-500g</td><td>Use with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Macron Chemicals</td><td>278408-500g</td><td>Use with eye protection and gloves.</td></tr><tr><td>Zinc Acetate Dihydrate ((CH&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;Zn&lt;sup&gt;2+&lt;/sup&gt;&amp;bull;2H2O)</td><td>Fisher Scientific</td><td>AC45180010 1 kg</td><td>Use with eye protection and gloves.</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>J.T. Baker</td><td>9070-05 4L</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>VWR Life Science Seradigm Premium Grade FBS</td><td>VWR</td><td>97068-085</td><td>Sterile filter. 5 mL FBS in 45 mL PBS</td></tr><tr><td>Mineral oil</td><td>CVS&amp;nbsp;</td><td>PLD-B280B</td><td>None</td></tr><tr><td>Round bottom flask</td><td>ChemGlass</td><td></td><td>N/A</td></tr><tr><td>Thermometer</td><td></td><td></td><td>N/A</td></tr><tr><td>Stir bar</td><td></td><td></td><td>N/A</td></tr><tr><td>Plain precleaned microscope slides 3&quot;x1&quot;x1&quot; mm thick</td><td>Thermo Scientific</td><td>420-004T</td><td>Spray with ethanol and let dry prior to use.</td></tr><tr><td>Glass pasteur pipets</td><td></td><td></td><td>N/A</td></tr><tr><td>1 mL rubber bulbs</td><td></td><td></td><td>N/A</td></tr><tr><td>Plastic 100 mm petri dishes</td><td></td><td></td><td>N/A</td></tr><tr><td>Sterile forceps</td><td></td><td></td><td>N/A</td></tr><tr><td>Silicone isolators</td><td></td><td></td><td>0.8 mm thick</td></tr><tr><td>Polydimethylsiloxane (PDMS) punches</td><td></td><td></td><td>N/A</td></tr><tr><td>Glass bottles</td><td></td><td></td><td>N/A</td></tr><tr><td>6 well plates</td><td>Cellstar</td><td>657 160</td><td>N/A</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Stirrer-hot plate</td><td>VWR Dya-Dual</td><td>12620-970</td><td>Use with eye protection and gloves.</td></tr><tr><td>2,2-Dimethoxy-2-phenylacetophenone (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;COC(OCH&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>24650-42-8</td><td>Use with eye protection and gloves.</td></tr><tr><td>1-Vinyl-2-pyrrolidone (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;9&lt;/sub&gt;NO)</td><td>Aldrich</td><td></td><td>Use with eye protection and gloves.</td></tr><tr><td>Polyethylene Glycol 10,000 (H(OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;10,000&lt;/sub&gt;OH)</td><td>Fluka</td><td>81280-1kg</td><td>Use with eye protection and gloves.</td></tr><tr><td>RGDS</td><td>Life Tein</td><td>180190</td><td>Use with eye protection and gloves.</td></tr><tr><td>Blak-Ray long wave UV lamp</td><td>UVP</td><td>Model B 100AP</td><td>N/A</td></tr><tr><td>Eppendorf tubes</td><td>USA Scientific</td><td>1615-5500</td><td>N/A</td></tr></tbody></table>

ultrathin porated elastic hydrogels as a biomimetic basement membrane for dual cell culture

基底膜是细胞双层的一个重要组成部分, 它的刚度、组成、结构和孔隙度都不同。体外内皮上皮双层的研究传统上依赖于允许双层培养的渗透性支持模型, 但渗透性支持在复制人类基底膜多样性的能力上是有限的。相比之下, 需要化学合成的水凝胶模型是高度可调谐的, 允许通过加入仿生肽或蛋白质来修改材料的硬度和生物化学成分。然而, 传统的水凝胶模型是有限的功能, 因为他们缺乏孔隙的细胞接触和功能的体外迁移研究。此外, 由于传统的水凝胶的厚度, 合并毛孔, 跨越整个厚度的水凝胶一直是挑战。在本研究中, 我们使用聚乙二醇 (PEG) 水凝胶和一种新颖的氧化锌模板化方法来解决仿生水凝胶以前的缺点。因此, 我们提出了一个超薄, 基底膜状水凝胶, 允许融合细胞双层的文化在一个可定制的脚手架, 具有可变的孔隙结构, 机械性能和生物化学成分。

RGD 水凝胶是在两个牺牲氧化锌层之间夹聚合物溶液形成的, 用氧化锌针创建孔隙模板。然后用盐酸除去牺牲的氧化锌组分, 生成具有连续孔隙的超薄 PEG 水凝胶 (图 1)。用扫描电镜 (SEM) 对氧化锌针的形貌进行了验证, 平均长度和宽度分别确定为3.92 ±0.089 µm 和0.43 ±0.02 µm (图 2A-2B)。在除去氧化锌针头后, 用 SEM 对水凝胶孔隙进行了可视化, 并进行了表征 (图 2C-2E)。水凝胶的平均孔隙密度为176863±49532孔隙。SEM 还用于可视化和计算标准的3µm 孔聚碳酸酯透水支架插入物的孔隙密度, 发现孔隙密度明显较低, 每平方毫米约2.51 ±0.05 孔 (图 2D,表 2)。氧化锌模板化 PEG 水凝胶的平均孔径为0.19 ±0.01 µm, 明显小于渗透支持插入 (图 2E,表 2) 中观察到的3.47 ±0.21 µm 孔。光学相干断层扫描是用来获取2和3维图像, 并确定水凝胶的厚度漂浮在 1x PBS (图 3A-3B)。平均凝胶厚度为18.89 ±3.47 µm (表 2)。凝胶力学也通过测量弹性模量, 如前所述, 和平均杨氏模量被发现为96.78 ±23.92 帕斯卡 (表 2)。14
免疫荧光显微镜显示内皮细胞和上皮细胞在超薄 PEG-RGD 水凝胶培养后保持其表型特征。内皮细胞维持 PECAM-1 的表达, 上皮细胞染色阳性的 anti-A549 抗体 (图 4A-4B)。两种细胞类型也保持了形成紧密接合的能力。内皮细胞表达 VE 钙黏附素在细胞连接, 和上皮细胞表达 e-钙黏蛋白 (图 4C-4D)。PEG-RGD 水凝胶还支持细胞双层, 并允许细胞的联系, 形成在多孔结构, 通过荧光成像证明笔染色样本 (图 4E)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56384/56384fig1.jpg"> 
图 1.水凝胶制备方案概述。A)合成和收集 ZnO 针。B)制作玻璃玻片;一个牺牲的 ZnO 层的创作。C)硅胶隔离器的制备和灭菌。D)用 ZnO 针制备 PEG 溶液。E)在 ZnO 涂层玻片之间聚合 PEG 凝胶。白线代表牺牲 ZnO 针和涂层。F) HCl 冲洗用于去除针头和从幻灯片中释放凝胶。暗线代表由溶解的 ZnO 针产生的孔隙。G)将凝胶和硅胶隔离器转移到6个好的盘中, 准备进行细胞播种。H)单元格播种。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56384/56384fig1large.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/56384/56384fig2.jpg"> 
图 2.锌针特性和水凝胶孔隙度。A)锌针 (刻度条5µm) 的 SEM 图像。B)锌针的长度和宽度。点代表单个针头的测量;线条表示平均值和标准错误。C)多孔水凝胶 (刻度条2微米) 的 SEM 图像。D)孔隙密度和E)聚碳酸酯透水支架和 PEG 水凝胶的平均孔径。条形表示平均值和标准错误。p & #60; 0.0001 通过 T 检验。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56384/56384fig2large.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/56384/56384fig3.jpg"> 
图 3.水凝胶厚度。光学相干断层扫描显示A) 3 维和B) 2 维水凝胶漂浮在 PBS 中的图像。箭头代表液体表面, 星号代表水凝胶。缩放条是100µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56384/56384fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56384/56384fig4.jpg"> 
图 4.水凝胶支持单层和双层培养.内皮细胞和上皮细胞都能在 PEG 水凝胶上形成汇合的单分子膜, 如荧光染色A) CD31 (CD31 红、DAPI 蓝) 和B) A549 (A549 红、DAPI 蓝) 分别显示。完整的钙黏蛋白结的形成表现为C)内皮细胞 (VE 钙黏附素绿色, DAPI 蓝色) 和D)上皮细胞 (E 钙黏蛋白绿色, DAPI 蓝色) (鳞片100微米)。E)通过笔染色 (笔红、DAPI 蓝) (比例条10µm) 演示细胞双层的两个例子。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56384/56384fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>问题</td>
			<td>潜在解决方案</td>
		</tr>
		<tr>
			<td>凝胶有大孔。</td>
			<td>在使用锌针之前, 一定要将其充分分解。</td></tr></tbody></table>用迫击炮和杵把它们分开可能会有帮助。确保在稀 FBS 中, 在悬浮液中快速旋转后, 大团聚体被移除。
		
		<tr>
			<td>凝胶在播种细胞后粘在6井板的底部。</td>
			<td>在凝胶下面加入一滴培养基, 然后再转入6井板进行播种。</td>
		</tr>
		<tr>
			<td>凝胶覆盖了硅胶隔离器的顶部和底部。</td>
			<td>对于较长的凝胶, 将任何多余的凝胶包裹在硅酮隔离器周围, 以确保在中心处只有一层细胞被播种。</td>
		</tr>
		<tr>
			<td>细胞层从凝胶中弹出。</td>
			<td>细胞可能 over-confluent。种子在较低的密度。要非常小心, 所有冲洗步骤。你也可以增加肽浓度, 提高细胞黏附力。</td>
		</tr>
		<tr>
			<td>细胞没有迅速增殖, 不会达到汇合。</td>
			<td>种子细胞在高密度, 使它们在播种时接近汇合。</td>
		</tr>
		<tr>
			<td>凝胶刚度高于预期。</td>
			<td>减少凝胶在 HCl 中保存的时间。</td>
		</tr>
		<tr>
			<td>凝胶厚度高于预期。</td>
			<td>确保在浇注 PEG 凝胶之前, 将所有的 ZnO 聚合体从玻片上擦掉, 以确保幻灯片平滑。沿主轴移动幻灯片, 以扩大 PEG 解决方案。</td>
		</tr>
	

表1。针对疑难解答问题的建议解决方案。
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>凝</td>
			<td>Transwell</td>
			<td>细胞外基质</td>
		</tr>
		<tr>
			<td>孔隙密度 (孔隙/mm2)</td>
			<td>1.77 x 105 ± 4.9 x 104</td>
			<td>2.51 ±0.05</td>
			<td>8.50 x 102到 7.0 x 104 214</td>
		</tr>
		<tr>
			<td>孔直径 (µm)</td>
			<td>0.19 ±0.01</td>
			<td>3.47 ±0.21</td>
			<td>0.47 到 3.86 214</td>
		</tr>
		<tr>
			<td>厚度 (µm)</td>
			<td>18.89 ±3.47</td>
			<td>10</td>
			<td>0.05 到 0.10 17</td>
		</tr>
		<tr>
			<td>杨氏模量 (人民军) 5</td>
			<td>96.78 ±23.92</td>
			<td>和 #62; 1000</td>
			<td>3.5 14</td>
		</tr>
	</tbody>
</table>
表2。水凝胶的性能与标准渗透性支持插入和胞外基质的比较 (值代表平均值 ±标准错误)

Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

目前的双层培养模式不允许功能性的体外研究, 模仿体内环境。使用聚乙二醇和氧化锌模板化方法, 该协议描述了一个超薄仿生基底膜的发展, 具有可调谐的刚度, 孔隙率和生物化学组成, 密切模仿在体内细胞外基质。

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

ultrathin-porated-elastic-hydrogels-as-biomimetic-basement-membrane

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.7K 次观看.  Yale University. 目前的双层培养模式不允许功能性的体外研究, 模仿体内环境。使用聚乙二醇和氧化锌模板化方法, 该协议描述了一个超薄仿生基底膜的发展, 具有可调谐的刚度, 孔隙率和生物化学组成, 密切模仿在体内细胞外基质。

视频: Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

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 ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

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

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

工程双层凝胶控制升序分化

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

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

<meta charset="utf8"></head><body>使用 Vessels-on-a-Plate 快速制造血管网络的 3D 生物打印

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

<meta charset="utf8"></head><body>作者聚焦:用于高通量血管模型制造的自动化生物打印

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

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微机械动态细胞培养平台

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

工程双层凝胶控制升序分化

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分层海藻酸钠构造:一个异质细胞群的共培养平台

可调水凝胶由肺细胞外基质的三维细胞培养

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性