作者所做的研究部分是由欧洲联盟第七框架方案 (FP7/2007-2013) 根据229239号赠款协议提供的赠款, 并由 AO 基金会、探索性研究合作研究项目急性软骨损伤/损伤/缺损 (CRP) 在项目生物活性和仿生支架的软骨再生 (BIOCART)。

1. 自组装肽支架 (sap) 的制备
<ol><li>在微离心管中, 用 20% (w/v) 蔗糖制备0.5% 肽 RAD16-I 溶液。油脂实验在超声波浴中以最大功率和室温 (RT) 为20分钟的肽溶液, 以确保均匀混合。 注: 要准备的解决方案数量将取决于所需胶囊的数量。RAD16-I 肽在这些超声波条件下不受降解。</li>
	<li>稀释在步骤1.1 中制备的多肽溶液, 所需量为 10% (瓦特/v) 蔗糖, 以获得所需的最终浓度。 注意: 要考虑的一个重要因素是初始肽浓度的调整, 因为最终的矩阵刚度将取决于这个参数。RAD16-I 肽溶液应比3D 结构所期望的最终浓度高2x。通常, 使用的最终 RAD16-I 肽浓度介于 0.15–0.5% (v.) 之间。</li>
	<li>将含有肽溶液的微离心管放入20分钟的超声波浴中, 确保均匀混合。 注: 肽溶液, 如果不立即使用, 可以储存几个月在4摄氏度。</li>
</ol>2. 细胞悬浮制剂
<ol><li>离心机细胞悬浮液 (在胰蛋白酶治疗后获得) 在 60 x g 和 RT 为5分钟。</li>
	<li>使用连接到真空线的巴斯德吸管去除上清液, 用微将 10% (3–5) 蔗糖中的细胞颗粒悬浮。使用手动或自动单元格计数器进行计数单元格。 注: 10% (w/v) 蔗糖溶液是一种等渗和非离子介质, 允许细胞维护和避免肽凝胶在混合过程中。</li>
	<li>离心机细胞悬浮在 60 x g 和 RT 为5分钟. 取出上清液, 再将细胞颗粒悬浮在必要体积 10% (瓦特/v) 蔗糖中, 以获得双倍的预期最终细胞浓度 (可推荐约 4 x 106细胞/毫升)。</li>
	<li>在6井板的每个井中放置所需数量的组织培养插入物 (每个封装一个插入)。</li>
	<li>油脂实验在步骤1中制备的多肽溶液, 最大功率和 RT 为5分钟。</li>
	<li>吸管并且安置40µL 细胞悬浮 (获得在 2.3) 为每个被要求的封装加大约10–15% 额外容量入标准微离心管。</li>
</ol>3. 3D 细胞封装到 sap 中
<ol><li>将 RAD16-I 溶液的等量体积混合在10% 蔗糖 (w/v) (40 µL 每封装), 并在步骤2中制备的细胞悬浮量 (每封装40µL) 相等, 以获得所需肽浓度的细胞悬浮 (10% 蔗糖)。混合吹打慢慢上下大约10次。在混合过程中避免气泡形成。 注意: 这一步是至关重要的, 必须非常小心地执行, 因为细胞是在敌对条件下由于低 pH 肽 (大约 3.5–4.0)。</li>
	<li>使用微, 仔细加载80µL 的悬浮到每个插入。</li>
	<li>将0.5 毫升培养基添加到井底湿润插入膜, 使介质扩散促进肽自组装和凝胶形成。让肽凝胶在流动柜中大约20分钟。 注: 在设定时间内对样品的操纵可能以凝胶破裂告终。</li>
	<li>非常缓慢地添加40µL 中的插入, 让它向下滑动内壁到凝胶。 注意: 通常, 此卷的加载应在20–30之间进行。</li>
	<li>将板材放在孵化器中 (37 °c, 5% CO2, 湿润气氛) 为20分钟。这一步骤和连续培养基添加将有利于浸出的蔗糖和平衡细胞与培养基。</li>
	<li>将60µL的介质添加到插入的内壁 , 使其沿着墙向动。吸入富含蔗糖的井中的培养基, 加入0.5 毫升的新鲜培养基。</li>
	<li>添加60µL 的培养基慢慢地插入和放置在孵化器 (37 °c, 5% CO2, 湿润的气氛) 的板块10分钟。</li>
	<li>吸入井中的介质, 并在井中加入2.5 毫升的新鲜培养基。在插入中添加200µL 介质。将盘子放在孵化器中 (37 °c, 5% CO2, 湿润气氛), 以维持细胞在适当的条件下。</li>
	<li>每天改变媒体。从井底取出旧介质。添加2.5 毫升的新鲜培养基到井和200µL 到插入。 注: 如果细胞分化继续, 特定的诱导培养基将被添加到文化中, 导致所需的细胞类型承诺 (见下文,细胞分化)。</li>
</ol>4. 3 个 d 型结构的细胞存活能力
<ol> <li>注: 细胞生存能力检测被称为活/死-生存能力/细胞毒性试验。</li>
	<li>在15毫升管中, 十年代 homodimer-1 在 PBS 中准备2µM calcein AM 和2µM 溴 EthD-1 (涡流) 的新溶液的必要体积. 保持在黑暗中。 注: 每个样品所用的体积取决于井身尺寸: 6 多井板每井2毫升;1毫升为每井12多井板和0.5 毫升为每井24多井板材。</li>
	<li>使用微, 用2毫升的 PBS 冲洗 3 d-结构调整器样本3次, 然后用步骤4.1 中准备的溶液覆盖。在黑暗中孵化40分钟。</li>
	<li>使用微, 冲洗2毫升 PBS 5 倍的结构。</li>
	<li>使用10X 或20X 放大器在荧光显微镜下可视化样品。</li>
</ol>5. 细胞分化
<ol><li>培养细胞 (37 °c, 5% CO2, 湿润大气) 在软骨基底培养基 (煤层气) 与生长补充剂。 注: 介质的准备工作在制造商的说明书中描述 (见材料表)。</li>
	<li>在2天诱导细胞分化的软骨培养基含有重组人转化生长 factor-b1 [TGFb1] (10 ng/毫升), l-抗坏血酸 2-磷酸盐 [AA2P] (25 µg/毫升) 和地塞米松 (100 nM)。</li>
	<li>在37摄氏度和 5% CO2的恒温环境中保持4周的培养。每隔一天更换软骨介质。</li>
</ol>6. 甲苯胺蓝 (结核) 染色
<ol><li>通过添加并移除2毫升的 PBS 与微, 洗净 3 d-结构的文化。通过在 PBS 中加入2毫升 2% (瓦特/v) p甲醛, 在 RT 中增加2小时。</li>
	<li>在 PBS (PBST) 中洗两次, 2 毫升0.1% 海卫 X-100。</li>
	<li>在2毫升 0.05% TB 的水中孵育20分钟。</li>
	<li>清洗几次 (至少3次) 与2毫升 pbs, 直到 pbs 解决方案清除是明确的。</li>
	<li>在11、13、14的立体显微镜下, 通过目视检查分析样品。 注: 结核表型与阴离子糖复合物在细胞外基质中的复合物, 如软骨 (PG) 和多糖 (插科打诨), 主要由软骨细胞样体细胞分泌。阳性样品被染成蓝色。</li>
</ol>7. 西方污点
注: 本议定书中使用的程序以前在参考11中作了说明。
<ol><li>溶解 3 d-马国贤缓冲液中含有蛋白酶抑制剂鸡尾酒的吹打。</li>
	<li>根据凝胶体积要求 (特定凝胶运行设备) 制备丙烯酰胺凝胶, 并在 150 V16裂解物90分钟的电池。 注: 丙烯酰胺凝胶通常是在 7.5–10% (w/v) 的浓度下制备的, 这取决于蛋白质的分子量。</li>
	<li>将凝胶中的蛋白质转移到聚偏聚氟 (PVDF) 膜中, 在 RT 上应用 40 V 2 小时。</li>
	<li>在一个单独的容器中, 用足够的容积覆盖膜, 在2小时的阻断缓冲器 (PBST 中的 4% (瓦特/v) 脱脂奶粉中) 孵化 PVDF 膜。</li>
	<li>在 PBST 的最后浓度为1毫克/毫升时, 在 RT 中孵育1小时的膜。请参阅材料表。</li>
	<li>添加二级抗体浓度为1毫克/毫升。请参阅材料表。</li>
	<li>用化学发光基底16制备 hP 检测膜。请参阅材料表。</li>
	<li>以化学发光图像16。</li>
</ol>

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作者没有什么可透露的。

以前, 我们的小组和其他人已经描述了使用三维 (3D) 文化平台与不同的细胞系统3,4,5,6,7,8 ,9,10,11,12,13,14,15。在目前的工作中, 我们描述了一种简单可靠的方法来获得3D 培养系统, 适用于任何类型的哺乳动物细胞, 包括任何类型的功能细胞, 胚胎或成体干细胞, 或最终, 功能失调的细胞分离活检或肿瘤等.另外, 独立地, 如果干细胞是胚胎或成人起源他们将有更好的血统承诺容量在3D 环境比在古典2D 文化菜10,11,12, 13,14,15。因此, 该系统的细胞培养将导致分化为功能性组织样结构, 可用于不同的应用, 从修复或再生生物医学的毒理学和药理学平台。
由于细胞微环境与自然组织在基质结构和生物力学、生物物理和生物学参数等方面的相似, 我们在细胞功能上显示出明显的增益。然而, 随着系统变得更加复杂, 需要调节的参数数量也在增加, 其中包括需要一个外部支撑平台 (如主动灌注系统以避免传质现象-相关问题).三维培养细胞在调节基本活动, 如移徙、增殖和分化方面表现较好。他们可以形成复杂的网络, 允许增强的细胞串扰, 这是一个重要的结果, 在3D 增长和区别。事实上, 结构调整可以创造出类似于那些细胞外基质蛋白的条件, 这是一个优势, 因为一个合理的研究所产生的影响每一个组成部分添加到脚手架 (生长因子, 多糖或信号肽) 可以很容易地进行。
与其他天然支架 (如胶原 i 型和胶) 相比, 使用该结构的明显优点如下: 1) 结构调整是一种合成生物材料, 从分批到分批生产的变化最小;2) 结构调整能力具有特定的肽图案功能;和 3) 结构在体外具有低生物降解性, 允许在一段时间内以相同的生物物理性、力学和结构特性维持3D 构造。然而, 在封装步骤中发现使用结构调整器与其他支架的限制, 因为 pH 值低, 细胞处于敌对的环境中。因此, 这是所述方法中的一个关键步骤。此外, 在开始任何实验之前, 确定每个特定细胞类型的 sap 浓度是很重要的。这实际上是必不可少的, 当细胞被培养或进入这些生物材料, 因为每一个特定的细胞类型将呈现最佳的生物力学生长条件。
最后, 我们相信, 设计和制造这种类型的生物材料脚手架将加强开发更多的生理和可靠的3D 组织模型, 以帮助制药业发展更好的治疗方法, 以再生医学, 癌症或任何医疗。

几十年来, 哺乳动物细胞培养是在实验条件下使用经典的二维 (2D) 文化系统, 由于实际和经济问题, 无论非生理方面。虽然这种文化体系有助于研究和理解大多数分子和细胞机制, 但我们今天知道, 需要新的细胞培养范式来研究更复杂的细胞系统。因此, 需要三维 (3D) 培养系统来重建一种 biophysically、biomechanically 和生物学上与自然组织相似的微环境。近年来, 3D 的文化体系, 总的来说, 在研究人员和工业界中变得越来越普遍, 因为它们代表了一种新的研究或筛选模式, 细胞可以在空间中生长, 形成细胞对细胞或细胞对矩阵的相互作用, 迁移和最终分化为特定的细胞血统。
这种方法的总体目标是重建离体内微环境更近的体外细胞微环境。特别是合成自组装肽支架 (sap) 是一种具有独特性质的生物材料;它形成了一个纳米级的孔隙网络, 由多肽之间的弱相互作用组成, 其力学和结构性质类似于自然细胞外基质。换言之, 使用这种材料的合理性是, 它创造了一个真正的3维环境, 是理想的获得 pseudo-3D 组织或器官单位。然而, 最重要的是, 3D 上下文允许3D 结构获得新的生物学功能, 通常不存在于2D 文化平台, 如与组织结构, 质量转移现象, 细胞模式和最终的属性组织形态发生是未来功能性组织和器官研究和发展的关键因素1、2。此外, 在他们的天然对应物 (胶原蛋白, 胶) 的优点是, 它们在室温下非常稳定, 不需要特殊条件的后期生产, 分配或存储3,4,5,6,7,8,9,10,11,12,13,14,15. 在需要时易于处理结构调整;3D 凝胶可以通过增加离子强度或调整 pH 值到中性1,2来简单地得到。最后, 本文所描述的方法已广泛应用于体外促进多种细胞类型的维持、生长和分化, 包括软骨细胞、肝细胞、血管内皮、成骨细胞、神经细胞以及胚胎和体细胞干细胞3,4,5,6,7,8,9,10,11,12,13,14,15. 在目前的工作中, 我们描述了使用一个 3 d-培养系统, 以区分人的扩大关节软骨细胞 (hACh) 成软骨样组织如前所述11。
本文介绍了一种使用结构模型对3D 系统中的细胞进行培养的方法。在这种合成生物材料中, 细胞首先与肽溶液混合, 随后诱导自组装, 在细胞周围创建一个纳米尺寸网络, 从而创造出真正的3D 环境 (图 1)。重要的是要考虑到细胞的行为受到矩阵刚度值 (即扩散, 迁移和分化) 的影响。因此, 一个共同的方法控制是培养细胞上的肽脚手架上具有相同的刚度值 (文化在两个维度)。

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:371px;">Human articular chondrocytes (Ach)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">CC-2550</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RAD16-I peptide solution (PuraMatrix)</td>
			<td>Corning</td>
			<td>354250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose (tissue culture grade)</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture inserts (0.4 &#181;m pore, 12 mm diameter)</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chondrocyte Basal Medium (CBM)</td>
			<td>Lonza</td>
			<td>CC-3217</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SingleQuots of Growth Supplements</td>
			<td>Lonza</td>
			<td>CC-4409</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Lonza</td>
			<td>DE14-801F</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dexamethasone</td>
			<td>Sigma</td>
			<td>D8893</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-ascorbic acid 2-phosphate (AA2P)</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human transforming growth factor-&#946;1 (TGF-&#946;1)</td>
			<td>Millipore</td>
			<td>GF111</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:371px;">RIPA buffer</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R0278</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyvinylidene difluoride (PVDF) membrane</td>
			<td>Invitrogen</td>
			<td>LC 2005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:371px;">SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Actin</td>
			<td>SCBT</td>
			<td>sc-1615</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Collagen I</td>
			<td>Abcam</td>
			<td>ab138492</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Collagen II</td>
			<td>Abcam</td>
			<td>ab3092</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Anti-Collagen X</td>
			<td>Abcam</td>
			<td>ab182563</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Antigoat IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97100</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-mouse IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97023</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Anti-rabbit IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97051</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing mammalian cells in three-dimensional peptide scaffolds

介绍了一种在自组装碳纤维三维 (3D) 支架上培养细胞的有效方法。这种文化系统再现了一个紧密模仿非极化组织结构特征的环境。此外, 该脚手架的独特的内在碳纤维结构使其透明的视觉光, 这使得在显微镜下容易可视化的样品。这一优势主要用于研究细胞的迁移、组织、增殖和分化, 从而使其特定的细胞功能通过染色或探针来发展。此外, 在这项工作中, 我们描述了良好的性能, 这一系统, 以方便地研究扩大人关节软骨细胞分化软骨组织。细胞被封装成自组装肽支架, 并在特定条件下培养, 以促进软骨。在实验的4周内, 三维文化表现出良好的生存能力。如所料, 用软骨诱导剂 (相对于非诱导控制) 培养的样品对甲苯胺蓝 (在软骨细胞外基质中高度存在的污渍多糖 (堵嘴)) 染色强烈阳性, 并表达具体的分子标记, 包括胶原 i 型, II 和 X, 根据西方印迹分析。该协议易于执行, 可用于实验室、工业和实验课程的教育目的。

在目前的工作中, 描述了一个简单和快速的协议, 培养细胞在3D 使用的结构, 以获得可靠的定性和定量数据。sap 创建了一个3D 微环境, 具有独特的属性, 允许培养细胞在理想的条件下进行细胞维护, 增殖和/或分化3,4,5,6,7,8,9,10,11,12. 在我们的小组中, 已经进行了一些研究, 利用人脂肪组织的祖细胞 (hAPC), 扩大人关节软骨 (hACh) 和人正常真皮成纤维细胞 (hNDF) 的文化, 扩大和分化成软骨样组织10,11,12。在这里, 我们描述的3D 培养和重新分化的扩张 hACh 成软骨样组织, 如前所述11。因此, 我们将细胞封装在3维结构中, 并将它们培养成软骨诱导介质, 如图 1 (和协议中所述)。24小时后, 以活/死生存能力/细胞毒性试验评估样品的生存能力。结果表明, 在5天的封包后, 少量细胞死亡 (红色), 在4周的培养后几乎没有死亡细胞 (图 2)。接下来, 我们用甲苯胺蓝染色法将它们染成细胞外基质中的多糖 (插科打诨) 沉积物。正如预期的那样, 感应组对治疗做出了正确反应, 表现出强烈的染色效果 (图 2)。
最后, 用印迹 (图 3) 分析了软骨和肥大标记物的分子标记, 包括胶原 i 型、II. 和 X。在2D 和3D 系统中观察到胶原 i 型 (COL1), 但仅在3D 观察到 130 kDa (代表经加工成熟的 COL1 蛋白在细胞外基质中组装) 的较低波段。这一结果清楚地表明, 在三维度分化的细胞以更生理的方式进行。值得注意的是, 胶原蛋白 II 型 (COL2) 被发现-作为一个单一带的预期分子量出现在细胞外基质-仅在3D 构建在软骨条件下培养。最后, 在软骨条件下培养的2D 和3D 结构中检测到胶原蛋白 X (COL10), 表明4周的培养后有一定程度的肥大。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57259/57259fig1v2.jpg"> 图 1: 细胞封装的示意图表示形式为3维度结构.描述了 hACh 封装的示意图表示, 以获取随机分散在3维结构中的单元格。1. 自组装多肽支架 (sap) 的制备;2. 细胞悬浮制剂;和3. 3 d 细胞封装成结构调整。请注意, 示意图仅显示步骤3.1 到3.4。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57259/57259fig2.jpg"> 图 2: 3 种 d-软骨的细胞性能和分化.在荧光显微镜下检测到5天和4周的细胞活力染色 (绿色和死细胞中的活细胞为红色)。刻度条 = 200 µm. 甲苯胺蓝染色 (蓝硫酸多糖) 对3种 d 型结构在控制和软骨培养基中培养的一般软骨承诺。刻度条 = 500 µm。这一数字已从 l. Recha-桑丘等修改。11.<a href="/files/ftp_upload/57259/57259fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57259/57259fig3.jpg"> 图 3: hACh 的胶原 i、II 和 X 表达软骨.hACh 培养在 2 d-单层和 3 d-结构调整后4周的软骨媒体和控制被评估的表达胶原 i 型 (COL1), 胶原 II 型 (COL2) 和胶原蛋白类型 X (COL10)。肌动蛋白表达被用作内部控制。这一数字已从 l. Recha-桑丘等修改。11.<a href="/files/ftp_upload/57259/57259fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds at JoVE.com

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三维肽支架中哺乳动物细胞的培养

在这里, 我们提出了一个协议, 以获得 3 d-培养系统的自组装肽支架, 以促进分化去分化肉瘤人关节软骨细胞成软骨样组织。

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

culturing-mammalian-cells-in-three-dimensional-peptide-scaffolds

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.1K 次观看.  Ramon Llull University. 在这里, 我们提出了一个协议, 以获得 3 d-培养系统的自组装肽支架, 以促进分化去分化肉瘤人关节软骨细胞成软骨样组织。

视频: Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

三维肽支架中哺乳动物细胞的培养

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