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><tbody><tr><td>Human articular chondrocytes (Ach)</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>RAD16-I peptide solution (PuraMatrix)</td><td>Corning</td><td>354250</td><td></td></tr><tr><td>Sucrose (tissue culture grade)</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Cell culture inserts (0.4 &amp;micro;m pore, 12 mm diameter)</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Chondrocyte Basal Medium (CBM)</td><td>Lonza</td><td>CC-3217</td><td></td></tr><tr><td>SingleQuots of Growth Supplements</td><td>Lonza</td><td>CC-4409</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D8893</td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate (AA2P)</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>Human transforming growth factor-&amp;beta;1 (TGF-&amp;beta;1)</td><td>Millipore</td><td>GF111</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Polyvinylidene difluoride (PVDF) membrane</td><td>Invitrogen</td><td>LC 2005</td><td></td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34080</td><td></td></tr><tr><td>Anti-Actin</td><td>SCBT</td><td>sc-1615</td><td></td></tr><tr><td>Anti-Collagen I</td><td>Abcam</td><td>ab138492</td><td></td></tr><tr><td>Anti-Collagen II</td><td>Abcam</td><td>ab3092</td><td></td></tr><tr><td>Anti-Collagen X</td><td>Abcam</td><td>ab182563</td><td></td></tr><tr><td>Antigoat IgG-HRP</td><td>Abcam</td><td>ab97100</td><td></td></tr><tr><td>Anti-mouse IgG-HRP</td><td>Abcam</td><td>ab97023</td><td></td></tr><tr><td>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

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

癌症研究

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, ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

医学

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

<meta charset="utf8"></head><body>用于上皮细胞和成纤维细胞的先进双层 3D 共培养系统

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>作者聚焦:使用 PVA-PCL 纳米纤维膜推进 3D 共培养系统

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

使用纳米纤维膜建立上皮细胞的三维共培养模块

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

三维肽支架中哺乳动物细胞的培养

摘要

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此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

在三维自组装多肽水凝胶的人类神经祖细胞的培养

自我报告的支架3维细胞培养

使用胶原基支架的神经母细胞瘤三维体外仿生模型

工程三维上皮组织细胞外基质中嵌入

人垂体腺瘤细胞的三维海藻酸钠珠文化

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

三维肽支架中哺乳动物细胞的培养

摘要

探索更多视频

此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

在三维自组装多肽水凝胶的人类神经祖细胞的培养

自我报告的支架3维细胞培养

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

工程三维上皮组织细胞外基质中嵌入

人垂体腺瘤细胞的三维海藻酸钠珠文化

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用胶原基支架的神经母细胞瘤三维体外仿生模型