Name: self-reporting scaffolds for 3-dimensional cell culture
Uploaded: 2013-11-07T19:15:00
Description: Watch this Scientific Journal Video about Self-reporting Scaffolds for 3-Dimensional Cell Culture at JoVE.com

从BBSRC资金体谅（批准号BB H011293 / 1）。

概观第1章描述的pH值敏感的纳米传感器和纳米传感器响应利用荧光光谱和它们的大小用扫描电镜pH值表征的准备。 第2节描述的静电聚合物支架和它们的形态和大小用扫描电镜表征的准备。部2还描述了自我报告的支架这是电纺支架与夹杂物的pH值敏感的纳米传感器的制备方法。所得到的自我报告的支架的特征在于通过SEM以评估是否在电纺纤维的任何形态学变?...

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组织工程立志创建生物替代品，可以用来作为在体内像体外组织模型和组织替代疗法进行修复，替换，维持或提高特定组织或器官的功能。合成的替代物已经使用了很多年，以取代或组织抢修，但这些往往是由于与宿主组织和/或感染，最终导致拒绝或进一步修正手术融合不佳失败。生成植入前活组织，在实验室可以解决实现全面整合的问题，并减少需要翻修手术。然而，这个目标要...

在组织工程中的一个关键的策略是使用生物相容性的材料来制作支架，其形态类似于组织，它要替代，也能够支持细胞生长和功能1,2。脚手架允许细胞附着和增殖但允许在整个三维细胞结构的空隙细胞迁移提供机械支撑。该支架还必须考虑到的细胞营养物质传输，而不是抑制代谢废物清除3。 静电纺丝已经成为一种很有前途的方法，能够支持细胞生长4-6聚合物支架的制造。生产的非织造电纺纤维是适合于细胞生长，因为它们往往是多孔的，并允许在整个三维细胞构建体7的间隙中的细胞-细胞相互作用，以及细胞迁移。它吨期间监测细胞存活力是重要他培养期间，确保细胞活力保持在整个三维构建体。例如，培养条件，如氧和pH值需要小心控制，因为在分析物的浓度梯度可以在三维结构内的存在。生物反应器或灌注系统可受聘于间质流体内条件，因此增加营养转移和代谢废物清除8模仿。这种系统是否正在确保恒定的微环境条件的问题可以通过评估在实时细胞微环境加以解决。 这可以实时监测关键微环境指标包括：温度，细胞培养基的化学成分，溶解的氧和二氧化碳的浓度，pH和湿度。这些指标中，温度可最容易使用的探针原位监测。方法监测通常在余下的上市指标沃尔沃去除等分取样，因此干扰了细胞培养和增加污染的风险。连续，实时的方法正在追捧。目前的监测方法通常依赖于物理上探测细胞结构，如pH监测仪或氧探头的仪器。然而，这些侵入性的方法可以破坏细胞结构，扰乱正在进行的实验。无创监测的三维结构中的分析物浓度可以使各种环境因素，如营养枯竭9实时监控。这将使营养供给的评估，以结构内更深的区域，并确定是否代谢废物得到有效10,11去除。试图解决侵袭的问题的系统一般涉及使用已灌注腔的传递培养基同时通过培养容器和外部传感器来监控pH值，氧气和葡萄糖12。该再是在发展中国家，可以直接集成到培养容器中，不需要除去的等分试样进行采样的，因此会在原位监测传感器提供越来越大的兴趣。 为解决现场和非侵入性的监测，我们已经把分析物响应纳米传感器为静电支架，产生自我报告的支架13微环境条件等缺点。即通过监测荧光活性充当传感装置的支架已预先制备的，其中所述感测装置是要么通过电或通过使用分析物响应性染料被加入到聚合物中之前，形成支架14,15的所产生的实际的聚合骨架。然而，这些传感装置必须给由从其他分析物可能的干扰错误的光输出的潜力。使用比例传感装置的su通道的那些在所描述的方案制备持有以消除这些可能的不利影响，并提供特定于所讨论的分析物的响应的可能性。 这里介绍的静电支架乃从合成的共聚物的聚（乳酸-共-乙醇酸）（PLGA），由于选择具有食品和药物管理局（FDA）批准的，由于其可生物降解和生物相容特性和轨道支持各种细胞类型16-18的生长和功能的记录。所制备的比例分析物响应纳米传感器的响应pH值。纳米传感器包括两个荧光染料成生物相容的溶胶 - 凝胶基质中，其中一种染料，FAM是响应于pH和其他，TAMRA作为内标，因为它是不响应的pH值。另外两个FAM和TAMRA的荧光可以单独进行分析，因为它们不显著重叠。确定所述荧光EMI的比例两种染料的特定波长裂变给出的pH响应独立于其它环境条件。自报支架可以让pH值的重复评估， 原位和实时而不破坏开发的3D模型。我们已经证明，这些支架能够支持细胞附着和增殖，并保持响应到有问题的分析。的酸性副产物的工程化构建体的动力学保持充分研究，因此使用对pH值敏感的支架可以大大促进这些研究19。此外，使用了自行申报组织工程支架的应用提供了机会，以充分了解，监控和优化的三维模型的组织结构在体外生长，无创性和实时性。 对pH值敏感的纳米传感器也已交付给成纤维细胞在静电PLGA scaffol培养的细胞内环境DS。荧光发射的从染料的比率被用来监测和pHi值相比，自我报告的支架结合的pH纳米传感器。纳米传感器的细胞在3D环境中培养的递送可以使深部以非破坏性的方式构造内的监测的分析物浓度的影响。因此，纳米传感器可能是一个可行的成像工具，非破坏性评估整个3D细胞行为的构造允许长期分析。筛选三维结构中的单个细胞的分析物浓度可以确保它们得到足够的营养物和氧的浓度。监测过程参数可有助于标准化的技术的发展为氧气和营养物质的有效质量传输。纳米传感器对细胞内环境和纳米传感器为高分子支架结合的交付可以合并，允许在3D常量评估细胞活力以及支架的性能在组织生长过程ructs。这可能会导致这些结构增加了知识和进步的生物学相关的组织替代品的制造。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Ethanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">32221</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Anhydrous dimethylformamide (DMF)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">270547</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Ammonium hydroxide 50% (v/v) aqueous solution</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:137px;">35574</td>
			<td style="width:79px;">diluted to 30% (v/v) with pure water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">TEOS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">13190-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">3-Aminopropyltriethoxysilane (APTES)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">A3648</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">5-(and-6)-carboxyfluorescein, succinimidyl ester (FAM-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1311</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1171</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate monobasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate dibasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-0876</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">NaOH</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S8045</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Trypsin/EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T4174</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">P0781</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">PBS</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D8537</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DMEM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D6046</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">FBS</td>
			<td style="width:71px;">Source Bioscience</td>
			<td style="width:137px;">Batch-213-101992</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">L-Glutamine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">G7513</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Lipofectamine 2000</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11668-019</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Optimem</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11058-021</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">LysoTracker Red</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">L-7528</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Draq5</td>
			<td style="width:71px;">Biostatus Ltd</td>
			<td style="width:137px;">DR50050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Nitrogen gas</td>
			<td style="width:71px;">BOC</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DCM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">320269</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">TFA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T6508</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:195px;">Confocal microscope</td>
			<td style="width:71px;">Leica TCS-SP</td>
			<td style="width:137px;"></td>
			<td style="width:79px;">equipped with argon and krypton lasers and a 63X 0.9NA water immersion lens</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">UV light</td>
			<td style="width:71px;">UVLS28 UVP, USA</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Stirrer plate</td>
			<td style="width:71px;">SB161-3 Jencons-PLS</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">pH meter</td>
			<td style="width:71px;">Jenway model 3510</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Rotary Evaporator</td>
			<td style="width:71px;">Buchi Rotary Evaporator R200</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Centrifuge (nanosensors)</td>
			<td style="width:71px;">Hermle Z300</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:195px;">Centrifuge (cell culture)</td>
			<td style="width:71px;">Thermo Scientific Heraeus Biofuge Primo</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Vortex</td>
			<td style="width:71px;">Whirlimixer Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Ultrasonicator</td>
			<td style="width:71px;">FB11021 Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Aluminum sheet</td>
			<td style="width:71px;">Nottingham University</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">35mm cell culture plate</td>
			<td style="width:71px;">Iwaki</td>
			<td style="width:137px;">3000035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">10 ml syringe</td>
			<td style="width:71px;">Becton Dickenson</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">3T3 Fibroblast cells</td>
			<td style="width:71px;">European Collection of Cell Cultures</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">PLGA</td>
			<td style="width:71px;">Lakeshore Biomaterials</td>
			<td>7525 DLG 7E</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pyridinium formate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>P8535</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trypan blue</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>T8154</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium phosphate monobasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium phosphate dibasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S5136</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>320331</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

self-reporting scaffolds for 3-dimensional cell culture

培养细胞在三维上合适的支架材料被认为是更好的模拟体内微环境和提高细胞间的相互作用。所得到的三维蜂窝结构往往可以更相关的研究的分子事件和细胞间的相互作用比研究中的2D类似的实验。在整个支架创建具有高细胞活性的有效三维培养的培养条件，如氧和pH值必须仔细控制在分析物的浓度梯度可以在整个三维结构存在。这里我们描述了制备生物相容的pH响应性溶胶 - 凝胶纳米传感器和它们掺入到聚（乳酸 - 共 - 乙醇酸）（PLGA）电纺支架以及它们的用于哺乳动物细胞的培养以后的制剂的方法。将pH响应性支架材料可以用作工具，以一个三维蜂窝结构内确定微环境的pH值。此外，我们详细的pH响应nanose交付nsors哺乳动物细胞，其增长主要由电纺PLGA支架支撑的细胞内环境。对pH值敏感的纳米传感器的细胞质位置可以利用正在进行的实验过程中监测细胞内pH（PHI）。

利用SEM，其中纳米传感器成像的人口进行测定，发现具有纳米尺寸在240-470纳米（ 图1A）的范围内制得的pH值敏感的纳米传感器的尺寸分布进行了表征。实现一个狭窄的，合理的小直径与使用施托贝尔法制备纳米粒子是一致的。已经发现，纳米粒子的合成过程中使用碱性pH环境即纳米颗粒制备使用施托贝尔方法，允许良好控制的大小的，而使用酸性条件制剂生产中颗粒大小广泛分...

Watch this Scientific Journal Video about Self-reporting Scaffolds for 3-Dimensional Cell Culture at JoVE.com

Self-reporting Scaffolds for 3-Dimensional Cell Culture

生物相容的pH响应性溶胶 - 凝胶纳米传感器可并入聚（乳酸 - 共 - 乙醇酸）（PLGA）电纺支架。所产生的自我报告的支架可用于在微环境条件原位监测，同时在所述支架中培养细胞。这是有益的，因为在三维细胞构建体可以在实时而不干扰试验来监测。

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 ...

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

科研

生物工程

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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

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 ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

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

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

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