Name: preparation of chitosan-based injectable hydrogels and its application in 3d cell culture
Uploaded: 2017-09-29T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

这项研究得到了中国国家科学基金会 (21474057 和 21604076) 的支持。

警告: 请在使用前查阅所有相关的材料安全数据表 (MSDS)。在进行化学实验时, 请使用适当的安全做法, 包括使用油烟罩和个人防护设备 (安全眼镜、防护手套、实验室大衣、 等 )。该协议要求标准的细胞处理技术 (杀菌, 细胞恢复, 细胞传代, 细胞冻结, 细胞染色, 等 ). 
 1. 水凝胶的制备 <ol> <li> 合成苯甲醛终止的 di 官能化聚乙二醇 (DF peg) <ol> 预干燥...

<ol>
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该协议中提出的水凝胶 (图 1) 有两个主要组成部分: 天然高分子乙二醇壳聚糖和合成苯甲醛终止聚合物胶 DF PEG, 两者都是生物相容性的材料。采用 one-step 改性反应合成 DF PEG。在溶解度、改性效率以及水凝胶刚度等方面, 选择了分子量4000的 PEG。采用不同的固体含量和乙二醇壳聚糖与 DF PEG 的配比, 制备了一系列不同机械强度的水凝胶。均匀水凝胶通过在室温下混合凝胶溶液, ?...

水凝胶是一种交联高分子材料, 具有大量的水分和软机械性能, 已在许多生物医学应用中使用1,2。水凝胶可以提供一个柔软和潮湿的环境, 这是非常类似的生理环境的细胞在体内。因此, 水凝胶已成为最受欢迎的3D 细胞培养支架之一3,4。3D 细胞培养与2D 培养皿细胞培养基相比, 具有先进的细胞外基质 (ECM) 的模拟微环境, 为增殖和分化目的而进行接触和组装.5。此外, 含有天然高分子的水凝胶可以为细胞增殖和分化提供生物相容和促进的环境, 从而使3。合成聚合物的水凝胶是首选的简单和明确的成分, 排除复杂的影响, 如动物起源的蛋白质或病毒。在所有的水凝胶候选3D 细胞培养中, 容易制备和具有一致的特性的凝胶总是首选。调整水凝胶的性质, 以适应不同的研究要求的设施是重要的, 以及6。
在这里, 我们介绍了一个简便的糖水凝胶使用动态胺化学, 这成为一个多功能的水凝胶平台的3D 细胞培养7。在这种方法中, 众所周知的生物相容的乙二醇壳聚糖是用来建立框架的水凝胶的网络。它的氨基是反应与苯甲醛终止聚乙二醇作为聚合物胶形成动态胺债券作为 crosslinkages 水凝胶8。动态胺键可可逆和回应地形成和分解, 使水凝胶具有机械可调的交联网络9,10,11。由于其高含水量, 生物相容材料和可调节的机械强度, 水凝胶成功地应用于3D 细胞培养中的 L929 细胞的支架12,13。这里的协议详细的程序, 包括聚合物胶合成, 水凝胶制备, 细胞嵌入, 和3D 细胞培养。
水凝胶还显示了其他一些特点, 由于其动态胺 crosslinkages, 包括其 multi-responsive 各种生物刺激 (酸/pH, 维生素 B6 衍生物哆, 蛋白木瓜蛋白酶,等), 表明水凝胶可以在生理条件下诱发分解8。水凝胶还 self-healable 和注射, 这意味着水凝胶可以通过微创注射法, 并获得优势的药物和细胞交付14,15。通过添加功能性添加剂或特定的预先设计的聚合物胶, 水凝胶可以获得诸如磁性、温度、pH 响应、等16、17等特定属性, 从而实现广泛的研究要求。这些特性表明, 水凝胶的潜在能力是可注射的多种药物和细胞在体外和体内生物医学研究和应用。

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

该协议提出了一种简便, 高效, 通用的方法来制备糖水凝胶使用动态胺化学。将乙二醇壳聚糖与合成的苯甲醛终止聚合物胶混合溶液制备水凝胶, 在室温下在几分钟内有效地获得凝胶。通过对乙二醇壳聚糖、高分子胶和含水量的不同配比, 得到了不同凝胶时间和硬度的多功能水凝胶。当损伤时, 由于动态胺键的可逆性, 水凝胶可以恢复其外观和模量, crosslinkages。这种 self-healable 的性质, 使水凝胶是可注射的, 因为它可以 self-healed 从压缩件的整体散装水凝胶后, 注射过程。由于动态胺键的不同平衡状态, 水凝胶还 multi-responsive 许多生物活性刺激。该水凝胶被证实为生物相容, L929 小鼠成纤维细胞被植入遵循标准的程序和细胞增殖容易评估的3D 细胞培养过程。水凝胶可以为不同的研究提供一个可调节的平台, 在这种情况下, 对细胞的3D 环境进行生理模拟是有益的。随着它的 multi-responsive, self-healable, 和可注射性的性质, 水凝胶可以作为多种载体用于药物和细胞在未来的生物医学应用。

在图 1中提供了有关水凝胶制剂的示意图, 并将其用作3D 细胞培养。在表 1中总结了不同机械强度的水凝胶的含量和配比的信息。在图 2中, 水凝胶的 self-healable 和流变性通过存储模量与频率测试来显示水凝胶的刚度。在图 3中, 在水凝胶中培养的细胞共焦图像和细胞数, 并通过3D 细胞培养确认细胞的活力和细胞增殖。<str...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

在这里, 我们描述了一个轻便的制备糖注射水凝胶使用动态胺化学。介绍了水凝胶的机械强度调整方法及其在3D 细胞培养中的应用。

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通过超性能液相色谱对细菌细胞壁进行隔离和制备，进行组成分析

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

掺硼金刚石电极质量和应用，以评估<em&gt;原位</em&gt;本地pH值通过水电改造

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

在EOB-DTPA和镓（三）复杂的调查其<sup&gt; 68</sup&gt;嘎放射性标记模拟

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

纳米金刚石的生物激发多巴胺表面改性及其对银纳米粒子的还原

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization

n-杂环卡本的光生成在光致环开环转移聚合中的应用

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

一种用于射击残留量推定检测的新型细菌生物传感器的研制与应用

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...