Name: preparation of chitosan-based injectable hydrogels and its application in 3d cell culture
Uploaded: 2017-09-29T14:00:00.000+00:00
Duration: 8 min 5 s
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 peg 聚合物 <li> <ol> 重4.00 克 peg (兆瓦 4000, 1.00 摩尔),将其转成圆底烧瓶 (250 毫升), 并加入甲苯 (100 毫升). 
			注: 其他具有不同分子量的销子可用于水凝胶的形成, 但会导致不同的刚度. </li>
			<li> 用热枪 (或热板大约40和 #176 C) 加热溶液, 温和地帮助溶解所有的聚合物。所有的聚合物溶解后, 用蒸发器除去所有的溶剂。重复此溶解和干燥过程两次, 使干 PEG 聚合物. 
			注意: 这应该是在油烟罩中进行的, 要格外小心。甲苯是易燃的. </li>
		</ol> </li> <li> peg 的苯甲醛终止反应 <ol> <li> 在烧瓶中添加磁力搅拌器和四氢呋喃 (呋喃, 100 毫升), 并使用搅拌器将 peg 溶解。将 4-甲醛 (0.90 克, 6.0 摩尔) 和 4-吡啶 (0.07 g, 0.6 摩尔) 依次添加到解决方案中, 以完全溶解所有固体. </li>
			<li> 添加 n、n 和 #39;-dicyclohexylcarbodiimide (DCC, 1.25 克, 6.0 摩尔) 的解决方案, 并添加一个干燥管, 充满无水 CaCl 2 到烧瓶。使反应在室温 (〜20和 #176; C) 下搅拌12小时左右. </li>
		</ol> </li> <li> 后反应过程 <ol> <li> 在反应结束后通过真空过滤出溶液中产生的白色固体。将溶液倒入冷二乙醚 (500 毫升) 搅拌下沉淀白色固体。收集所有的白色固体过滤器和干燥的固体在一个油烟罩. </li>
			<li> 再次将白色固体溶解到呋喃中, 过滤掉任何不溶性的白色固体, 将溶液倒入新鲜的冷二乙醚中沉淀白色固体, 并将其晒干。重复这个过程二到三次, 然后安置白色固体在真空干燥烤箱 (20 和 #176; C, 0.1 毫巴) 完全地烘干他们。收集白色粉末作为最终产品: 苯甲醛终止 DF PEG. </li>
		</ol> </li> </ol> </li> <li> 水凝胶的制备 <ol> 重量不同量的 DF PEG (0.11 克, 0.028 摩尔; 0.22 克, 0.055 摩尔; 0.44 克, 0.110 摩尔) 在管中 (10 毫升), 添加去离子水 (5.0 毫升), 并使用涡流或磁力搅拌器来帮助 溶解聚合物. </li>
		<li> 溶解乙二醇壳聚糖 (0.495 克, 6.18 x 10 -3 摩尔) 在去离子水 (15.0 毫升) 在一个管 (50 毫升), 并使用一个漩涡几分钟质壳聚糖溶液 (3%). </li>
		<li> 在试管 (2.0 毫升) 中依次添加乙二醇壳聚糖溶液 (0.2 毫升、3%) 和 DF PEG 溶液 (0.2 毫升)。使用涡流混合的解决方案, 形成水凝胶在几分钟内。按照 表 1 中的比率来制作不同机械强度的水凝胶. 
		注: 使用管倒置法确定水凝胶是否已经形成. </li>
	</ol> </li> <li> 流变分析 <ol> <li> 用平行钢板 (直径:20 mm) 对旋转流变仪进行流变分析。对于凝胶试验, 将乙二醇壳聚糖溶液 (0.2 毫升, 3%) 涂在下板上。然后, 加入 DF PEG 水溶液 (0.2 毫升, 2%) 均匀滴到壳聚糖溶液表面, 并迅速与吸管混合。从上到下板开始测试. </li>
		<li> 对于水凝胶和 #39 的刚度测试, 将水凝胶切割成一个圆 (直径:20 mm), 并测量存储模数 (G 和 #39;) 值与1% 应变频率分析。典型的 G 和 #39; 在 表 1 中列出了 6.3 rad 和 #8722; 1 中的值. 
		注: 对水凝胶形成后 0.5 h 的水凝胶进行流变学分析, 确保水凝胶的动态粘结稳定. </li>
		<li> 水凝胶和 #39 的 self-healable 性能测试, 将水凝胶切成碎片, 然后将这些碎片聚在一起, 自我成一个完整的部分。然后将水凝胶片切割成一个圆 (直径:20 mm), 并将其放在流变仪上进行流变分析. </li>
	</ol> </li> </ol> 2. 水凝胶中的3D 细胞培养 <ol> <li> 凝胶凝胶溶液的制备 <ol> <li> 在无菌管 (4.0 毫升) 中称 DF PEG (0.44 克, 0.11 摩尔), 在细胞培养基中添加 (RPMI-1640, 2。0毫升), 并使用涡流或搅拌器来帮助溶解聚合物获得聚合物溶液 (20%)。将溶液装入注射器 (10.0 毫升) 中, 然后通过微米级细菌保留过滤器 (0.22 和 #181; m) 将其消毒. </li>
		<li> 在无菌管 (15.0 毫升) 中称量乙二醇壳聚糖 (0.165 克, 2.06 x 10 -3 摩尔), 加入细胞培养基 (RPMI-1640, 4.0 毫升) 和涡流, 以帮助溶解聚合物获得乙二醇壳聚糖溶液 (4.0%)。在注射器 (10.0 毫升) 中装入溶液, 并用0.22 和 #181 过滤。
	</ol> </li> <li> 细胞在水凝胶中培养 警告: 在组织培养罩中执行所有与细胞相关的过程。有无菌技术的基本知识。
	<ol> <li> 细胞悬浮液的制备 <ol> <li> 培养 RPMI-1640 培养基中的 L929 细胞, 辅以 10% FBS、5% 青霉素 (10 毫升) 在 Petri 盘 (直径10厘米), 孵育在37和 #176; C, 5% CO 2 。使用前每天更换介质. </li>
			<li> 用含有胰蛋白酶 (0.025 瓦特/五%) 和 EDTA (0.01% w/v) 的 PBS L929 细胞, 然后离心机 (70 x g, 5 分钟) 和 re-suspend RPMI-1640 培养基中的细胞 (1.0 毫升)。使用血计数板的标准操作进行细胞计数。重新挂起细胞, 调整细胞浓度为 ~ 3.75 和 #215; 10 6 单元格/mL. </li>
		</ol> </li> <li> 水凝胶中的细胞封装 <ol> <li> 将 L929 细胞悬浮液 (0.4 毫升, 3.75 x 10 6 细胞 mL -1 ) 与乙二醇壳聚糖溶液 (0.4 毫升) 在试管 (4.0 毫升) 中由涡旋混合。将 L929/glycol 壳聚糖溶液 (0.8 毫升) 移入共焦培养皿 (直径2.0 厘米) 的中心。吸管的 DF PEG 解决方案 (0.2 毫升) 到同一盘, 并轻轻吸管混合溶液和诱导水凝胶的形成. 
			注: 通过倾斜培养皿来评估水凝胶的形成. </li>
			<li> 对于直接的细胞培养, 在水凝胶的顶部添加额外数量的 RPMI-1640 培养基 (1.0 毫升)。把细胞嵌入水凝胶 (1.0 毫升, 1.5% 乙二醇壳聚糖, 4.0% DF PEG, 1.5 和 #215; 10 6 细胞 mL -1 ) 在孵化器 (37 和 #176; C, 5% CO 2 ), 并改变媒体每天。在单元封装后1、3、5和7天准备细胞成像. </li>
		</ol> </li> <li> 对于在注射后的水凝胶中的3D 细胞 post-culture, 在注射器 (10.0 毫升, 48 克针) 上准备细胞载入的凝胶 (1.0 毫升, 见步骤 2.2.2)。水凝胶形成后, 将水凝胶慢慢注入培养皿中进行共聚焦成像。在水凝胶的顶部添加额外的培养基 (1.0 毫升), 每天更换。将培养皿放入孵化箱 (37 和 #176; C, 5% CO 2 ), 然后准备成像. 
		注意: 请检查并遵守注射器的安全操作协议. </li>
	</ol> </li> <li> 单元可行性分析 <ol> <li> 共聚焦观测 <ol>
<li> 用 PBS (1.0 毫升) 冲洗水凝胶两次。用荧光素双乙酸 (FDA, 0.5 毫升, 0.05 毫克/毫升) 和碘碘化物 (PI, 0.5 毫升, 0.08 毫克/毫升) 溶液对水凝胶进行染色, 15 分钟。染色后, 去掉所有的溶剂. </li>
			<li> 在 488 nm 和 543 nm 的激发波长下使用共聚焦显微镜观察水凝胶, 分别显示活细胞和死单元。采取 z 栈通过每2和 #181; m 深度的水凝胶, 以验证整个细胞均匀分布. 
			注: FDA 对活细胞进行染色, 而 PI 染色死细胞. </li>
		</ol> </li> <li> 将水凝胶 (1.0 毫升) 与乙酸 (醋酸、3 v%、1.0 毫升) 降解为5分钟, 将吸管放入试管 (4.0 毫升)。通过离心机收集细胞 (70 x g, 5 分钟) 和 re-suspend 细胞在 RPMI-1640 细胞培养基 (1.0 毫升)。使用血球计数板进行细胞计数. </li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

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><tbody><tr><td>Glycol chitosan</td><td>Wako Pure Chemical Industries</td><td>39280-86-9</td><td>90% degree of deacetylation</td></tr><tr><td>4-Carboxybenzaldehyde</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>619-66-9</td><td>99%</td></tr><tr><td>N, N&#039;-dicyclohexylcarbodiimide</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>538-75-0</td><td>99%</td></tr><tr><td>Calcium chloride anhydrous</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>10043-52-4</td><td>96%</td></tr><tr><td>4-dimethylamiopryidine</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>1122-58</td><td>99%</td></tr><tr><td>Polyethyleneglycol</td><td>Sino-pharm Chemical Reagent</td><td>5254-43-7</td><td>99%</td></tr><tr><td>Tetrahydrofuran</td><td>Sino-pharm Chemical Reagent</td><td>109-99-9</td><td>99%</td></tr><tr><td>Toluene</td><td>Sino-pharm Chemical Reagent</td><td>108-88-3</td><td>99%</td></tr><tr><td>Ethyl ether</td><td>Sino-pharm Chemical Reagent</td><td>60-29-7</td><td>99%</td></tr><tr><td>Acetic acid</td><td>Sino-pharm Chemical Reagent</td><td>64-19-7</td><td>99%</td></tr><tr><td>Anhydrous CaCl2</td><td>Sino-pharm Chemical Reagent</td><td>10043-52-4</td><td>99%</td></tr><tr><td>Fluorescein diacetate</td><td>Sigma</td><td>596-09-8</td><td>99%</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>25535-16-4</td><td>94%</td></tr><tr><td>RPMI-1640 culture media</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td></td><td>0.25%</td></tr><tr><td>PBS</td><td>Solarbio</td><td></td><td>0.01 M</td></tr><tr><td>Penicillin streptomycin solution</td><td>Hyclone</td><td></td><td>10,000 U/mL</td></tr><tr><td>Rheometer</td><td>TA Instrument</td><td></td><td>AR-G2</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>710-3channel</td></tr><tr><td>L929 Cells</td><td>ATCC</td><td></td><td>NCTC clone 929; L cell, L929, derivative of Strain L</td></tr><tr><td>Evaporator</td><td>EYELA</td><td></td><td>N-1100</td></tr><tr><td>48 guage needle</td><td>ShanghaiZhiyu Medical Material Co., LTD</td><td></td><td>48-guage</td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>DM3000 B</td></tr><tr><td>Microscope software</td><td>Imaris</td><td></td><td></td></tr><tr><td>Heat gun</td><td>Confu</td><td></td><td>KF-5843&amp;nbsp;</td></tr><tr><td>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。

糖注射水凝胶的制备及其在3D 细胞培养中的应用

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Research

JoVE Journal

Chemistry

19.0K 次观看.  Tsinghua University. 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。

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

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

科研

生物化学

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

生物工程

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

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 ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

奇托桑膜上人类牙周韧带细胞球体的形成

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

发育生物学

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 ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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 ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...