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, 在几分钟内迅速形成, 虽然凝胶速度也会因稀溶液而减慢。采用流变学试验方法评价不同水凝胶的力学强度。这些水凝胶的存贮模量 (G) 发现了近似地不同从 900 pa (软), 2100 pa (中等) 到 4700 pa (刚性) (表 1和图 2A)。通过在水凝胶网络中加入更多的聚合物胶, 从而提高了交联密度, 从而提高了贮存模量 (更高的刚度)。水凝胶的弹性模量 (图 2B) 的恢复也证实了 self-healable 的特性。众所周知, ECM 的刚度不同的组织, 因此, 水凝胶可以提供可调的机械提示, 以满足不同的研究要求的变化率乙二醇壳聚糖和 DF PEG 聚合物胶18。
L929 细胞, 一个典型的小鼠成纤维细胞系与在体内环境组织刚度的〜 5600 pa19, 被用作一个细胞模型中嵌入水凝胶。在水凝胶中的细胞包封后, 可以很容易地观察到, 在整个培养过程中, 凝胶中的细胞表现出极高的生存能力 (和 #62; 99%), 证实了糖凝胶的优良生物相容性。明显增加的细胞密度在水凝胶暗示, 这种水凝胶可以支持增殖的 L929 细胞即使没有 extra-added 生长因子 (图 3A)。经过7天的培养, 在水凝胶, 细胞数量增加了 300% (图 3B)。据报道, 在支架培养的细胞可以区分以下机械提示20,21, 这可能意味着进一步研究使用这种3D 水凝胶培养方法。
注射用水凝胶植入细胞具有无与伦比的优势, 大大提高了植入细胞的分娩效率和生存能力14。在一个动态的胺交联网络中, 水凝胶可以在注射后自我。self-healable 化学性质使水凝胶可以注射, 这意味着潜在的应用凝胶作为可注射多载体。为了进一步评价这种水凝胶作为一种可注射的细胞载体, 进行了注射模拟和 post-culturing 实验。该细胞内嵌的水凝胶被装入注射器, 并通过48克针挤压到一个培养皿, 随后7天 post-culturing 过程的细胞活力和增殖率研究。
如图 4A所示, 压扁的水凝胶件可以自我在注射后1小时内进行综合水凝胶的改造, 这对细胞的传递很有帮助。对于水凝胶中的细胞, 与刚封装后的图像 (图 4A, 左插入) 相比, 细胞密度在7天后急剧增加 (图 4A, 中间插入)。在扩大的视野中, 一个更详细的细胞体式过程, 其中一些细胞被发现被分为两个 (图 4A, 右插入), 直接证实由3D 细胞增殖在水凝胶。图 4B显示了注射和 post-culture 实验后活死细胞测定的共焦图像。注入细胞的密度明显增加, 细胞活力增高 (与 #62; 99%), 表明3D 水凝胶注射后细胞增殖成功。为了了解注射是否影响细胞增殖率, 对水凝胶中的或不注射的细胞增殖率进行定量统计分析的比较 (图 4C)。注射后细胞增殖率虽略有下降, 但仍处于高水平 (约 75%), 细胞数在水凝胶培养7天后增加 145%, 表明注射过程中的剪切力具有可观察对细胞增殖的负面影响有限。
我们描述了这个水凝胶应用的典型过程, 但研究人员可以修改该协议, 以适应特定的研究要求。聚合物胶对水凝胶有显著影响, 易于修改。例如, 这种水凝胶的刚度可以很容易地被不同比例的乙二醇壳聚糖和 DF PEG 胶。同时, 使用其他分子量的 DF PEG 也能达到这个目标。我们使用了 peg 4K 在这个协议, 但 peg 2K 到10K 可能也工作。当使用其他的销子, 它是重要的监测凝胶时间和刚度具体。其他功能高分子胶也可以工作, 如果研究员有合成技能, 准备特定功能苯甲醛终止聚合物17。
此协议有几个限制。首先, 由于动态结构, 与共价键交联水凝胶相比, 该水凝胶的刚度有限。一个人可能会遇到失败, 准备水凝胶太高的模数设计。其次, 这种水凝胶是生物降解, 从而限制了长期细胞培养内的水凝胶。第三, 水凝胶提供了一个3D 的交联种植环境, 具有扩散关注。到目前为止, 大多数的生物分析是基于2D 文化。3D 结构可能会阻碍进一步的细胞相关的体内研究。
使用此协议时, 需要遵循几个关键步骤。首先, 在聚合物胶 DF PEG 的合成过程中, 脱水是非常重要的。其次, 在执行细胞相关程序时, 无菌操作是至关重要的。第三, 在水凝胶中应充分控制染色时间, 以获得良好的共焦图像。
总之, 该协议介绍了一个轻便的制备糖注射水凝胶, 这是作为一个平台的3D 细胞培养, 可以提供可调的机械提示, 为各种研究。它已经被应用在有关药物传递、细胞治疗和肿瘤化疗的领域, 这不仅证明了它的性能, 而且也增加了它在生物医学 ap 中的潜力。1999年10月20日.

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。图 4提供了显微镜、共焦图像, 并分析了在注射和 post-culture 的水凝胶中嵌入的细胞的增殖速率。高细胞活力和细胞增殖表明, 嵌入在水凝胶的细胞没有破坏性的伤害, 并可以恢复 post-culture, 表明水凝胶的可。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56253/56253fig1.jpg"> 
图 1: 水凝胶的制备及其在3D 细胞培养中的应用.(A) 水凝胶制剂;(B) 细胞悬浮制剂;(C) 3D 细胞培养的水凝胶有或不注射。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56253/56253fig2.jpg"> 
图 2:水凝胶刚度.存储模量 G 与频率 (A) 原和 (B) self-healed 水凝胶。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56253/56253fig3.jpg"> 
图 3: 在水凝胶中的3D 细胞培养.(A) 3D 在硬强度水凝胶中嵌入的 L929 细胞共聚焦图像 (绿色: 活细胞;红色: 死细胞);(B) 细胞计数结果的 L929 细胞嵌入在水凝胶后细胞封装在指示时间。缩放条 = 300 µm. 数据以平均值为± SD.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56253/56253fig4.jpg"> 
图 4: 注射后水凝胶中的3D 细胞 post-culture.(a) 一个细胞载水凝胶的注射和 post-culture 的示意图图像。插入: 在注射前 (左) 和细胞 post-culture 后7天内嵌入水凝胶细胞的显微图像 (中和右)。刻度栏 = 100 µm;(B) 注入硬强度水凝胶和 post-cultured 后的 L929 细胞共聚焦图像 (绿色: 活细胞;红色: 死的细胞), 比例酒吧 = 300 µm;(C) 比较在水凝胶中培养的细胞在包封后有无注射的增殖速率。数据显示为平均± SD. (改编自参考12;版权所有©2017唯)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>水凝胶刚度</td>
			<td>软</td>
			<td>中</td>
			<td>僵硬</td>
		</tr>
		<tr>
			<td>乙二醇壳聚糖 (mg)</td>
			<td>6。6</td>
			<td>6。6</td>
			<td>6。6</td>
		</tr>
		<tr>
			<td>DF (mg)</td>
			<td>4。4</td>
			<td>8。8</td>
			<td>17。6</td>
		</tr>
		<tr>
			<td>聚合物胶含量 (%)</td>
			<td>1</td>
			<td>2</td>
			<td>4</td>
		</tr>
		<tr>
			<td>去离子水 (毫升)</td>
			<td>0。4</td>
			<td>0。4</td>
			<td>0。4</td>
		</tr>
		<tr>
			<td>Gelaion 时间 (分钟)</td>
			<td>7。5</td>
			<td>5</td>
			<td>3。5</td>
		</tr>
		<tr>
			<td>G ' (Pa)*</td>
			<td>900</td>
			<td>2100</td>
			<td>4700</td>
		</tr>
		<tr>
			<td>*在频率 6.3 rad s-1下测试, 使用旋转流变仪上的平行板 (直径 20 mm) 进行应变1%。</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
表 1: 用不同机械强度配制的水凝胶信息。

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

18.9K 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

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

科研

化学

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

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 face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

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 ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

羰基饰卡宾的白色磷的活化制备方法和用途

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

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

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

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

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

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

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) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

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

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

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) ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

C 型60/石墨烯混合纳米结构的制备与表征

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

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

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

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

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 and determine the corresponding photochemical mechanism. Then, we describe a protocol to perform ring-opening metathesis polymerization (ROMP) in solution and in miniemulsion using this NHC-photogenerating system. To photogenerate IMes, a system comprising 2-isopropylthioxanthone (ITX) as the sensitizer and 1,3-dimesitylimidazolium tetraphenylborate (IMesH+BPh4-) as the protected form of NHC is employed. IMesH+BPh4- can be obtained in a single step by anion exchange between 1,3-dimesitylimidazolium chloride and sodium tetraphenylborate. A real-time steady-state photolysis setup is described, which hints that the photochemical reaction proceeds in two consecutive steps: 1) ITX triplet is photo-reduced by the borate anion and 2) subsequent proton transfer takes place from the imidazolium cation to produce the expected NHC IMes. Two separate characterization protocols are implemented. Firstly, CS2 is added to the reaction media to evidence the photogeneration of NHC through formation of the IMes-CS2 adduct. Secondly, the amount of NHC released in situ is quantified using acid-base titration. The use of this NHC photo-generating system for the ROMP of norbornene is also discussed. In solution, a photopolymerization experiment is conducted by mixing ITX, IMesH+BPh4-, [RuCl2(p-cymene)]2 and norbornene in CH2Cl2, then irradiating the solution in a UV reactor. In a dispersed medium, a monomer miniemulsion is first formed then irradiated inside an annular reactor to produce a stable poly(norbornene) latex.

We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system.

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 ...

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

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, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

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, ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

石墨烯支持的微井液体细胞制备,用于原位透射电子显微镜

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...