Name: hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells
Uploaded: 2018-08-24T14:30:00
Description: Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

这项工作得到了 NIH (R21NS093199) 和加州大学洛杉矶分校 3 r 奖的资助。我们衷心感谢哈利科恩布卢姆博士实验室提供的 HK301 和 HK157 细胞系。我们还感谢加州大学洛杉矶分校组织病理学核心实验室 (TPCL) 为 cryosectioning, 先进的光镜/光谱学核心设施 (施舍) 加利福尼亚 Nanosystems 研究所 (CNSI) 在加州大学洛杉矶分校使用共聚焦显微镜, 加州大学洛杉矶分校克伦普研究所为使用 IVIS 成像系统的分子成像, 加州大学洛杉矶分校分子仪器中心 (MIC) 提供磁共振光谱学, 和流式细胞术核心在琼森综合癌症中心 (JCCC) 在加州大学洛杉矶分校提供检测流程细胞.

所有人类组织收集步骤都是根据体制上批准的议定书进行的。
1. 透明质酸的 Thiolation
注: 除另有规定外, 对羧酸盐组总数量的摩尔比率进行了说明。
<ol><li>溶解500毫克的透明质酸钠 (HA, 500-750 kDa) 在10毫克/毫升在去离子, 蒸馏水 (蝶和2O) 在蒸压灭菌, 250 毫升锥形瓶。搅拌溶液 (约 200 rpm) 在室温下2小时完全溶解 HA。在 thiolation 过程中, 使用搅拌棒和?...

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使用此3D 区域性系统生成可重现的数据需要: 1) 一致的批对批 thiolation, 2) 实践, 以实现水凝胶前体的有效混合和水凝胶培养的处理, 防止破坏和 3) 优化播种使用的每个单元格线的密度。
当水凝胶中需要 ha 的特定重量百分比时, ha 的 thiolation 程度决定了交联密度。我们建议为每个 thiolation 反应使用一致的 HA 量, 以减少批处理的变化。我们建议, 至少300毫克的 ha 用于每个 thiolation ...

三维 (3D) 培养系统在本机组织中重述细胞与其周围胞外基质 (ECM) 之间的相互作用, 比它们的二维 (2D) 对应1,2要好。组织工程的进步已经产生了复杂的3D 文化平台, 能够控制调查到 1) 基质微环境的化学物质成分如何影响细胞行为和 2) 新疗法的疗效包括2癌症在内的一系列疾病的战略。虽然体外模型不能解释系统的因素, 如内分泌和免疫信号, 从而不能完全取代体内模型, 它们提供了一些好处, 包括重现性, 实验控制,负担能力和速度。在这里, 我们描述了大脑模拟水凝胶的使用, 其中3D 培养的患者源性脑肿瘤细胞捕获肿瘤生理学的许多方面, 特别是获得治疗阻力的动力学3。与其他体外方法相比, 这些培养物更能代表体内肿瘤模型和临床观察3。
胶质瘤 (肾小球) 是最常见和致命的癌症, 起源于大脑, 平均存活1-2 年4,5。近年来, 许多研究都集中于肿瘤基质环境对肾小球6、7、8的影响。独特的脑 ECM 已被报道影响肾小球细胞的迁移, 增殖和治疗耐药性6,7,8,9,10,11,12. 透明质酸 (HA) 是大脑中的一种丰富的多糖 (插科打诨), 它与其他的堵嘴和软骨相互作用, 形成水凝胶状的网状13。许多研究报告了 HA 在肾小球肿瘤中的过度表达及其随后对癌症进展的影响8、9、13、14、15、16 ,17。其他 ECM 成分也影响肾小球瘤的生长和侵袭6,7,15,18。例如, 纤维连接蛋白和及其与受精, 通常抗原在肾小球, 诱导 heterodimerization 细胞表面整合素受体通过绑定到 "" 序列, 并启动复杂的信号叶栅, 促进肿瘤生存19 ,20,21。除了生物化学的影响, 组织基质的物理性质也影响肾小球的进展22,23。
连续获得的抗性治疗是肾小球杀伤力4的主要驱动因素之一。在2D 或 gliomasphere 模型中显示出有希望的结果的药物在随后的动物研究和临床案例3中失败, 表明微环境因素的影响显著促进了肾小球瘤的反应1。动物模型可以提供 3D, 生理上适当的微环境移植患者细胞和产生临床相关结果24,25, 大脑微环境的复杂性在体内使它具有挑战性, 以确定哪些功能, 包括细胞基质相互作用, 是关键的具体生物学结果。确定新的治疗目标将受益于使用简化的文化平台, 其中生化和生物物理属性的定义。
不同于以前报告的生物材料模型的肾小球肿瘤微环境26,27没有实现真正的正交控制的个别生化和物理特性的 ECM, 材料平台这里的报告使多重独立特征对肾小球细胞表型的贡献脱钩。在这里, 我们提出了一个基于 HA, 正交可调谐, 水凝胶系统的3D 培养的患者衍生的肾小球细胞。水凝胶由两个高分子成分组成: 1) 生物活性 HA 和 2) 生物惰性聚乙二醇 (PEG)。PEG 是一种广泛应用的生物相容性和亲水性的材料, 低蛋白质吸附和最小免疫原性28。在这里, 大约5% 的醛酸酸基团在 HA 链是功能性的硫醇组, 以使交联到一个商业上可用的4臂 PEG 终止与 maleimides 通过迈克尔型加法。在体内最常见的形态中, HA 存在于高分子量 (大分子) 链中。在这里, 低的修饰的高分子量传感器 ha (500-750 kDa) 有助于保持本机相互作用的 ha 及其细胞受体, 包括 CD4429。用 PEG-硫醇代替 HA 硫醇, 同时保持总硫醇到 maleimides 的恒摩尔比, ha 浓度可以与由此产生的水凝胶的力学性质分离。此外, 化学计量控制可用于将半胱氨酸终止的肽与在每4臂 PEG 上定义的平均 maleimide 终止的手臂结合在一起。结合 ECM 衍生的, 粘合剂肽, 使与整合的相互作用的培养细胞, 通过它的生物化学和化学信号是转基因1。在生理条件下, Maleimide-硫醇的增加发生得非常快, 最大限度地减少细胞封装所需的时间, 并最大限度地提高患者的细胞存活率。此外, 水凝胶的培养可以像典型的组织标本, 并符合标准的表征技术, 包括西方印迹, 流式细胞术, 免疫荧光染色。下面的协议描述了制造水凝胶的过程, 建立了患者衍生的肾小球细胞的3D 培养和生化分析技术。

<table border="0" cellpadding="0" cellspacing="0" style="width:2204px;" width="2203">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">pH meter</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td style="width:1579px;">Any pH meter that has pH 2-10 sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Stir plate</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>General lab equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Erlenmeyer flask (125mL)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">FB-501-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">dialysis tubes</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">21-152-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2L polypropylene beaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">S01916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">sodium hyaluronan</td>
			<td style="width:136px;">Lifecore</td>
			<td style="width:121px;">HA700k-5</td>
			<td>500-750 kDa range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">N-hydroxysuccinimide (NHS)</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">130672-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hydrochloric acid (HCl)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SA48-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Sodium hydroxide (NaOH)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SS266-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cystamine dihydrochloride</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC111770250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dithiolthreitol (DTT)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP172-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Ellman&#39;s test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">D218200-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Deuterated water (deuterium oxide)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC166301000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.22&#181;m vacuum driven filter</td>
			<td style="width:136px;">CellTreat</td>
			<td style="width:121px;">229706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Phosphate buffered saline (PBS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">P32080-100T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hanks&#39; balanced salt saline (HBSS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">MT-21-022-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-maleimide</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7029-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-thiol</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7039-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">L-Cysteine&#160;</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">C7880-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RGD ECM mimetic peptide</td>
			<td style="width:136px;">Genscript Biotech</td>
			<td style="width:121px;">N/A</td>
			<td>Custom peptide with sequence &#34;GCGYGRGDSPG&#34;, N-terminal should be acetylated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">silicone molds</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">GBL664201-25EA</td>
			<td>Use razor blade to cut into single pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">complete culture medium</td>
			<td style="width:136px;">Various</td>
			<td style="width:121px;">Various</td>
			<td>DMEM/F12 (Thermofisher) with non-serum supplement (G21 from GeminiBio), epidermal growth factor 50ng/mL (Peprotech), fibroblast growth factor 20ng/mL (Pepro Tech) and heprain 25&#181;g/mL (Sigma Aldrich), culture medium varies in different labs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">patient derived GBM cell</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">20G needle</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">305175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1mL syringe</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">14-823-434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">10mL syringe</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">302995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RIPA Buffer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">protease/phosphatase inhibitor mini tablet</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">5892970001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">vortex shaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-814-5Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">TrypLE express</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">70&#181;m cell strainer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Paraformaldehyde</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC416785000</td>
			<td>Dissolve 4% (w/v) in PBS, keep pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">D-sucrose</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP220-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Optimal Cutting Temperature (O.C.T.) compound</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">NC9373881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cell culture incubator</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General One with 5% CO2 and 37C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">fridge/freezer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General Lab equipment with -20C and -80C capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Disposable embedding molds</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lyapholizer</td>
			<td style="width:136px;">Labconco</td>
			<td style="width:121px;">N/A</td>
			<td>Any -105C freeze dryers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">HEPES</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Amber vial</td>
			<td style="width:136px;">Kimble Chase</td>
			<td style="width:121px;">60912D-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Wide orifice pipette tips</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">9405120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2-methylbutane</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">03551-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dry Ice</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells

胶质瘤 (肾小球) 是最常见的, 但最致命的中枢神经系统癌症。近年来, 许多研究都集中在细胞外基质 (ECM) 的独特的大脑环境, 如透明质酸 (HA), 促进肾小球的进展和入侵。然而, 大多数体外培养模型包括在 ECM 的上下文之外的肾小球细胞。小鼠移植的肾小球细胞也普遍使用。然而,在体内模型中, 很难分离复杂肿瘤微环境的个体特征对肿瘤行为的贡献。这里, 我们描述了一种基于 ha 水凝胶的三维 (3D) 培养平台, 使研究人员能够独立地改变 HA 浓度和刚度。高分子量 HA 和聚乙二醇 (PEG) 包括水凝胶, 在活细胞存在的情况下通过迈克尔型加法交联。3D. 从患者衍生的肾小球细胞中提取的水凝胶培养物的生存能力和增殖率均优于标准 gliomaspheres。水凝胶系统还能将 ECM 模拟的多肽结合起来, 分离出特定细胞 ECM 相互作用的效应。水凝胶是光学透明的, 所以活细胞可以在3D 文化中成像。最后, HA 水凝胶培养符合分子和细胞分析的标准技术, 包括 PCR、西方印迹和 cryosectioning, 其次是免疫荧光染色。

对于每批 thiolated HA, 应使用 H1核磁共振或埃尔曼测试验证 thiolation 的程度。HA 修改使用这里描述的过程始终如一地产生〜 5% thiolation (定义为摩尔比的硫醇到 HA 双糖) (图 1)。
建立这个新的文化平台将要求每个实验室进行严格的测试, 以确保良好的文化活力, 在实施大规模实验之前。我?...

Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

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

在这里, 我们提出了一个协议, 三维培养病人的胶质瘤细胞在正交可调谐生物材料设计, 以模仿大脑基质。这种方法提供了一种体外实验平台, 它能维持体内胶质瘤细胞的许多特征, 通常在培养中丢失。

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 ...

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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 ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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 ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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 ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...