Name: hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells
Uploaded: 2018-08-24T14:30:00.000+00:00
Duration: 8 min 35 s
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, 使用搅拌棒和磁搅拌板保持反应搅拌。</li>
	<li>使用0.1 米盐酸 (HCl), 调整 HA 溶液的 pH 值为5.5。称69.6 毫克 (0.25x 摩尔比) 1-乙基 3-(3-dimethylaminopropyl) carbodiimide (EDC)。 注意: 尽管难溶可以直接添加到 ha 解决方案中, 但通常更容易在1毫升的蝶和2O 中先将其溶解, 然后快速地将 edc 溶液添加到 ha 溶液中。前溶解在1毫升的蝶和2O 也可以做与 n-琥珀 (NHS) 和半胱胺 dihydrocholoride, 然后添加到反应中的步骤1.3 和1.4。</li>
	<li>加入17.9 毫克 (0.125x 摩尔比) 的 NHS 的反应。用0.1 米 HCl 将反应溶液的 pH 值调整为 5.5, 在室温下孵育45分钟。</li>
	<li>将盐酸半胱胺的70.0 毫克 (0.25x 摩尔比) 加入反应溶液中。用0.1 米氢氧化钠 (氢氧化钠) 将 pH 值调整为6.25。搅拌时, 在室温下一夜间孵化反应。</li>
	<li>第二天, 用1米氢氧化钠将反应溶液的 pH 值调整到8左右。加入192毫克 (4x 摩尔比) 的 dithiothreitol (德勤) 的反应。将 pH 值调整到8后, 再加上德勤, 在室温下保持1-2 小时的搅拌。</li>
	<li>用1米 HCl 将 pH 值调整为4。将整个反应混合物转化为预浸泡的透析膜 (分子量切断约 13 kDa) 和透析对蝶和2O 预调整为 pH 4。 注: 透析蝶和2O 的体积应至少是反应 HA 溶液容积的40倍 (例如, 50 毫升样品在2升的蝶和2O, pH 4)。</li>
	<li>在室温下, 每天两次更换透析液 (蝶和2O, pH 值 4), 3 天。</li>
	<li>过滤透析溶液通过0.22 µm 膜, 真空驱动过滤器。thiolated HA 在液氮中的闪光冷冻溶液。使用冻干机冷冻干燥样品超过2天。Thiolated HA 在干燥形式可以被存放在真空密封的干燥在-20 °c 至少6月。</li>
	<li>为了确定 thiolation 的程度, 使用埃尔曼的测试和/或质子核磁共振光谱学30,31。</li>
</ol>2. 交联材料的制备
<ol><li>溶解 thiolated HA 在所需浓度 (在此例中, 13.3 毫克/毫升) 在 HEPES 缓冲盐水 (20 毫米 HEPES 在汉克的平衡盐水 (HBSS) 缓冲, 调整到8-9 的 pH 值) 在琥珀瓶, 以尽量减少暴露在周围的光线。使溶液不断搅拌。 注: 如果 pH 值在8以上, 溶液浓度过高, 或溶液在凝胶前过长, 则硫醇与 HA 之间的二硫化物键形成可能发生。为避免这一问题, 使用低于15毫克/毫升的 thiolated ha 和溶解 thiolated ha 在2小时内形成水凝胶。</li>
	<li>在磷酸盐缓冲盐水 (PBS) 中溶解4臂钉 maleimide (peg, 20 kDa) 和4臂 peg 硫醇 (peg 硫醇, 20 kDa), pH 为 7.4, 准备50毫克/毫升 peg 和 peg-硫醇库存溶液。 注: 完全溶解 PEG 试剂需要至少1小时。</li>
	<li>如果加入 ECM 衍生肽, 在 PBS 中溶解含半胱氨酸的多肽, 以制备一种库存溶液。 注: 使用库存浓度为2-4 毫米。</li>
	<li>将硅橡胶模具浸泡在乙醇中至少20分钟来清洗, 然后蒸压灭菌。</li>
</ol>3. 水凝胶交联和细胞封装
注: 举例来说, 四个人, 80 µL 水凝胶与 0.5% (w/v) HA 和压缩模量1帕的封装被描述3。请参见表 1 , 例如, 产生不同性质的水凝胶的食谱: 两个水凝胶结合整合素结合肽和两个水凝胶结合半胱氨酸帽作为阴性控制肽活性。患者衍生的肾小球细胞的播种浓度为50万细胞/毫升。
<ol><li>将库存解决方案的40µL 添加到120µL PBS 中, 稀释 PEG (50 毫克/毫升, 从步骤 2.2) 到12.5 毫克/毫升。将稀释溶液分成两个1.5 毫升的离心管, 使每管有80µL。</li>
	<li>将16µL (2.8 毫米, 从步骤 2.3) 添加到一管和16µL 的库存半胱氨酸溶液 (2.8 毫米, 从步骤 2.3) 到第二管。漩涡混合。将 peg-不胱氨酸或 peg-不适合的解决方案放在冰上, 直到步骤3.9 中使用。 注: 这里描述的过程产生凝胶与140µM 肽。这相当于每8可用的 PEG 武器中大约有1被用肽占据。这可以通过改变半胱氨酸终止肽的摩尔比到可用的 maleimide 组来变化。一般而言, 最大浓度的280µM 肽可以达到, 同时仍留下足够数量的 maleimide 组可用于水凝胶交联。</li>
	<li>将 PEG-硫醇库存溶液 (50 毫克/毫升, 从步骤 2.2) 稀释为5毫克/毫升, 通过混合4µL 50 毫克/毫升库存溶液与36µL 的 PBS。添加120µL 溶解, thiolated HA 从步骤2.1 到混合物。 注: 高分子量谷蛋白的溶液粘性很大。因此, 我们建议使用宽孔微尖端来转移解决方案。吸管慢慢地避免溶液粘在微尖端的壁上, 提高测量精度。</li>
	<li>将清洁、干燥的模具 (在步骤2.4 中准备) 放入非组织培养的每个井中处理 12-井板。使用吸管尖端的清洁, 钝端, 按凝胶模具和双检查密封的模具和底部的井板。 注: 在封装前再次检查密封以防止泄漏。</li>
	<li>通过培养的肾小球细胞, 离解到单细胞和确定细胞浓度如前所述3。 注意: 某些肾小球系不能与单个细胞分离。在这种情况下, 可以封装整个 gliomaspheres。然而, 在精确的细胞计数后, 准备分离的单细胞, 以提高重现性。gliomasphere 传代的协议在不同的实验室中有所不同, 其中许多可能与水凝胶封装兼容。<ol>
<li>建议离心机 gliomaspheres 在 500 x g 5 分钟, 取出上清液, 并将1毫升细胞离解酶添加到细胞颗粒中。然后, 孵育5分钟, 轻轻地敲管搅拌。</li>
		<li>在 DMEM/F12 中加入4毫升完整培养基 (50 毫升 EGF, 20 FGF-2, 25 µg/毫升肝素, G21 补充剂和1% 青霉素/链霉素)。</li>
		<li>离心机再以 500 x g 为5分钟, 取出上清液。最后, 并用重悬细胞颗粒在1毫升的完整培养基和通过悬浮细胞通过70µm 细胞过滤器。(建议) 用另外4毫升的完整培养基清洗过滤器, 以最大限度地恢复细胞数量。</li>
	</ol>
</li>
	<li>用 hemocytometer 估计悬浮液中细胞的浓度。将细胞悬浮液分成2个离心管, 其中每管含有 ~ 8万个细胞。离心机在 500 x g 5 分钟;一般情况下, 8万个细胞将组成 2 80 µL 水凝胶。</li>
	<li>在80µL 的 peg 溶液中除去上清和并用重悬一个颗粒, 在80µL 的 peg-不胱氨酸溶液中, 第二个颗粒 (在步骤3.1 中制备)。</li>
	<li>使用200µL, 宽孔微尖端, 免除40µL 的 HA 解决方案 (从步骤3.3 以上) 到每个橡胶硅胶模具 (如在步骤3.4 中准备)。</li>
	<li>使用200µL, 宽孔微尖端, 混合40µL 的 peg-不胱氨酸或 peg-不符合的细胞溶液与 HA 溶液在模具中。吸吸管迅速上下不超过10次。重复为每一个凝胶培养准备。 注意: 这一步需要实践, 因为最初的凝胶发生迅速 (在三十年代)。吸管向上和向下移动的尖端到不同的位置在模具, 以确保均匀混合。始终保持低于液体水平的尖端, 以避免形成气泡。不要把溶液搅拌太多次, 因为凝胶可能会在微尖端形成。peg-胱氨酸和 peg-不均可在不同比例下进行组合, 以达到预期的 RGD 肽浓度。</li>
	<li>将含有凝胶封装细胞的钢板放入37°c 细胞培养孵化器中5-10 分钟, 以确保反应完成。</li>
	<li>添加 2-2. 5 毫升培养基, 每个井与形成凝胶。使用无菌, 2 µL 吸管尖端或微铲, 轻轻地分开的模具和凝胶。使用预灭菌钳从井板中取出模具。将井板放回细胞孵化器 (37 °c 和 5%CO2) 用于培养和未来的实验。 注: 垂直向上拉模具, 以避免损害胶凝体的培养。一般而言, 在凝胶培养培养基中应每3-4 天更换一次。去除培养基时, 注意不要吸水凝胶。如果这是一个问题, 我们建议使用塑料转移吸管。如果计划的生物荧光成像跟踪细胞数, 必须转基因与慢病毒北疆编码组成性荧光素酶表达前, 如前所述3。对于生物发光成像, 在发光成像前添加1毫米的 d-荧光素到培养基 1 h。</li>
</ol>4. 用于西方印迹的裂解制剂
<ol><li>在冰上冷却1.5 毫升离心管 (每凝胶样品 1)。冷却离心机到4°c。</li>
	<li>从井板中取出介质。将凝胶转移到 prechilled 离心管中。</li>
	<li>用1x 蛋白酶/磷酸酶抑制剂添加100µL 马国贤缓冲液。</li>
	<li>使用1毫升注射器与20克针, 打破了凝胶通过推动整个混合物通过针至少20次。</li>
	<li>闪光旋转的样品使用工作台顶离心, 并把样品放回冰上。</li>
	<li>涡流样品每5分钟一次, 总共20分钟。</li>
	<li>离心机样品在 14000 x g 15 分钟在4摄氏度。</li>
	<li>转移上清到新的, 预冷的离心管和存储在-20 °c (短期) 或-80 摄氏度 (长期)。使用标准程序进行凝胶电泳, 如前所述3。</li>
</ol>5. 从水凝胶培养中提取单细胞流式细胞术
<ol><li>去除培养基和转移凝胶培养 (从步骤 3.11) 成1.5 毫升离心管。</li>
	<li>孵育凝胶与500µL 细胞离解酶 (例如, TrypLE 表达) 在37°c 5 分钟, 偶尔地弹管鼓动。</li>
	<li>将混合物转化为50毫升离心管, 5 毫升的完全培养基。把管子放在冰上。</li>
	<li>将20G 针连接到10毫升注射器上。轻轻地将细胞悬架向上和向下通过针8次。</li>
	<li>将混合物通过70µm 细胞过滤器流进一个新的50毫升离心管。通过过滤器再应用5毫升的完整培养基, 收集其余的细胞。</li>
	<li>离心样品在 400 x g 为5分钟. 移除上清液, 并在所需的缓冲器中并用重悬颗粒, 用于流式细胞术 (使用标准协议3)。</li>
</ol>6. 切片用水凝胶的超低温保存
<ol><li>从凝胶培养中移除培养基 (从步骤 3.11)。</li>
	<li>孵化凝胶培养与2毫升4% 多聚甲醛 (粉煤灰) 在4°c 过夜。 注意: 粉煤灰是有毒的, 必须小心处理。</li>
	<li>第二天, 去掉煤灰。在 PBS 中加入2毫升5% 蔗糖, 在室温下凝胶和孵育1小时。</li>
	<li>在 PBS 中更换5% 蔗糖溶液, 2 毫升20% 蔗糖, 孵育30分钟。</li>
	<li>在 PBS 中用2毫升新鲜20% 蔗糖取代蔗糖溶液, 再孵化30分钟。</li>
	<li>第三次, 在 PBS 中用2毫升新鲜20% 蔗糖取代蔗糖溶液。孵化在4°c 过夜。</li>
	<li>在最佳切削温度 (OCT) 化合物中制备20% 蔗糖。 注: 由于 OCT 的粘度, 蔗糖的溶解可能需要一段时间 (到一夜之间)。我们建议在溶解过程中把混合物放在振动筛上以保持搅拌。</li>
	<li>第二天, 去掉20% 蔗糖溶液, 并轻轻地将20% 蔗糖在 OCT 溶液中的凝胶上倒入。确保覆盖整个凝胶。孵育4°c 为3小时。</li>
	<li>使用大的平铲将凝胶转移到嵌入模具的中心;这必须小心地做, 以避免损坏的凝胶。</li>
	<li>凉爽的 2-丁烷内的 cryochamber 与过剩的干冰。</li>
	<li>用纯 OCT (无蔗糖) 填充嵌入模具。填充到正下方的模具的顶边。</li>
	<li>冷冻水凝胶样品浸泡在冷却 2-丁烷, 然后进行切片。</li>
	<li>使用恒温器对样品进行切片;推荐10-18 µm 和切片温度约-26 摄氏度的断面厚度。</li>
	<li>对切片凝胶培养执行标准染色程序, 如前所述3。 注: 粉煤灰是已知的刺激性和致癌物质。请根据当地适用的标准和规定处理和处置粉煤灰。</li>
</ol>7. 水凝胶样品中总 RNA 的提取
注: 在这里, 我们描述了一个协议使用商业套件 (见材料表), 以提取总 RNA 从水凝胶培养细胞。缓冲区和所有材料都在使用的套件中可用。
<ol><li>移除培养基。使用宽孔尖端的 P1000 转移吸管将水凝胶培养成1.5 毫升离心管。</li>
	<li>添加350µL 的缓冲 RLT 到水凝胶培养。</li>
	<li>使用20克针连接到1毫升注射器, 粉碎凝胶, 通过针推整个混合物至少20次。</li>
	<li>将整个混合物转移到2毫升收集管中的均质机柱上。离心机在 1.3万 x g 2 分钟。</li>
	<li>将上清液转移到干净的1.5 毫升离心管中。小心不要扰乱底部的凝胶沉淀。</li>
	<li>将裂解样品与350µL 100% 乙醇混合。把所有的样品转移到一个旋转柱 (从套件) 的地方在2毫升收集管。离心机为1分钟, 在 1.3万 x g。</li>
	<li>有关 RNA 纯化的后续步骤, 请遵循套件制造商提供的常规协议。 注: 提取 RNA 后, 可使用 PCR 标准协议。</li>
</ol>

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作者声明他们没有竞争的财政利益。

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><tbody><tr><td>pH meter</td><td>Thermo Fisher</td><td>N/A</td><td>Any pH meter that has pH 2-10 sensitivity</td></tr><tr><td>Stir plate</td><td>Thermo Fisher</td><td>N/A</td><td>General lab equipment</td></tr><tr><td>Erlenmeyer flask (125mL)</td><td>Thermo Fisher</td><td>FB-501-125</td><td></td></tr><tr><td>dialysis tubes</td><td>Thermo Fisher</td><td>21-152-14</td><td></td></tr><tr><td>2L polypropylene beaker</td><td>Thermo Fisher</td><td>S01916</td><td></td></tr><tr><td>sodium hyaluronan</td><td>Lifecore</td><td>HA700k-5</td><td>500-750 kDa range</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td><td>Thermo Fisher</td><td>PI-22980</td><td></td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>sigma aldrich</td><td>130672-5G</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Thermo Fisher</td><td>SA48-500</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Thermo Fisher</td><td>SS266-1</td><td></td></tr><tr><td>Cystamine dihydrochloride</td><td>Thermo Fisher</td><td>AC111770250</td><td></td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Thermo Fisher</td><td>BP172-25</td><td></td></tr><tr><td>Ellman&#039;s test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid</td><td>Sigma Aldrich</td><td>D218200-1G</td><td></td></tr><tr><td>Deuterated water (deuterium oxide)</td><td>Thermo Fisher</td><td>AC166301000</td><td></td></tr><tr><td>0.22&amp;micro;m vacuum driven filter</td><td>CellTreat</td><td>229706</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher</td><td>P32080-100T</td><td></td></tr><tr><td>Hanks&#039; balanced salt saline (HBSS)</td><td>Thermo Fisher</td><td>MT-21-022-CV</td><td></td></tr><tr><td>4-arm-PEG-maleimide</td><td>JenKem Technology</td><td>A7029-1</td><td>molecular weight around 20kDa</td></tr><tr><td>4-arm-PEG-thiol</td><td>JenKem Technology</td><td>A7039-1</td><td>molecular weight around 20kDa</td></tr><tr><td>L-Cysteine&amp;nbsp;</td><td>sigma aldrich</td><td>C7880-100G</td><td></td></tr><tr><td>RGD ECM mimetic peptide</td><td>Genscript Biotech</td><td>N/A</td><td>Custom peptide with sequence &quot;GCGYGRGDSPG&quot;, N-terminal should be acetylated</td></tr><tr><td>silicone molds</td><td>Sigma Aldrich</td><td>GBL664201-25EA</td><td>Use razor blade to cut into single pieces</td></tr><tr><td>complete culture medium</td><td>Various</td><td>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&amp;micro;g/mL (Sigma Aldrich), culture medium varies in different labs</td></tr><tr><td>patient derived GBM cell</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>20G needle</td><td>BD medical</td><td>305175</td><td></td></tr><tr><td>1mL syringe</td><td>Thermo Fisher</td><td>14-823-434</td><td></td></tr><tr><td>10mL syringe</td><td>BD medical</td><td>302995</td><td></td></tr><tr><td>RIPA Buffer</td><td>Thermo Fisher</td><td>PI-89901</td><td></td></tr><tr><td>protease/phosphatase inhibitor mini tablet</td><td>sigma aldrich</td><td>5892970001</td><td></td></tr><tr><td>vortex shaker</td><td>Thermo Fisher</td><td>12-814-5Q</td><td></td></tr><tr><td>TrypLE express</td><td>Thermo Fisher</td><td>12604013</td><td></td></tr><tr><td>70&amp;micro;m cell strainer</td><td>Thermo Fisher</td><td>22-363-548</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher</td><td>AC416785000</td><td>Dissolve 4% (w/v) in PBS, keep pH 7.4</td></tr><tr><td>D-sucrose</td><td>Thermo Fisher</td><td>BP220-1</td><td></td></tr><tr><td>Optimal Cutting Temperature (O.C.T.) compound</td><td>Thermo Fisher</td><td>NC9373881</td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo Fisher</td><td>N/A</td><td>Any General One with 5% CO2 and 37C</td></tr><tr><td>fridge/freezer</td><td>Thermo Fisher</td><td>N/A</td><td>Any General Lab equipment with -20C and -80C capacity</td></tr><tr><td>Disposable embedding molds</td><td>Thermo Fisher</td><td>12-20</td><td></td></tr><tr><td>Lyapholizer</td><td>Labconco</td><td>N/A</td><td>Any -105C freeze dryers</td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Amber vial</td><td>Kimble Chase</td><td>60912D-2</td><td></td></tr><tr><td>Wide orifice pipette tips</td><td>Thermo Fisher</td><td>9405120</td><td></td></tr><tr><td>2-methylbutane</td><td>Thermo Fisher</td><td>03551-4</td><td></td></tr><tr><td>Dry Ice</td><td>N/A</td><td>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 ...

hyaluronic-acid-based-hydrogels-for-3-dimensional-culture-patient

Research

JoVE Journal

Bioengineering

12.9K 次观看.  University of California, Los Angeles. 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。

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

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

科研

癌症研究

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

生物工程

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

医学

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

在 PEG 水凝胶中制备肿瘤球状体后进行 3D 封装

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

作者聚焦:胶质母细胞瘤微环境研究的体外水凝胶模型

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

<meta charset="utf8"></head><body>使用透明质酸-胶原蛋白 I 水凝胶系统复制胶质母细胞瘤侵袭

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

<meta charset="utf8"></head><body>作者聚焦：用于研究通过间质液流浸润胶质母细胞瘤的患者知情 3D 模型

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

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

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此视频中的章节

胶质母细胞瘤的3D球形模型

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型