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 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 ...

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

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

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

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

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

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

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 ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

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

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

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 ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

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

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

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

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

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 ...

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 ...