Name: 3d microtissues for injectable regenerative therapy and high-throughput drug screening
Uploaded: 2017-10-04T11:45:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening at JoVE.com

这项工作得到了中国国家自然科学基金的资助 (赠款: 81522022, 51461165302)。作者想感谢所有杜实验室的成员提供一般的帮助。

动物实验遵循了清华大学生物医学分析中心动物伦理委员会批准的严格的协议。在伦理委员会的批准下, 从北京协和医院整形外科部门获得了患者的知情同意, 得到了人体脂肪组织. 
 1. 制作 3D Microcryogels <ol> <li> microstencil 阵列芯片的设计和制作 <ol> <li> 使用商用软件设计特定几何图形的数组, 如圆、椭圆、三角形或草 14 , 具体取决于后续应用程序. 
		注: 请参阅《再生医学议定书》2节和《药物筛选议定书》3节的设计细节. </li>
		<li> 将设计导入激光雕刻机随附的激光雕刻软件中。根据工厂推荐的激光雕刻机, 将进口的设计用激光刻在聚 (methymethacrylate) (PMMA) 片上. </li>
		<li> 用去离子水清洗 microstencil 阵列芯片, 清除激光雕刻的碎片。在60和 #176 中烘干 microstencil 阵列芯片; c. 在室温下存放几个月的密封袋. </li>
	</ol> </li> <li> 制作 microcryogel 阵列芯片 <ol> <li> 将 4 microstencil 阵列芯片放在样品托盘上, 并插入等离子清洗器中。关闭清洁器的门, 打开真空泵2分钟, 然后打开最大的射频功率 (18 W), 以处理等离子清洗器中的 microstencil 阵列芯片在室温下3分钟以提高亲水性. </li>
		<li> 添加0.06 克明胶至1毫升去离子水, 形成 6% (重量/卷) 明胶前驱体溶液 (足以制造5芯片)。在60和 #176 温暖; C 在水浴中充分溶解。在冰上孵育5分钟. </li>
		<li> 添加3和 #181; L 戊二醛进入明胶前驱溶液, 最终浓度为0.3%。完全混合. </li>
		<li> 吸管200和 #181; 在 microstencil 阵列芯片的上表面上有戊二醛的前驱溶液. 
		注: 200 和 #181; L 足够75毫米和 #215; 25 毫米芯片不管设计。增加芯片表面面积时的比例. </li>
		<li> 通过将2到3倍的弯曲的玻璃棒来回刮, 将溶液均匀地分布在芯片上. 
		注: microstencil 芯片上的每一个微井都可以用前驱体溶液填充, 因为等离子体处理后它们具有亲水性. </li>
		<li> 立即将前体解决方案填充阵列芯片放置在-20 和 #176 中; C 型冰箱为 cryogelation 16 小时. </li>
		<li> 将冻调整为-40 和 #176; C 为30分钟, 将阵列芯片放在 cryogelation 冻和 lyophilize 阵列芯片中, 在真空中放置2小时. 
		注: 明胶 microcryogels 与相互连接的孔隙形成, 因为冰形成期间, cryogelation 升华在冻. </li>
		<li> 进行2节以治疗严重肢体缺血 (CLI) 和3节的高通量药物筛选. </li>
	</ol> </li> </ol> 2。收集单个 Microcryogels 以形成可注射的 3D Microtissues, 用于处理 CLI <ol> <li> 收集单个 Microcryogels <ol> <li> 设计阵列 (75 mm 和 #215; 25 mm) 的600个圆圈, 每一个都有400和 #181; m 直径, 在可注射 3D microtissue 结构的商用软件。使用300和 #181 的 PMMA 制造 microstencil 阵列芯片的厚度, 详见1.1 节. </li>
		<li> 制造 microcryogel 阵列芯片, 如1.2 节所示. </li>
		<li> 用标准的软光刻制作一个与步骤2.1.1 相同的设计的芯片顶针阵列 16 , 17 . </li>
		<li> 将 microcryogel 阵列芯片叠加在集成电路顶针阵列的顶部, 将每个 microcryogel 与阵列上的顶针引脚对准。按 microcryogel 阵列芯片对顶针阵列, 以推动 microcryogels 出的 microwells 与突出的引脚上的硅橡胶顶针阵列, 以收获个人 microcryogels. </li>
		<li> 将 microcryogel 收获到水中, 并借助细胞过滤剂收集它们。使用一个过滤器从一个芯片收集 microcryogels ( 即 , 600 microcryogels). </li>
		<li> 用0.1 米 NaBH 清洗 microcryogels 4 在冰上20分钟以淬火任何 uncross 链醛残渣。每芯片使用5毫升的0.1 米 NaBH 4 。丢弃 NaBH 4 , 用5毫升去离子水冲洗3至5次, 每次15分钟. </li>
		<li> 将水从电池过滤器中丢弃, 并将 microcryogels 收集到每个单元格过滤器的一个簇中, 并使用弯曲的镊子将一个簇放入一个 35 mm 的 Petri 盘中。添加 50-70 和 #181; 我去离子水到每个星团, 盖上培养皿的盖子。轻轻地在桌面上轻敲培养皿, 以使所有的 microcryogels, 所以没有 microcryogels 是躺在另一个 microcryogel 的顶部形成单层 microcryogels 在培养皿的表面. </li>
		<li> 将收获的 microcryogels 冻结在-20 和 #176; C 为4到 16 h, 然后冻为 2 h (请参见步骤 1.2.7)。在室温下将 microcryogels 储存在真空中直到进一步使用. </li>
	</ol> </li> <li> 特性 microcryogels <ol> 通过扫描电子显微镜 (SEM) 成像来评估 microcryogels 的孔隙。
		<ol> <li> 将收获的和冻干的 microcryogels 固定到一个样品持有人的双面胶带和大衣与镀金的溅射涂布九十年代, 在成像之前的 SEM 11 。利用图像分析软件对七种不同扫描电镜图像中 microcryogel 孔直径的分布进行评价和分析. </li>
		</ol> </li> <li> 在 35 mm 的培养皿中称一簇600冻干 microcryogels, 用于干明胶 microcryogels (GMs) 的重量。添加60和 #181; 我去离子水到冻干 microcryogels, 等待三十年代 microcryogels 完全吸收水和膨胀。重 microcryogels 肿。
		<ol> <li> 确定 microcryogels 的平衡溶胀率和孔隙率, 因为肿胀的 microcryogels 与干明胶 microcryogels 的重量之比以及 microcryogels 中的保持水与水合 microcryogels 的重量之比,分别为 13 . </li>
		</ol> </li> <li> 使用与数字测力仪集成的可编程注射器泵来测量可 microcryogels 14 。
		<ol> <li> 用台盼蓝染色600片 microcryogels, 并在600和 #181 中悬浮; L 15% 明胶溶液, 达到均匀分布。负载1毫升 microcryogel 悬浮在1毫升注射器。使用可编程注射器泵在1毫升/分钟的流速下, 通过一个27口径的针头连接到1毫升注射器注入混合物. </li>
			<li> 通过数字测力仪检测 real-time 注入力, 绘制力-时曲线。注射后, 观察显微镜下 microcryogels 的完整性. 
			注: 在 15% (重量/卷) 明胶溶液中悬浮的 GMs 可以顺利注射, 并在注射后保持完好. </li>
		</ol> </li> <li> 描述 microcryogels 的可降解性。
		<ol> <li> 首先, 测量一组600冻干 microcryogels 在 35 mm 培养皿中作为干重的重量。然后, 浸泡600冻干 microcryogels 在2毫升 0.025% (卷/卷) 胰蛋白酶/EDTA. </li>
			<li> 在不同时间点使用单元过滤器从解决方案中收集 microcryogels ( 即 、10、20、30、40、50、60和 70 min)。用纸巾把多余的溶液吸走。测量 microcryogels 在不同时间点的重量, 并计算降解程度, 因为 microcryogel 在一定时间点的重量与 microcryogel 在时间上的比值为零. </li>
		</ol> </li> </ol> </li> <li> 将单元格自动到 microcryogels 中, 以形成 3D microtissues <ol> <li> microcryogels 从步骤2.1.8 到 12 h 气体暴露的环氧乙烷灭菌系统, 然后在 vac 下进行12小时的脱气真空. </li>
		<li> 选择人脂肪间质基质细胞 (hMSCs) 治疗小鼠缺血性后肢。按照先前报告的 18 的步骤隔离单元格. </li>
		<li> 培养基中含有2% 胎牛血清 (FBS) 的生长培养基中的 hMSCs, 10 ng/毫升表皮生长因子 (EGF), 10 ng/毫升血小板衍生生长因子 bb (bb), 1X 胰岛素转铁蛋白硒 (其), 10 -8 M 地塞米松, 10 -4 M 抗坏血酸 2-磷酸酯, 100 U/毫升青霉素, 以及100和 #181; DMEM/F12 中的链霉素。通过细胞的比例为1:3 时汇合。在下面的实验中使用3到5的单元格. </li>
		<li> 收获 hMSCs 与胰蛋白酶和量化的细胞数使用一个福斯计数室和重密度 8 x 10 6 细胞/毫升在 hMSCs 生长培养基. </li>
		<li> 吸管60和 #181; L hMSCs 悬浮到 600 microcryogels 的单层, 直径为400和 #181; m 在35毫米的菜从2.1 节。细胞被自动吸收到多孔微结构的 microcryogels. </li>
		<li> 保持在湿润的室内, 孵育在37和 #176; C 为2小时以允许单元格附加。经过 2 h 的孵化, 加入2毫升培养基。每2天更换一次培养基。文化 hMSCs 加载 microcryogels 2 天, 形成 3D microtissues. </li>
		<li> 经过2天的培养, 吸管 100 hMSCs microcryogels 每井在一个96井板上, 添加120和 #181; 根据制造商和 #39 的指示, 对每个井进行了天青的工作解决方案。在37和 #176 孵育2小时;
		<ol> <li> 检测天青在微读卡器中的可存活细胞代谢的荧光, 其激发光波长为 560 nm, 发射光波长为 590 nm。根据试剂盒协议 13 , 建立细胞数与荧光的标准曲线, 用 hMSCs 从荧光强度中插入细胞数. </li>
		</ol> </li> <li> 从0天到4天的步骤2.3.7 中所述, 使用天青从0天到4天评估 microcryogels 中的 hMSCs 的数量. </li>
		<li> 在磷酸盐缓冲盐水 (PBS) 中, 用1:500 稀释素 AM 和1:250 稀释碘碘 (即活/死染色) microcryogels 染色细胞, 并在荧光显微镜或共聚焦荧光显微镜下观察. </li>
	</ol> </li> <li> 注射 3D microtissues 在体内 在鼠标模型中处理 CLI <ol> <li> 建立女性 balb/c/c 裸鼠的严重后肢缺血报告 11 , 19 以确定 3D microtissue-based 治疗的疗效. </li>
		<li> 使用5毫升吸管将 microtissues 从步骤2.3.6 转移到细胞过滤器中, 并过滤掉培养基. </li>
		<li> 重 hMSCs 3D microtissues 在15% 明胶溶液中, 密度为 100 microcryogels 每 100 #181; L 解决方案. </li>
		<li> 手术前, 用高压釜消毒手术工具, 在动物设施中心的动物手术室里进行手术. </li>
		<li> 将鼠标放入麻醉诱导室中, 其中含有1-3% 氟烷100% 氧气, 流速为1升/分. 将红霉素涂抹在老鼠的眼睛上以防止干燥。通过一个1厘米长的皮肤切口, 结扎股动脉及其分支与5-10 丝绸缝合和消费税 19 . </li>
		<li> 注射哚绿色 (0.1 毫升100和 #181; g/毫升) 通过尾静脉注射监测血流。在反射几何 11 中使用荧光成像系统 (这里是自制的系统) 执行组织的荧光成像. </li>
		<li> 注注射 microtissues 到三个部位的股薄肌周围的动脉切口使用1毫升注射器与23口径针. </li>
		<li> 手术后, 在恢复笼中用加热垫将鼠标保持温暖。注射皮下昔, 以减轻疼痛和持续监测, 直到清醒. </li>
		<li> 28 天后, 用荧光成像技术监测 3D microtissue 治疗肢体的疗效 11 . 
		#160; 注意: 注射皮下昔, 以减轻当小鼠出现自发肢体截肢时的疼痛. </li>
		<li> 在实验完成后, 用二氧化碳安乐老鼠. 
		注: 在这里, 在清华大学生物医学分析中心的动物伦理学委员会批准的严格的协议中, 使用二氧化碳进行了安乐死的 . </li>
	</ol> </li> </ol> 3。用于高通量药物筛选的 Microtissue 阵列芯片的组装在片上单元格区域性的 microcryogel 数组的 <ol> <li> 程序集 <ol> <li> 根据常规井板尺寸修改1.1 节中的设计, 即 , 对于 384-井格式, 设计一个16和 #215 的数组; 24 口井 (行按列), 每个 2 mm 直径和 4.5 mm 中心间距. </li>
		<li> 激光雕刻到500和 #181; m 厚的 PMMA 板材, 如1.1 节所述. </li>
		<li> 使用从步骤3.1.2 获得的384井阵列芯片来制造 microcryogels, 如1.2 节所述。这个包含 microcryogel 的阵列被指定为 microcryogel 阵列芯片. </li>
		<li> 用50毫升0.1 米 NaBH 清洗 microcryogel 阵列芯片, 以使任何残留醛 uncross 连接, 然后用50毫升去离子水反复冲洗3次, 每次2小时. </li>
		<li> 丢弃水, 将 microcryogel 阵列芯片冻结在-20 和 #176; C 为4到16小时, 冻根据步骤 1.2.7. </li>
		<li> 在步骤3.1.1 中修改384井阵列设计, 以包含16和 #215; 24 口井, 每3毫米直径和 4.5 mm 中心间距. </li>
		<li> 从一张超薄 (10 & #181; m) 的双面胶粘带上取下背面的一侧, 并将其粘贴到3毫米厚的有机玻璃片的一侧。激光雕刻的设计, 从步骤3.1.4 到这个3毫米厚的有机玻璃板根据1.1 节。该数组被指定为储层阵列. </li>
		<li> 将 microcryogel 阵列芯片与储能阵列芯片对齐, 并紧密结合在一起, 组装 3D microcryogel 阵列芯片, 用于芯片上的细胞培养。1小时紫外线辐射灭菌 </li>
		<li> 在室温下在真空中存储 3D microcryogel 阵列芯片, 以进行进一步的实验. </li>
	</ol> </li> <li> 3D microtissue 阵列上的药物筛选 <ol> <li> 将25毫升无菌去水的湿箱填满, 作为细胞培养的湿度室。预热在湿润的 5% CO 2 孵化器到37和 #176; C. </li>
		<li> 从组织培养皿中采集非小细胞肺癌细胞 (NCI-H460) 和肝癌细胞 (HepG2), 根据标准协议和培养基中的 re-suspend, 最终密度为 1.0 x 10 6 细胞/毫升。完全混合. 
		注: RPM1640 10% FBS, 100 U/毫升青霉素和100和 #181; 链霉素用于 NCI-H460。使用 DMEM 与 10% FBS, 100 U/毫升青霉素和100和 #181; 链霉素 HepG2。
		<li> 从孵化箱中取出湿度室。使用镊子小心地将组装的 3D microcryogel 阵列芯片从台阶3.1.9 在湿度室。注意不要在房间里用水浸湿 microcryogels. </li>
		<li> 将电池悬浮液彻底混合, 然后分3和 #181; 将细胞悬浮液的 L 直接放到 microcryogel 上. 
		注: 在排出细胞悬浮液前, 吸管尖端应轻触 microcryogel 表面。细胞通过吸收 auto-loaded 进入 microcryogel。不要在外围油井中播种细胞. </li>
		<li> 添加10和 #956; 在单元格 microcryogel 使用多通道吸管 (例如96通道的液体处理器) 后, 将培养基中的每一个井。介质也被添加到外围油井中. 
		注: 使用 RPM1640 介质与 10% FBS, 100 U/毫升青霉素和100和 #181; g/毫升 streptomycin 为 H460 细胞。使用 DMEM 介质与 10% FBS, 100 U/毫升青霉素和100和 #181; 用于 HepaG2 细胞的链霉素. </li>
		<li> 将细胞负载的 microcryogel 阵列芯片放在湿度室中, 在湿湿 5% CO 2 孵化器37和 #176; C 形成 3D microtissue 阵列. </li>
		<li> 溶解阿霉素和 IMMLG-8439 9 在二甲基亚砜 (亚砜) 中形成一个10毫米的股票溶液。用培养基稀释药物, 形成10倍稀释浓度梯度, 从2纳米到200和 #181; m. </li>
		<li> 在每个井中添加10和 #181; 我的药物解决方案。使用0.1% 甲基亚砜 (稀释中) 作为对照。孵育药物负载 3D microtissue 阵列在湿 5% CO 2 孵化器在37和 #176; C 为 24 h. </li>
		<li> 添加4和 #181; 天青库存解决方案。孵育在37和 #176; C 为 2 h. 在微读取器中放置 3D microtissue 阵列, 并在 560 nm 的激发光波长和 590 nm 的发射光波长上检测可活细胞代谢天青的荧光. </li>
		<li> 在进一步数据处理之前, 从所有井中减去天青基线信号。通过控制井的平均荧光信号除以其荧光信号, 计算出各井的细胞活力分数. </li>
		<li> 在绘图软件中绘制剂量-响应曲线, 以细胞活力分数为 Y 轴, 以药物浓度的基10对数作为 X 轴。将50% 抑制浓度 (IC50) 插在细胞活力分数 0.5. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

再生医学和体外药物筛选模型是组织工程的两个重要应用:5,6,7,8,9。虽然这两种应用有着截然不同的需求, 但它们之间的共同点在于需要一个更具仿生的培养条件来增强细胞功能19。只有改进的细胞功能研究才能更好的治疗疾病20,<...

传统的细胞培养在平坦的 two-dimensional (2D) 表面, 如培养皿或井板, 很难引出细胞行为接近他们的本土状态。在三维 (3D) 体系结构中包含各种细胞类型、胞外基质和生物活性可溶性因子的环境的精确重述1,2,3 ,4, 对于组织工程、再生医学、基础生物学研究和药物发现的应用 (5、6、7),在体外构建 biomimicking 组织是必不可少的. ,8,9。
在2D 细胞培养中, 3D 细胞培养被广泛应用于培养细胞的仿生微结构和功能性特征的体外。流行的3D 单元格区域性方法是将单元格聚合为椭球7、8、9、10。细胞椭球可以注射到受伤的组织, 增强细胞的保留和生存相比, 注射的分散细胞。然而, 在注射过程中, 流体剪切力对细胞的非均匀球体大小和不可避免的机械损伤导致细胞治疗效果不佳11,12,13。同样, 在椭球聚合过程中固有的不均匀性使得他们的翻译为3D 细胞高通量药物筛选挑战10。
另一种3D 细胞培养方法是在生物材料的帮助下实现的, 它通常将水凝胶或多孔支架中的细胞封装起来。它允许在构建3D 体系结构时具有更大的灵活性。对于治疗, 细胞包裹在散装支架通常被送到动物身体通过外科植入, 这是侵入性和创伤, 因此限制其广泛的翻译到床边。另一方面, 水凝胶可以通过注射悬浮在水凝胶前体溶液中的细胞进入动物体内进行微创治疗, 使原位凝胶通过热, 化学或酶交联的11。然而, 当细胞被交付, 而水凝胶前体仍然在水状态时, 他们也暴露于机械剪在注射期间。不仅如此, 化学或酶交联过程中, 在原位凝胶的凝胶体也可能造成损害的细胞内。在药物筛选中, 生物材料辅助细胞培养面临着均匀性、可控性和吞吐量等问题。使用水凝胶, 细胞通常涉及在凝胶过程中, 这可能会影响细胞的活力和功能。在细胞播种过程中, 凝胶也会阻碍大多数高通量设备的使用, 因为水凝胶可能需要在冰层上保存, 以防止在细胞播种前凝胶, 而水凝胶可能堵塞配药技巧, 这通常非常薄, 以确保准确性高通量筛选。预成形的支架可能会将生物材料的制造过程从细胞培养中分离出来, 但是大多数 scaffold-based 的产品都可以作为相对较低吞吐量14的散装材料。
为了克服目前3D 文化方法的一些缺点, 我们开发了一种微加工-cryogelation 集成技术来制造现成的和 user-friendly 的 microcryogel 阵列芯片15。在本协议中, 选择明胶作为 microcryogel 制造技术的例证, 因为它具有生物相容性、可降解、cost-effective, 并且不需要对细胞附着进行进一步的修改。其他天然或合成来源的聚合物也可用于制造, 这取决于应用。通过这一技术, 我们可以制造小型化和高弹性 microcryogels 的大小, 形状和布局可控。当加载各种细胞类型, 3D microtissues 可以形成各种应用。这些独特的功能使理想的可, 细胞保护和现场定向保留后, 注入在体内, 以加强治疗效果。不仅如此, microcryogels 可以进一步处理, 形成 3D microtissue 阵列, 这是兼容的通用实验室设备和仪器, 以实现高通量细胞培养多功能药物筛选和其他细胞化验。在这里, 我们将详细介绍 microcryogels 及其后处理作为单独的 3D microtissues 或 3D microtissue 阵列用于两个重要的应用, 细胞治疗和药物筛选, 分别为10,15.

<table><tbody><tr><td>Gelatin</td><td>sigma</td><td>G7041</td><td>All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.</td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>J&amp;amp;K</td><td>902042</td><td>Used as crosslinker in preparation of material.</td></tr><tr><td>Glass cover slip (24 x 50 mm)</td><td>CITOGLASS, China</td><td>10212450C</td><td>To scrape prcursor solution onto microstencils array chips.</td></tr><tr><td>Sodium&amp;nbsp;borohydride, NaBH&lt;sub&gt;4&lt;/sub&gt;</td><td>Beijing Chemical Works</td><td>116-8</td><td>To wash remaining glutaraldehyde away after gelation.</td></tr><tr><td>Vacuum jar</td><td>asperts, China</td><td>VC8130</td><td>To preserve microgels under vacuum.</td></tr><tr><td>Polymethylmethacrylate (PMMA) sheets&amp;nbsp;</td><td>Sunjin Electronics Co., Ltd, China</td><td></td><td>Ordinary PMMA sheets.</td></tr><tr><td>Rayjet laser system</td><td>Rayjet, Australia</td><td>Rayjet 50 C30</td><td>To engrave PMMA sheets to form wells.</td></tr><tr><td>Plasma Cleaner</td><td>Mycro Technologies, USA</td><td>PDC-32G</td><td>To make PMMA hyphophilic.</td></tr><tr><td>Lyophilizer</td><td>Boyikang, China</td><td>SC21CL</td><td>To lyophilize materials.</td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Zhongkekeao, China</td><td>DA0065</td><td>To dye microgels.</td></tr><tr><td>Doxorubicin hydrochloride</td><td>ENERGY CHEMICAL, China</td><td>A01E0801360010</td><td>To test drug resistance of cells in 2D or 3D microgel.</td></tr><tr><td>Live/dead assay</td><td>Dojindo Molecular Technologies (Kumamoto, Japan)</td><td>CS01-10</td><td>To distinguish alive and dead cells.</td></tr><tr><td>Cell Titer-Blue</td><td>Promega (Wisconsin, USA).</td><td>G8080</td><td>To test cell viability.</td></tr><tr><td>Cell strainer</td><td>BD Biosciences, USA</td><td>352360</td><td>To collect microgels.</td></tr><tr><td>D-Luciferin</td><td>SYNCHEM (Germany)</td><td>s039</td><td>To tack cells.</td></tr><tr><td>Scanning electron microscope</td><td>FEI, USA</td><td>Quanta 200</td><td>To characterize microgel morphology.</td></tr><tr><td>&amp;nbsp;Mechanical testing machine</td><td>Bose, USA</td><td>3230</td><td>To measure mechanical features.</td></tr><tr><td>Programmable syringe pump&amp;nbsp;</td><td>World Precision Instruments, USA</td><td>ALADINI 1000</td><td>To test injactabiliy.</td></tr><tr><td>Digital force gauge</td><td>HBO, Yueqing Haibao Instrument Co., Ltd., China</td><td>H-50&amp;nbsp;</td><td>To test injactabiliy.</td></tr><tr><td>Ethylene oxide sterilization system</td><td>Anprolene, Anderson Sterilization, Inc., Haw River, NC</td><td>AN74i</td><td>To sterilize microgels with ethylene oxide gas.</td></tr><tr><td>Microplate reader</td><td>Molecular Devices,USA</td><td>M5</td><td>To measure fluorescence intensity in micro-array.</td></tr><tr><td>Confocal microscope</td><td>Nikon, Japan</td><td>A1Rsi</td><td>To observe cell distribution in 3D.</td></tr><tr><td>Xenogen&amp;nbsp; Lumina II imaging system</td><td>Caliper Life Sciences, USA</td><td>IVIS</td><td>To track cell in animals.</td></tr><tr><td>Liquid work stataion</td><td>Apricot design,USA</td><td>S-pipette</td><td>To load medium or cell suspension high-throuputly.</td></tr></tbody></table>

3d microtissues for injectable regenerative therapy and high-throughput drug screening

为了将传统的2D 细胞培养技术升级到3D 细胞培养, 我们将微加工与 cryogelation 工艺相结合, 生产出大孔微尺度 cryogels (microcryogels), 可加载多种细胞类型, 形成 3D microtissues。在此, 我们提出了制造多功能 3D microtissues 及其在再生疗法和药物筛选中的应用的协议。尺寸和形状可控 microcryogels 可以在一个阵列芯片上制作, 可以被捕获片作为单独的细胞负载载体的可注射再生疗法或进一步组装在芯片上 3D microtissue 阵列的高通量药物筛选。由于这些微型 cryogels 的高弹性性质, 3D microtissues 在注射过程中保护细胞免受机械剪切力的侵袭, 为微创细胞治疗提供了巨大的可。这就保证了小鼠肢体缺血模型中细胞存活率的提高和治疗效果。同时, 以标准的 384-井格式组装 3D microtissue 阵列, 便于使用通用的实验室设施和设备, 使高通量的药物筛选在这个多才多艺的3D 细胞培养平台。

3D microtissue 形成的 microcryogels 的制备和表征。
根据该协议, microcryogels 是捏造的, 形成 3D microtissues 和个人 microcryogels 或 microcryogel 阵列, 并分别用于再生疗法和药物筛选 (图 1)。用 PMMA 制备的 Microstencil 阵列芯片作为 microcryogel 阵列芯片的 micromolds。microstencil 阵列芯片可进行可变几何设计。我们...

Watch this Scientific Journal Video about 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening at JoVE.com

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

本协议描述了通过将微细加工与 cryogelation 技术相结合的方法制备弹性3D 大孔 microcryogels。在加载与细胞, 3D microtissues 生成, 这可以很容易地注入在体内, 以促进再生治疗或组装成阵列的体外高通量药物筛选。

3D Microtissues 用于可注射再生疗法和高通量药物筛选

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

3d-microtissues-for-injectable-regenerative-therapy-high-throughput

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Tsinghua University. 本协议描述了通过将微细加工与 cryogelation 技术相结合的方法制备弹性3D 大孔 microcryogels。在加载与细胞, 3D microtissues 生成, 这可以很容易地注入在体内, 以促进再生治疗或组装成阵列的体外高通量药物筛选。

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

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

科研

生物工程

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

癌症研究

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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 ...