3D multicellular tumor spheroids (MCTSs) have recently emerged as a landmark for cancer research due to their inherent traits that are physiologically relevant to primary tumor microenvironments. A facile approach–laser‐ablated micro U‐wells–has been widely adopted in the past decade. However, the differentiation of microwell uniformities and the construction of arrays have all remained elusive. Herein, an improved laser‐ablated microwell array technique is proposed that can not only achieve arrayed MCTSs with identical sizes but can also perform high‐throughput drug assessments in situ. Three critical laser ablation parameters, including frequency, duty cycle, and pulse number, are investigated to generate microwells flexibly with a range from 170 to 400 μm. The choice of microwells is optimally arranged into an array via precise control of horizontal spacing (d_x) and vertical spacing (d_y) amenable of cell‐loss‐free culture during cell seeding. Harvested T24, A549 and Huh‐7 MCTSs from the microwell array correspond to approximately 75 to 140 μm in diameter. Anticancer drug screening of cisplatin validated IC50 values in 2D and MCTS conditions are 3.5 versus 9.1 μM (T24), 11.8 versus 277.7 μM (A549) and 33.5 versus 52.8 μM (Huh‐7), and the permeability is measured to range from 0.042 to 0.58 μm min⁻¹.

中文翻译：

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高度可重复的微型 U 型孔阵列板促进高通量肿瘤球体培养和药物评估

3D 多细胞肿瘤球体 (MCTS) 最近已成为癌症研究的里程碑，因为它们具有与原发肿瘤微环境生理相关的固有特征。一种简便的方法——激光烧蚀微型 U 形孔——在过去十年中已被广泛采用。然而，微孔均匀性的差异和阵列的构建仍然难以捉摸。在此，提出了一种改进的激光烧蚀微孔阵列技术，该技术不仅可以实现具有相同尺寸的阵列MCTS，而且还可以进行原位高通量药物评估。研究了三个关键的激光烧蚀参数，包括频率、占空比和脉冲数，以灵活地生成范围为 170 至 400 μm 的微孔。通过精确控制水平间距 ( d _x ) 和垂直间距 ( d _y ) ，微孔的选择被最佳地排列成阵列，以适应细胞接种过程中的无细胞损失培养。从微孔阵列中收获的 T24、A549 和 Huh-7 MCTS 的直径约为 75 至 140 μm。顺铂抗癌药物筛选验证在 2D 和 MCTS 条件下的 IC50 值分别为 3.5 与 9.1 μM (T24)、11.8 与 277.7 μM (A549) 和 33.5 与 52.8 μM (Huh-7)，测得的渗透性范围为 0.042 至0.58μm最小值^-1。