Sarkar Sreerupa, Peng Chien-Chung, Kuo Chiung Wen, Chueh Di-Yen, Wu Hsiao-Mei, Liu Yuan-Hsuan, Chen Peilin, Tung Yi-Chung
Department of Engineering and System Science, National Tsing Hua University Hsinchu 30013 Taiwan.
Research Center for Applied Sciences, Academia Sinica Taipei 11529 Taiwan
RSC Adv. 2018 Aug 28;8(53):30320-30329. doi: 10.1039/c8ra05505j. eCollection 2018 Aug 24.
Three-dimensional cell spheroid culture using microfluidic devices provides a convenient model for studying tumour spheroid structures and internal microenvironments. Recent studies suggest that oxygen deprived zones inside solid tumors are responsible for stimulating local cytokines and endothelial vasculature proliferation during angiogenesis. In this work, we develop an integrated approach combining microfluidic devices and multi-photon laser scanning microscopy (MPLSM) to study variations in oxygen tension within live spheroids of human osteosarcoma cells. Uniform shaped, size-controlled spheroids are grown and then harvested using a polydimethylsiloxane (PDMS) based microfluidic device. Fluorescence live imaging of the harvested spheroids is performed using MPLSM and a commercially available oxygen sensitive dye, Image-iT Red, to observe the oxygen tension variation within the spheroids and those co-cultured with monolayers of human umbilical vein endothelial cells (HUVECs). Oxygen tension variations are observed within the spheroids with diameters ranging from 90 ± 10 μm to 140 ± 10 μm. The fluorescence images show that the low-oxygenated cores diminish when spheroids are co-cultured with HUVEC monolayers for 6 hours to 8 hours. In the experiments, spheroids subjected to HUVEC conditioned medium treatment and with a cell adherent substrate are also measured and analyzed to study their significance on oxygen tension within the spheroids. The results show that the oxygenation within the spheroids is improved when the spheroids are cultured under those conditions. Our work presents an efficient method to study oxygen tension variation within live tumor spheroids under the influence of endothelial cells and conditioned medium. The method can be exploited for further investigation of tumor oxygen microenvironments during angiogenesis.
使用微流控设备进行三维细胞球体培养为研究肿瘤球体结构和内部微环境提供了一个便捷的模型。最近的研究表明，实体瘤内部的缺氧区域在血管生成过程中负责刺激局部细胞因子和内皮血管系统增殖。在这项工作中，我们开发了一种结合微流控设备和多光子激光扫描显微镜（MPLSM）的综合方法，以研究人骨肉瘤细胞活球体中氧张力的变化。使用基于聚二甲基硅氧烷（PDMS）的微流控设备培养出形状均匀、大小可控的球体，然后进行收获。使用MPLSM和市售的氧敏染料Image-iT Red对收获的球体进行荧光实时成像，以观察球体以及与人类脐静脉内皮细胞（HUVEC）单层共培养的球体中的氧张力变化。在直径范围为90±10μm至140±10μm的球体中观察到了氧张力变化。荧光图像显示，当球体与HUVEC单层共培养6至8小时时，低氧核心会减少。在实验中，还对接受HUVEC条件培养基处理并带有细胞粘附底物的球体进行了测量和分析，以研究它们对球体中氧张力的影响。结果表明，在这些条件下培养球体时，球体内的氧合作用得到了改善。我们的工作提出了一种有效的方法，用于研究在内皮细胞和条件培养基影响下活肿瘤球体中的氧张力变化。该方法可用于进一步研究血管生成过程中的肿瘤氧微环境。
