Ong Louis Jun Ye, Zhu Liang, Tan Gabriel Jenn Sern, Toh Yi-Chin
Department of Biomedical Engineering, National University of Singapore, 4, Engineering Drive 3, E4-04-10, Singapore 117583, Singapore.
Institute for Health Innovation and Technology, National University of Singapore, 14 Medical Drive, #14-01, Singapore 117599, Singapore.
Micromachines (Basel). 2020 Jul 9;11(7):669. doi: 10.3390/mi11070669.
Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.
微流控3D组织培养系统对于体外药物测试应用具有吸引力，因为这些平台能够生成3D组织模型并在非常小的规模上进行药物测试。然而，微小的细胞数量和液体体积给使用传统比色法或荧光法（如MTS或Alamar Blue）进行定量细胞活力测量带来了重大技术挑战。同样，活死染色方法通常使用代谢染料来标记活细胞的细胞质，这使得在紧密的3D组织培养中难以分割和计数单个细胞。在本文中，我们提出了一种基于图像的定量细胞活力（QuantICV）检测技术，该技术规避了在微流控3D组织培养中进行定量细胞活力检测的当前挑战。使用一对细胞非渗透性核染料（EthD-1和DAPI）分别依次标记坏死细胞群体和总细胞群体的细胞核。采用共聚焦显微镜和图像处理算法来可视化和量化3D组织体积中的细胞核。QuantICV检测经过验证，在静态2D和3D肿瘤细胞培养中与传统的批量MTS检测显示出良好的一致性。最后，在微流控3D培养装置中，将QuantICV检测用作芯片上的读数，以确定亲本和转移性3D口腔鳞状细胞癌（OSCC）对吉非替尼的不同剂量反应。这种提出的技术在微流控细胞培养以及传统细胞活力检测不可用的情况下可能会有用。
