Department of Physics, University of Cambridge, Cavendish Laboratory, JJ Thomson Avenue, Cambridge, CB3 0HE, United Kingdom.
Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, United Kingdom.
Sci Rep. 2018 Aug 22;8(1):12604. doi: 10.1038/s41598-018-30407-8.
Live cell Raman micro-spectroscopy is emerging as a promising bioanalytical technique for label-free discrimination of a range of different cell types (e.g. cancer cells and fibroblasts) and behaviors (e.g. apoptosis). The aim of this study was to determine whether confocal Raman micro-spectroscopy shows sufficient sensitivity and specificity for identification of primary human bronchial epithelial cells (HBECs) to be used for live cell biological studies in vitro. We first compared cell preparation substrates and media, considering their influence on lung cell proliferation and Raman spectra, as well as methods for data acquisition, using different wavelengths (488 nm, 785 nm) and scan protocols (line, area). Evaluating these parameters using human lung cancer (A549) and fibroblast (MRC5) cell lines confirmed that line-scan data acquisition at 785 nm using complete cell media on a quartz substrate gave optimal performance. We then applied our protocol to acquisition of data from primary human bronchial epithelial cells (HBEC) derived from three independent sources, revealing an average sensitivity for different cell types of 96.3% and specificity of 95.2%. These results suggest that Raman micro-spectroscopy is suitable for delineating primary HBEC cell cultures, which in future could be used for identifying different lung cell types within co-cultures and studying the process of early carcinogenesis in lung cell culture.
活细胞拉曼微光谱技术正逐渐成为一种很有前途的生物分析技术，可用于对多种不同细胞类型（如癌细胞和成纤维细胞）和行为（如凋亡）进行无标记区分。本研究旨在确定共聚焦拉曼微光谱技术是否具有足够的灵敏度和特异性，可用于识别原代人支气管上皮细胞（HBEC），从而可用于体外活细胞生物学研究。我们首先比较了细胞制备底物和培养基，考虑了它们对肺细胞增殖和拉曼光谱的影响，以及使用不同波长（488nm、785nm）和扫描方案（线、面）进行数据采集的方法。使用人肺癌（A549）和成纤维细胞（MRC5）细胞系评估这些参数，证实了在石英基底上使用完整细胞培养基进行 785nm 线扫描数据采集可获得最佳性能。然后，我们将我们的方案应用于从三个独立来源获得的原代人支气管上皮细胞（HBEC）的数据采集，结果表明不同细胞类型的平均灵敏度为 96.3%，特异性为 95.2%。这些结果表明，拉曼微光谱技术适合描绘原代 HBEC 细胞培养物，将来可用于识别共培养物中的不同肺细胞类型，并研究肺细胞培养物中早期癌变过程。
