Autofluorescence spectroscopy in redox monitoring across cell confluencies
Autofluorescence spectroscopy in redox monitoring across cell confluencies
复制标题
自发荧光光谱在细胞汇合氧化还原监测中的应用
DOI：
10.1371/journal.pone.0226757
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发表时间：
2019
期刊：
影响因子：
3.7
通讯作者：
May Win Naing
中科院分区：
文献类型：
--
作者：
D. Yong;A. A. Abdul Rahim;C. Thwin;Sixun Chen;Wei;May Win Naing
患者特异性疗法需要以多批小批量生产细胞，这对传统的质量控制模式构成了挑战。固有的变异性（即使在批次内）增加了复杂性，需要不断监测，以确保可比的最终产品。因此，开发新的非破坏性细胞监测模式至关重要。在这里，我们研究，第一次，使用光谱学在确定细胞的氧化还原跨细胞汇合，通过利用细胞内发现的分子的自发荧光特性。这是通过对具有光谱仪输出和适当的荧光滤光立方体的标准倒置荧光显微镜进行简单改造来实现的。通过对获得的自发荧光光谱进行光谱分解，我们能够进一步辨别不同分子的相对贡献，即黄素腺嘌呤二核苷酸（FAD）和还原型烟酰胺腺嘌呤二核苷酸（NADH）。然后，这可量化为氧化还原比（RR），其表示基于光学测量的FAD和NADH的量的氧化至还原的程度。结果表明，RR随着细胞融合度的增加而降低，这归因于几个相互关联的细胞过程。我们通过生物化学和活力测定验证了RR、代谢和细胞融合之间的关系。进一步进行了活死和DNA损伤研究，以证实我们的测量过程对细胞的影响可以忽略不计。在这项研究中，我们证明了自发荧光光谱衍生的RR可以作为一个快速，非破坏性和无标记的替代细胞代谢测量。这被进一步用于建立细胞代谢和细胞融合过程中细胞氧化还原之间的关系，并且可能被用作细胞疗法制造中的质量指标。
Patient-specific therapies require that cells be manufactured in multiple batches of small volumes, making it a challenge for conventional modes of quality control. The added complexity of inherent variability (even within batches) necessitates constant monitoring to ensure comparable end products. Hence, it is critical that new non-destructive modalities of cell monitoring be developed. Here, we study, for the first time, the use of optical spectroscopy in the determination of cellular redox across cell confluencies by exploiting the autofluorescence properties of molecules found natively within cells. This was achieved through a simple retrofitting of a standard inverted fluorescence microscope with a spectrometer output and an appropriate fluorescence filter cube. Through spectral decomposition on the acquired autofluorescence spectra, we are able to further discern the relative contributions of the different molecules, namely flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH). This is then quantifiable as redox ratios (RR) that represent the extent of oxidation to reduction based upon the optically measured quantities of FAD and NADH. Results show that RR decreases with increasing cell confluency, which we attribute to several inter-related cellular processes. We validated the relationship between RR, metabolism and cell confluency through bio-chemical and viability assays. Live-dead and DNA damage studies were further conducted to substantiate that our measurement process had negligible effects on the cells. In this study, we demonstrate that autofluorescence spectroscopy-derived RR can serve as a rapid, non-destructive and label-free surrogate to cell metabolism measurements. This was further used to establish a relationship between cell metabolism and cellular redox across cell confluencies, and could potentially be employed as an indicator of quality in cell therapy manufacturing.
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影响因子：
2.2
作者：
Heikal AA
通讯作者：
Heikal AA
DOI：
10.1152/nips.01448.2003
发表时间：
2003-10-01
期刊：
NEWS IN PHYSIOLOGICAL SCIENCES
影响因子：
--
作者：
Aw, TY
通讯作者：
Aw, TY
影响因子：
11.2
作者：
Sarita G. Menon;E. Sarsour;D. Spitz;R. Higashikubo;M. Sturm;Hannah J. Zhang;P. Goswami
通讯作者：
Sarita G. Menon;E. Sarsour;D. Spitz;R. Higashikubo;M. Sturm;Hannah J. Zhang;P. Goswami
影响因子：
3.4
作者：
Huang, SH;Heikal, AA;Webb, WW
通讯作者：
Webb, WW
影响因子：
2.6
作者：
Jones, Dean P.
通讯作者：
Jones, Dean P.