Yong D, Abdul Rahim AA, Thwin CS, Chen S, Zhai W, Win Naing M (2019) Autofluorescence spectroscopy in redox monitoring across cell confluencies. PLoS ONE 14 (12): e0226757.
Abstract:
Patient-specific therapies require that cells be manufactured in multiple batches of small vol- umes, making it a challenge for conventional modes of quality control. The added complex- ity 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 autofluores- cence properties of molecules found natively within cells. This was achieved through a simple retrofitting of a standard inverted fluorescence microscope with a spectrometer out- put 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 nicotin- amide adenine dinucleotide (NADH). This is then quantifiable as redox ratios (RR) that rep- resent 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 autofluores- cence spectroscopy-derived RR can serve as a rapid, non-destructive and label-free surro- gate 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.
License type:
http://creativecommons.org/licenses/by/4.0/
Funding Info:
The authors received no specific funding for this work, any form of funding was provided for by their employer - Agency for Science Technology and Research (A*STAR) Singapore.