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Autofluorescence spectroscopy in redox monitoring across cell confluencies

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....

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Autores principales: Yong, Derrick, Abdul Rahim, Ahmad Amirul, Thwin, Chaw Su, Chen, Sixun, Zhai, Weichao, Win Naing, May
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919590/
https://www.ncbi.nlm.nih.gov/pubmed/31851724
http://dx.doi.org/10.1371/journal.pone.0226757
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author Yong, Derrick
Abdul Rahim, Ahmad Amirul
Thwin, Chaw Su
Chen, Sixun
Zhai, Weichao
Win Naing, May
author_facet Yong, Derrick
Abdul Rahim, Ahmad Amirul
Thwin, Chaw Su
Chen, Sixun
Zhai, Weichao
Win Naing, May
author_sort Yong, Derrick
collection PubMed
description 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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spelling pubmed-69195902020-01-07 Autofluorescence spectroscopy in redox monitoring across cell confluencies Yong, Derrick Abdul Rahim, Ahmad Amirul Thwin, Chaw Su Chen, Sixun Zhai, Weichao Win Naing, May PLoS One Research Article 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. Public Library of Science 2019-12-18 /pmc/articles/PMC6919590/ /pubmed/31851724 http://dx.doi.org/10.1371/journal.pone.0226757 Text en © 2019 Yong et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Yong, Derrick
Abdul Rahim, Ahmad Amirul
Thwin, Chaw Su
Chen, Sixun
Zhai, Weichao
Win Naing, May
Autofluorescence spectroscopy in redox monitoring across cell confluencies
title Autofluorescence spectroscopy in redox monitoring across cell confluencies
title_full Autofluorescence spectroscopy in redox monitoring across cell confluencies
title_fullStr Autofluorescence spectroscopy in redox monitoring across cell confluencies
title_full_unstemmed Autofluorescence spectroscopy in redox monitoring across cell confluencies
title_short Autofluorescence spectroscopy in redox monitoring across cell confluencies
title_sort autofluorescence spectroscopy in redox monitoring across cell confluencies
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919590/
https://www.ncbi.nlm.nih.gov/pubmed/31851724
http://dx.doi.org/10.1371/journal.pone.0226757
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