Autofluorescence spectroscopy for determining cell confluency

TL;DR

This study introduces a non-destructive optical spectroscopy method using autofluorescence to determine cell confluency by analyzing metabolic markers, validated through biochemical assays and viability tests.

Contribution

It is the first to utilize autofluorescence spectral decomposition for quantifying cell confluency via redox ratios, offering a novel non-invasive monitoring technique.

Findings

Redox ratios are higher at lower confluencies (≤50%).

The method correlates well with biochemical assays.

Measurement process does not affect cell viability.

Abstract

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 cell confluency. We exploit the autofluorescence properties of molecules found natively within cells. By applying 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 is significantly higher for lower confluencies ($\leq$50$\%$), which we attribute to the different metabolic requirements as cells switch from individual survival to concerted proliferation. We validate this relationship through bio-chemical assays and autofluorescence imaging, and further confirmed through a live-dead study that our measurement process had negligible effects on cell viability.