Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability

Highlights

• Only live cells showed changes in refractive index of cytosol under osmotic change.

• The change of MCW sensor signal was linearly proportional to the cell viability.

• The MCW sensor has a potential for a label-free measuring tool of cell viability.

Abstract

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers.

Introduction

Cell viability is a fundamental physiological characteristic of a cell, and has been of general interest in various kinds of cell-based biological research and applications. In general, cell viability has been determined by staining cells with vital dyes such as trypan blue (TB) and Methylene Blue. However, these dyes have some problems that the number of blue-stained cells increases by lapse of time (Jones and Senft, 1985) and determining viability may be inaccurate when viability is <95%. Alternatively, fluorescent dyes such as Acridine Orange (AO), Alcian Blue (Fukudome et al., 2002), Congo Red (Kovács and Foote, 1992), ethidium bromide (EB) (Maria et al., 1997), and SYTOX Green (Breeuwer and Abee, 2000) have been used to assess cell viability. It is impossible to recover cells for further analysis and to continuously monitor over the entire culture procedure because all of these assays are cell invasive and require multiple steps and end-point detection (Hynes et al., 2003).

The release of specific molecules such as ⁵¹Chromium (Vennström et al., 2008), iododeoxyuridine (Schneiderman et al., 1991, Gee et al., 1985), [³H] praline (Vázquez et al., 2003), [⁷⁵Se] selenomethionine, and [³H] uridine (Hu et al., 2000) from cells can also be utilized to measure cell viability, which may be toxic to the cells and limit sensitivity of the assay. Based on cellular metabolic activity, the bioluminescence-based ATP assay using luciferase (Kanges et al., 1984, Cree, 1998) and the MTT/XTT assay (Carmichael et al., 1987) also suffer from the limitation such as the requirement of cell lysis and lower sensitivity, respectively.

An ideal method of measuring cell viability would not use chemicals that are toxic and hinder measurements of cell viability, but remain sensitive enough to measure viability accurately in real time. A number of assays have been developed that allow cell viability to be assessed label-free and noninvasively such as the assay based on monitoring the metabolic activity of living cells via consumption of oxygen has been developed (O'Riordan et al., 2000). However, it has potential problems such as the self-quenching of reagent at high concentrations and phototoxic effects.

The ultimate objective of our research including this work is development of a method for label-free and real-time measurement of cell viability using a surface-refractometric sensor, overcoming the limitations of existing cell viability assays. In case of using a surface-refractometric sensor, however, target cells should be contacted with the sensor surface for measurement. Nevertheless, this method could be applied to most of cell systems since all cells would sink to contact with the sensor surface due to gravity. In this report, as a preliminary result, we assessed the feasibility of label-freely estimating the viability of animal cells adhering to solid surfaces using a metal-clad waveguide (MCW) sensor in real time by monitoring changes in the refractive index (RI) of cytosol due to osmotic stimuli. A recent study showed that surface plasmon resonance (SPR) can be used to monitor volume changes caused by osmotic stimuli in a confluent monolayer of cells (Robelek and Wegener, 2010). Compared to SPR, the MCW sensor with the extensive penetration depth (>1.5 μm) of the optical field is comparatively efficient for monitoring of changes in cell volume (Zourob et al., 2003). Intentional imbalances between osmolarity of the cytosol and ambient media cause the diffusion of water through the cell membrane and consequent alterations in cell volume and the RI of cytosol, which could be observed only in live cells. We investigated the correlation between the changes in RI of cytosol in cells adhering to a surface measured using the MCW sensor and their viability.