Direct and Label‐Free Cell Status Monitoring of Spheroids and Microcarriers Using Microfluidic Impedance Cytometry

Abstract

cytotoxicity assessment due to close resemblance of intercellular and cellmatrix complexities in vivo. [1][2][3] Microcarriers are bead-based 3D matrix scaffolds (≈100-400 µm in diameter) used to upscale cell culture processes in tissue engineering [4,5] and cell therapies. [6,7] As these cultures may last from several days to months, [8] real-time monitoring of spheroid/microcarrier status in a label-free and non-invasive manner is of great significance in many bioprocesses. Conventional sensors for bioreactors probe changes in culture media properties (pH, dissolved oxygen, temperature etc.) that are affected by biomass. As quantification of cell metabolites in culture media indirectly indicates cell/spheroid growth, it is not suitable for real-time monitoring due to a delay between metabolites change and the cell state. [9] Unlike single cells, an inherent problem for optical imaging of 3D spheroids/microcarriers is their large sizes which limit light penetration and fluorescent staining. [10] Typically, cellular aggregates are dissociated into single cells and stained to determine viability as endpoint measurements. [11,12] Electrical impedance spectroscopy is a label-free approach for characterization of material dielectric properties in a static (non-flow) manner, [13] and has been recently applied in microfluidic systems to monitor 3D 3D cellular spheroids/microcarriers (100 µm-1 mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|Z LF | (60 kHz), |Z HF | (1 MHz)) which are optimal for spheroid growth and viability monitoring are identified. Using a closed-loop recirculation system, it is demonstrated that |Z LF | increases with breast cancer (MCF-7) spheroid biomass, while higher opacity (impedance ratio |Z HF |/|Z LF |) indicates cell death due to compromised cell membrane. Anti-cancer drug (paclitaxel)-treated spheroids also exhibit lower |Z LF | with increased cell dissociation. Interestingly, impedance characterization of adipose-derived mesenchymal stem cell differentiation on Cytodex microcarriers reveals that adipogenic cells (higher intracellular lipid content) exhibit higher impedance than osteogenic cells (more conductive due to calcium ions) for both microcarriers and single cell level. Taken together, the developed platform offers great versatility for multi-parametric analysis of spheroids/microcarriers at high throughput (≈1 particle/s), and can be readily integrated into bioreactors for long-term and remote monitoring of biomass and cell quality.

Dual-frequency impedance assays for intracellular components in microalgal cells

“…Besides, the cell membrane (10 nm) was modelled using the contact impedance approximation rather than the full-fidelity thin-layer geometry, which greatly simplified the numerical calculations while ensuring reliable analysis. 23,48 Additionally, organelles and biomolecules are all simplified as 2D circles (dimensions: 1 μm radius, 2 μm span) with twice the thickness of the cell membrane (20 nm), because chloroplasts have a double membrane envelope. The impedance signal can be obtained through calculating the current signal when particles move along the centreline under the stimulation of the two frequencies (i.e., 500 kHz and 6 MHz) simultaneously.…”

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges

“…Quality control tests include e purity, viability, genetic stability and, immunophenotype characterization [ 278 ]. Recently, Gong et al reported a novel microfluidic approach that label-free and continuous-flow monitoring of single microcarrier using co-planar Field's metal electrodes and can be integrated into bioreactors for long-term [ 279 ]. Taken together, by employing bioreactors and microcarriers, it is expected that production costs would decrease due to improved process monitoring and quality control leading to better consistency and process efficiency, and enabling economies of scale.…”

Microfluidic flow cytometry for blood-based biomarker analysis

“…However, imagingbased detection [81][82][83][84][85][86][87][88][89][90] (Fig. 3b) and impedance-based detection [91][92][93][94][95][96][97][98] (Fig. 3c) have become progressively universal in the past decade.…”