Optical Single Cell Resolution Cytotoxicity Biosensor Based on Single Cell Adhesion Dot Arrays

Abstract

Low cost easy to use cell viability tests are needed in the pharmaceutical, biomaterial and environmental industry to measure adverse cellular effects. Herein we present a new methodology to track cell death with high resolution. We achieved dynamic digital quantification of cell viability by simple optical imaging using "Single Cell Adhesion Dot Arrays" (SCADA). Fibronectin (FN) dot arrays were fabricated on cell culture multiwell plates. The dot array was designed to accomodate a single cell on each fibronectin dot. For cytotoxicity measurements, cell-filled SCADA substrates were exposed to K2CrO4, HgSO4 salts and dimethyl sulfoxide (DMSO). Adherent cells commonly detach from the surface when they die. Dynamic monitoring of the toxic effect of DMSO and K2CrO4 was done measuring cell detachment rate during more than 30 hours by quantifying the number of occupied dots in the SCADA array. HgSO4 inhibited cellular detachment from the surface, and cytotoxicity was monitored using Trypan Blue life/death assay directly on the surface.In all cases, the cytotoxicity effects were easily monitored with single cell resolution and the results were comparable to previous reports. Cytotoxicity SCADA tests require only a transparent substrate, with a patterned area of less than 1 mm 2 and a reduced number of cells. SCADA enabled dynamic measurements at the highest resolution due to the digital measuring of this methodology. Integrated into microfluidic platforms, SCADA will provide a practical tool that will extent to fundamental research and commercial applications.

Advances in Microtechnology for Improved Cytotoxicity Assessment

“…Surface chemistry modification at the micro-scale enables the precise positioning of cells, which following specific adhesion patterns, can lead to new functionalities. In a recent work, microcontact-printing, which creates defined patterns of molecules on surfaces by stamping a micropatterned PDMS, was used to create single cell adhesion dots arrays to address cell viability by optical microscopy (Garcia-Hernando et al, 2020). Cell adhesion spots made by fibronectin protein were designed to allocate single cells, attributing an individual value to each cell.…”

“…Optical microscopy and label-free monitoring of cytotoxicity is also possible for single-cells. In the recent work from Garcia-Hernando et al, previously mentioned, an array of individual cell adhesion fibronectin islets was used to create single cell arrays for the monitoring of cytotoxicity by optical microscopy (Garcia-Hernando et al, 2020). This system offers two approaches for the assessment of cell viability, depending on the mechanism of action of the toxic compound.…”

Continuous monitoring of cell transfection efficiency with micropatterned substrates

“…These cell arrays have been proven to be suitable for gene transfection, having the potential to overcome the limitations of conventional cell cultures (McConnell et al, 2011). Thanks to the versatility of the technique, great control over cell interactions can be achieved, including cell-material and cell-cell interactions, while also enabling the easy generation of a high number of data points in a small substrate area (Garcia-Hernando et al, 2020;Gonzalez-Pujana et al, 2019;Hamon et al, 2016;Hu et al, 2018;Kobel & Lutolf, 2010). Cell patterns can be directly transfected and monitored, without the need of detaching and transporting the cells to a different setup, as the pattern itself simplifies quantification due to the highly controlled localization of the cells.…”

“…This procedure facilitates the comparison of the transfection efficiency, in arrays of single cells and of small cell-colonies, the later resembling conventional cultures. This control over the absence or presence of cell-cell contact cannot be achieved in conventional cell culture, where factors such as cell confluence do not ensure the generation of a homogenous cell-cell contact scenario.Fibronectin was chosen as the adhesion protein as established in previous works(Garcia-Hernando et al, 2020;Gonzalez-Pujana et al, 2019).…”

“…GFP expressing cells were calculated using the number of cells presenting fluorescence after image processing at each specific time. Total number of alive cells for our patterns equals the number of cells that remain in the pattern at each specific time(Garcia-Hernando et al, 2020), S3. = × tx tx Transfection efficiency GPF expressing cells ( ) Total number of alive cells ( ) 100…”