Protein spot arrays on graphene oxide coatings for efficient single-cell capture

R. Kumar, S. Llewellyn, S. K. Vasantham, Kaiwen Nie, S. Sekula-Neuner, A. Vijayaraghavan, M. Hirtz
2022
Biomedical applications such as cell screening or cell-cell interaction studies require placement and adhesion of cells on surfaces with controlled numbers and location. In particular, single-cell arraying and positioning has come into focus as a basis of such applications. An ideal substrate would combine biocompatibility with favorable attributes such as pattern stability and easy processing. Here, we present a simple yet effective approach to single-cell arraying based on a graphene oxide
more » ... ) surface carrying protein (fibronectin) microarrays to define cell adhesion points. These capture NIH-3T3 cells, resulting in cell arrays, which are benchmarked against analogous arrays on silanized glass samples. We reveal significant improvement in cell-capture performance by the GO coating with regards to overall cell adhesion and single-cell feature occupancy. This overall improvement of cell-arraying combined with retained transparency of substrate for microscopy and good biocompatibility makes this graphene-based approach attractive for single-cell experiments. Single-and low-density cell arrays have garnered significant attention for their promising potential in biomedical research where selective and precise cell positioning is key 1, 2 . Experiments such as cell-cell communication or artificial cell-network assembly, necessary in drug screening or in vitro biomedical research, require sophisticated single-cell analysis to evaluate overall experimental components 3, 4 . Thus, the effective production of single-or few-cell arrays is vital to these efforts. To achieve this, several engineering approaches have been developed to generate suitable cell arrays. These commonly involve designing advanced microwells, followed by targeted cell dispensing e.g. through a microfluidic setup 5, 6 . Also, single cells can also be dispensed from a printer or spotter to formulate single-cell arrays 5, 7 . Recently, 3D printing has been utilized to even produce 3D tissues by singlecell dispensing 8 . While these methods effectively position cells with high accuracy to produce desired single-cell arrays, they involve complex and costly infrastructure. An alternative, simpler method to establish single-cell arrays involves engineering substrates used for cell incubation to define spatial-specific cellular adhesion. Examples of this strategy include chemically tuning the material or patterning the substrates with micro-scale protein arrays for cells to preferentially bind onto. The latter approach is possible through various spotting techniques, including printing (inkjet, microcontact) or stamping (polymer pen lithography, capillary spotting) methods. The different spotting techniques vary with respect to their pattern-spacing resolution and individual protein feature sizes. However, each technique has the capability to promote single-or few-cell occupancy in the patterned regions, thereby producing desired cell-arrays 5,9 . Crucially, spotting techniques separate the substrate fabrication step from the cell seeding procedure. This allows for complex array designs within basic lab environments and provides a versatile method where one can modify arrays on the fly in between experiments, through changing pattern designs. Moreover, it permits a variety of substrates or materials as basis for the printing or spotting process, which may be necessary depending on the experimental outcome, or desired application. Substrates suitable for spotting procedures in cell array production must allow a high density of protein to be deposited and adhere to the surface and maintain protein function 10 . Patterned substrates must also be susceptible to a blocking procedure to inhibit non-specific adhesion and prevent cells from attaching at unwanted areas outside or in-between array features. Furthermore, substrates must exhibit good biocompatibility, without adversely impacting cellular function and promote selective adhesion once patterned. From a fabrication
doi:10.5445/ir/1000143666 fatcat:arsgkcdfzbdplfgfqm2zexrooa