Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning

Abstract

Assays used for high-throughput screening often rely on viable cells to facilitate lead discovery of functional therapeutics of interest, such as neutralizing antibodies. The reliance on living cells has traditionally constrained the form factor of these assays to common microtitre plates (e..g., 96-, 384-, 1536-well plates). This form factor can constrain the total numbers of unique assays performed (number of plates examined) in part due to the costs of media and components used. We sought to develop a simple and efficient format to implement functional assays for screening biological responses that rely on cell-based signal readouts. We report a versatile and scalable method that uses dense arrays of subnanoliter wells (nanowells) for imparting defined patterns on monolayers of cells. The living cell arrays produced by nanowell-assisted cell patterning (NWAP) can easily be assayed for migration, proliferation, viability and accumulation of reporter signal. We also show that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutralizing antibodies against HIV. The flexible assay format allows for the interrogation of thousands of individual antibody secreting cells in parallel, making it well-suited for screening libraries of cells producing candidate lead antibodies based on functions like neutralizing activity.

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

“…4d); in their approach, the slide was removed from the nanowell array for imaging, and the positions of the positive secretion profiles on the slide reported which nanowells contained live cells with intended antibody specificities for subsequent cell recovery and clonal expansion. By coating the capping slide with cells that could be targeted by a pseudotyped HIV virus, Ozkumur et al 113 were able to test not only the antigen specificity of antibodies secreted by single cells in each well but also their ability to neutralize infection. Han and colleagues 114 extended this approach to monitor the kinetic expression of four cytokines over 17 hours from thousands of activated T cells, demonstrating that cytokine release kinetics can predict T cell effector phenotype.…”

Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

“…The wells are fabricated onto a suitable medium; a glass or a polymer, the fabrication strategy such that the wells at micron-scale have bottom side closed while top side opened and furthermore loaded by discontinuous dewetting, this strategy usually achieved with micromaching or lithography [56]. Cells or functionalized microspheres both can be applied to spatially segregate single proteins in micro-well arrays, and the applicability of these arrays include protein screening [57], engineering [58], or analysis of single molecule [59]. An example of platform based on micro-well arrays, in order to release the fluorescein exploited, the hydrolysis of fluorescein and result in engineering of a lipase enzyme [58].…”

Recent Advances in Microfluidic Techniques for Systems Biology

“…The first unique advantage offered by open-surface microfluidic arrays is that cells can be easily and precisely accessed during culturing. Benefiting from this feature, Ozkumur et al presented a high-throughput functional cell-based screening assay via a patterned 2D cell culture array . After coating a microscope slide with a monolayer of target cells, they used a high-density PDMS nanowell array to press the cell monolayer and selectively remove cells for the desired patterns.…”

“…Benefiting from this feature, Ozkumur et al presented a high-throughput functional cell-based screening assay via a patterned 2D cell culture array. 71 After coating a microscope slide with a monolayer of target cells, they used a high-density PDMS nanowell array to press the cell monolayer and selectively remove cells for the desired patterns. This process was termed as nanowell-assisted cell patterning (NWAP).…”