Student Poster Finalist: Laser micropatterned monolithic nanowell-in-microwell arrays

A common need in biology is to study single or small numbers of cells isolated in small volumes where it is possible to track cell behaviour over time (hours and days). Examples include secretion, proliferation, differentiation, and cell-cell interactions. A common approach is to confine individual cells in arrays of nanoliter wells (nanowells) fabricated using polydimethylsiloxane (PDMS). However, this approach cannot be easily integrated in standard microwell plates in order to take advantage of high-throughput infrastructure for automated and multiplexed analysis. Furthermore, it is often desirable to provide cells with a glass bottom surface for high-quality imaging.

Here, we used laser micropatterning to fabricate monolithic hydrogel nanowells inside a microwell plate (microwells) using polyethylene glycol diacrylate (PEGDA). The PEGDA prepolymer is sprayed onto a glass slide to coat a uniform thin-layer. The prepolymer is then laser patterned under an automated microscope to fit into any sized microwell. A standard 384-well square microwell can hold an average of 2,100 nanowells with sizes of down to 50 x 50 µm with heights of 30-70 µm. The slides are then attached to a bottom-less imaging plate by adhesive. These patterned hydrogel nanowells show stable adhesion to the glass slide, while minimizing swelling or protein absorption. This approach produces monolithic thin nanowells that can retain both cells and beads during reagent exchange, enabling simultaneous profiling of single cell secretion and phenotyping via immunostaining, without hindering viability or other biological processes.

We demonstrated the application of monolithic PEGDA nanowells to profile three distinct phenotypes. First, we surveyed single cell protein secretion by seeding cells and protein capture beads, which are constrained to the individual well with limited cross-contamination. We screened thousands of individual cells to reveal the heterogeneity of protein secretion. We then studied the heterogeneity of response in a model of CAR-T cell therapy by monitoring tumor cell killing by cytolysis. Finally, we examined cell proliferation in nanowells and established that cultured cells could be continuously to monitored for cell function, state and phenotype changes. Together, these capabilities suggest that micropatterning of PEGDA nanowells inside microwells could greatly expand the potential applications of existing high throughput microwell plate screening platforms.