Abstract: Microwell assays involve the cultivation of numerous cells in microwells to measure a collective response to stimuli. However, this approach often fails to detect unique behaviors exhibited by subpopulations due to the heterogeneous nature of individual cell responses. To elucidate single-cell heterogeneity, nanowell assays that confine cells in nanoliter chambers have been developed. Currently, these nanowells are fabricated using a single material, such as Polydimethylsiloxane or cyclic olefin copolymer, which results in plastic-like bottoms that limit the quality of microscopy imaging. Additionally, the use of a common material between well walls and bottom prohibits the use of an adhesive surface for cell growth as this would allow cells to crawl out of the nanowells. Moreover, fluorescent molecules can adsorb to the plastic-like material surfaces, which adversely affects fluorescence assays and further limits imaging quality.
Here, we developed a new material, named NanoBlack, which facilitates photolithographic microfabrication of microstructures directly onto a glass substrate. Using this material, we successfully created open nanowell arrays with a glass bottom and thin non-adhesive walls. These nanowells can be produced with dimensions as small as 25 x 25 µm and enable grids of nanowells to be housed in each standard microwell of a 384-well plate, resulting in up to 3 million nanowells on a single plate. The dimensions of the nanowells can also be made much larger to accommodate imaging of multi-cellular models such as spheroids or organoids. These nanowells provide a glass-bottom surface, exhibit no autofluorescence, and quench adsorbed fluorescent molecules to enable long-term, high-resolution image-based assays. Furthermore, the high contrast between the walls and the well bottoms helps simplify the downstream image processing. These features make our technology an ideal candidate for a broad range of applications due to its compatibility with different cellular models and delivery in a standard well plate format.
We demonstrate the use of these nanowells in numerous ongoing single cell experiments. First, we image cells, spheroids, and organoids in our nanowells to demonstrate the improved imaging quality and universal compatibility. Second, we have assessed the cytolytic activity of CAR-T cells by depositing single CAR-T cells with multiple target cells in nanowells. We have found a small subpopulation of CAR-T cells behave like serial killers while the vast majority of CAR-T cells exhibit limited activity. Third, we measured the motility of single cancer cells to measure their response to cytokine stimulants and anti-cancer drugs. Finally, our ongoing single-cell drug screening assay aims to quantify the efficacy of pharmaceutical drugs targeting a breast cancer cell line, at the single-cell level, and further investigate their methods of action.