Extended Data Fig. 1: Cell loading into nanovials, enrichment of single cell-loaded nanovials by FACS, and VEGF-A nanovial secretion assay validation.

a, Anchorage of single cells on nanovials coated with gelatin via integrin binding. Cell loading into nanovials is achieved by simple mixing. b, Flow cytometry histogram of cell-loaded nanovials stained with calcein AM viability dye (0.4 cell per nanovial loading shown here). Cells are sorted via FACS (SONY SH800S) based on calcein signal into ‘Multiple Cells’ and ‘Single-cell’ gates. The distribution of the calcein signal has one peak with a tail at higher intensities, which represents nanovials containing more than one cell. n = 14,934 single cells events and 7,586 multiple cell events. c, We tested three cell loading concentrations (0.4, 0.7, and 1 cell per nanovial) and analysed the fraction of nanovials carrying zero, single or multiple cells using the gates described in (b). The graph quantifies cell loading into nanovials for these conditions. When loading cells at the 1:1 cell-to-nanovial ratio, we achieved 23% single-cell loaded nanovials which could be separated by sorting for downstream approaches and analyses. d, Fluorescence microscopy images of nanovials sorted for the indicated gates as described in (b). By sorting nanovials in the ‘Single-cell’ gate, we enriched for nanovials carrying single cells as confirmed by Hoechst nuclei staining, whereas nanovials in the tail (‘Multiple Cells Gate’) represented mostly two or more loaded cells. Following sorting, we estimated that 95% of the ‘Single-cell’ gate sorted nanovials contained one cell based on image analysis. e, To isolate single cells on single nanovials, the following gating strategy was used for cell-loaded nanovial samples: i) All nanovials were gated based on FSC/SSC (n = 93,263), followed by ii) a single nanovial gate based on FSC-Width (n = 80,253), and iii) the ‘Single-cell’ gate based on calcein signal intensity (n = 9,527). iv) Flow cytometry fluorescence histogram of the fluorescent (AF647) anti-VEGF-A detection signal in single cell-loaded nanovials and empty nanovials isolated from the same nanovial cell-loading experiment after 12 hours of secretion incubation. Single cell-loaded nanovials have higher fluorescent (AF647) anti-VEGF-A signal than empty nanovials, showing low crosstalk. f, Stability of recombinant VEGF-A on nanovials over 24 hours. There is a 23% decrease in AF647 Anti-VEGF-A signal from 0 to 12 hours, and a 10% decrease in signal from 12 to 24 hours. g, Level of autofluorescence and VEGF-A detection antibody signal for cell-loaded nanovials without VEGF-A capture/detection antibodies and cell-loaded nanovials without the VEGF-A capture antibodies, respectively. h, Image of one well in the ELISpot assay measuring VEGF-A secretion from MSCs. An average of 99% of cells seeded formed spots across 3 wells. The range of integrated intensity of the spots across 3 wells, quantified in the histogram on the right, indicates that there is heterogeneity in secretion level. Schematics in (a) and (b) created with BioRender.com.