Development of a low cell density model for examining astrocyte reaction to 3D suspension using AVIFJ

Alternating viscous and inertial force jetting (AVIFJ) was used to print single-cells and position small numbers of cells precisely. The effect of nozzle size on single-cell encapsulation and droplet spacing ability was tested using astrocytes. Astrocytes were patterned in Matrigel and examined for 7 d after printing to assess viability and their response to being suspended in 3D at low cell density. The results showed AVIFJ had a single-cell printing efficiency around 30% using nozzles with outer diameters (ODs) of 63μm and 96μm at a 300 000 cells ml^-1concentration, and 30% for 143µm and 195µm OD nozzles at a 50 000 cells ml^-1concentration. Spacing using a 63μm nozzle tests printed droplets with a minimum center-to-center spacing of 199.53 ± 2.52μm on a culture well and cell spacing of 122.23 ± 21.69μm in 3D. Astrocyte viability was 93.60 ± .27% when printed into Matrigel. RNA sequence results showed astrocytes suspended Matrigel upregulated IGFBP3 compared to 2D controls. Results also showed that astrocytes suspended in 3D that contact the culture well react differently than those completely suspended over 4 d. Overall, this study showed the viability of AVIFJ printing for low cell number experiments and differences in astrocyte behavior in 3D suspension compared to 2D.