Extended Data Fig. 5: Cell viability study and immunofluorescence staining of neural microtissues before and after treatment of the droplet device.

a, Cell viability in neuron-containing droplets, which had been connected to droplet devices for 10 min and then cultured in medium for 4, 12, 24, and 48 h. The control group was neural microtissues embedded in agarose droplets and not placed in contact with a droplet device. Salt concentrations in the high-salt droplets (CaCl₂) were: 0.5 M (50-fold gradient, light blue); 1 M (100-fold gradient, dark blue). Data are presented as mean ± s.d. (n = 5). b, Fluorescence (bottom) and overlaid bright-field (top) live/dead imaging of microtissues embedded in agarose droplets. Calcein-AM (live, green) and PI (dead, red) staining were conducted after droplet device (0.5 M) modulation. Scale bars, 600 μm. c and d, RFP-labelled microtissues were stained for the neuronal cell marker TUJ1 and the apoptosis marker caspase 3. RFP is expressed by live cells; the TUJ1 staining reveals the neuronal cells including their processes; the caspase 3 staining reveals apoptotic cells. The control group (c, Day 10) was not contacted with a droplet device. The experimental group had been connected with the droplet device for 10 min and then cultured in medium for 48 h (d, Day 12). Salt concentrations in the high-salt droplets (CaCl₂) were 0.5 M (50-fold gradient). e, The number of apoptotic cells in an area of 100 × 100 μm². Data are presented as mean ± s.d. (n = 6).