Fig. 1

3D microniche preparation and single hMSC encapsulation. a Schematic of the method to encapsulate single cells in a 3D microniche. b Fluorescence image shows nuclear staining of single cells encapsulated in a 3D microniche with cylindrical geometries at different cell densities (2500 and 10,000 cells cm⁻²), scale bar: 100 µm. c Cell encapsulation efficiency at different cell densities in the 3D microenvironment with cylindrical geometry. d Quantification of cell viability (by live/dead staining) after 1 and 3 days of culture in a 3D microniche with different geometries; n ≥4 regions of interest (ROI) with a total of 80–100 cells analyzed. e Side view and fluorescent heat maps of actin organization in a microenvironment with and without lid, red: F-actin, scale bar: 20 µm. f 3D organization of actin cytoskeleton in a microenvironment with and without lid, red: F-actin, blue: nuclear, scale bar: 20 µm. g Quantification of cell volume after 24-h culture in a microenvironment with and without lid; n = 50–60 cells analyzed for each data point. The microniche volume was controlled by changing the height (from 7 to 30 µm), with a constant value for project area (400 µm²). Data are shown as mean ± s.d. for all panels, and *P < 0.05, **P < 0.01 (ANOVA using a Tukey post-test), compared to theoretical niche volume. Microniches with heights 23, 12, 9, and 7 µm are denoted as V ₁, V ₂, V ₃, and V ₄, respectively