A novel 3D differentiation method using sodium alginate encapsulation. (a) Permeability/of sodium alginate microspheres and sodium alginate–polylysine–sodium alginate microspheres was detected by 60–76 kDa FITC-dextran (scale, 100 μm). (b) Schematic diagram of the 3D cell differentiation procedure. (c) Calcein/PI apoptosis staining assay for cell viability on Day 3 after Day 0 encapsulation and during the complete process of differentiation (Day 8, Day 14, and Day 18) after Day 3 encapsulation (scale, 100 μm). (d) MTT assay for detecting cell viability during the complete differentiation process following Day 0 encapsulation and Day 3 encapsulation. (e) Microsphere morphology following microsphere encapsulation on Day 0 and Day 3 (scale, 500 μm). Data are the mean ± SD (n = 3) and analyzed by Student’s t test. ** p < 0.01, **** p < 0.0001.