Figure 3.

Rotational culture in a bioreactor accelerates the growth and maturation of suspension HIOs

(A) Schematic representation of suspension spheroid culture system encompassing static suspension and rotating suspension in a bioreactor.

(B) Image showing the rotating bioreactor composed of 10 mL culture vessels on bioreactor. Spheroids float in the culture vessel (inset).

(C) Bright-field images of s-Spheroids cultured in both conditions, static suspension and rotating bioreactor, on day 19. Both showed mesenchymal growth surrounding spheroids (inset). Scale bars, 1,000 μm.

(D) Growth rates of s-Spheroids in two culture conditions (static and rotation suspensions). The black boxes represent generated spheroid sizes on days 1 and 4 (Bindshut). False discovery rate (FDR)-adjusted t test (∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001).

(E and F) Whole-mount immunostaining of CDX2, E-cad, and Hoechst (E) and VIM, E-cad, and Hoechst (F) in s-HIOs cultured in rotational suspension with high-magnification inset at right. Scale bars, 1,000 μm.

(G) Expression analyses of CDX2, SOX2, FOXF1, and VIM in s-HIOs cultured in rotational suspension in each medium condition (day 30) relative to expressions at s-Spheroid formation. Bars are means ± SD for triplicate independent experiments. Student’s t test (∗∗p < 0.001, ∗∗∗p < 0.0001).