Figure 5.

rDM outperforms cDM in both 3%FBS and DMAD during long-term (28-day) 3D culture

(A) Illustration of the experimental design. Bovine SVC were encapsulated with alginate hydrogel and differentiated with 3%FBS or DMAD. cDM contained all 4 inducers during first media change followed by progression for the whole period of culture, rDM contained rosiglitazone and insulin for 28 days. DMAD contained HC/PR.

(B) Representative images of maximum intensity projection confocal microscopy. Differentiation with control (cDM) or reduced (rDM) differentiation medium in 3%FBS or DMAD at day 28. Blue, Hoechst; and yellow, Nile Red.

(C) PLIN1 immunohistochemistry of 3D hydrogel/cell constructs differentiated in cDM or rDM medium at day 28. Blue, Hoechst; red, PLIN1; and yellow, Nile red. (B and C) Scale bar, 100 μm.

(D) Quantitative analysis of glycerol release at day 0, 7 and 28. Data is representative of 3 independent experiments using 4 donors.

(E) Qualitative and quantitative assessment of the amount of fatty acids within triglycerides (normalized to the amount of DNA). Results are representative of 5 independent experiments using 6 donors. (D and E) Statistical differences were analyzed with a one-way ANOVA. Data are represented as mean ± SD; NS, not significant; ∗∗∗p < 0.001. See also Figure S5 and S6.