3D Spheroid Culture Enhances the Expression of Antifibrotic Factors in Human Adipose-Derived MSCs and Improves Their Therapeutic Effects on Hepatic Fibrosis

Figure 2

Protection of 3D spheroid-derived MSCs on hepatocyte injury in vitro. Hepatocytes were isolated from C57BL/6 mice and cultured in collagen-coated 6-well plates. After treatment with CCl₄ for 6 h, the medium of cells was replaced with normal medium, conditioned medium of 2D cultured MSCs, or conditioned medium of 3D spheroids of MSCs for 24 h before LDH assays, apoptosis analysis, and RT-PCR. (a) LDH assays. (b) Statistics of Annexin V staining. (c) Images of Annexin V staining. Annexin V staining was performed to evaluate cell apoptosis (red), with DPAI for nucleus staining (blue). (d) RT-PCR. The mRNA levels of Bax, Bcl-1, NFκB, and TGF-β were tested by RT-PCR, with GAPDH as the internal control. (e) Quantitative analysis of Bax/Bcl mRNA level. (f) Quantitative analysis of NFκB mRNA level. Band intensities of NFκB were normalized to those of the internal control. (g) Quantitative analysis of TGF-β mRNA level. Band intensities of TGF-β were normalized to those of the internal control. compared with normal; compared with CCl₄; compared with 2D MSC medium.

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