Characterization of 3D spheroid MDFs through low attachment culture. (A), Phase-contrast images of monolayer MDFs and 3D spheroid MDFs. (B-G), RT-PCR testing for NPC-specific genes including Sox2, Nestin, Brn2, Blbp, Gpm6a and Pax6 in monolayer MDFs and 3D spheroid MDFs (n = 3). (H, I), RT-PCR testing for Collagen 1A1 and α-SMA genes in monolayer MDFs and 3D spheroid MDFs (n = 3). (J-L), Western blotting for Nestin, Collagen 1A1 and α-SMA in monolayer MDFs and 3D spheroid MDFs. Immunostaining (M, N) and the corresponding fluorescence intensity quantification (O, P) (n = 6) of Sox2 and Nestin of monolayer MDFs and 3D spheroid MDFs. Data are mean ± SD. **P < 0.01. M means monolayer MDFs; S means spheroid MDFs.