Preparation, appearance and gene expressions of aggregates of human BM MSCs after hanging drop culture.

(A) Schema for the study. 2.5 × 105 human BM MSCs were cultured for 3 days in hanging drops. (B) Drops hanging on the cover of a 15 cm dish. (C) Macroscopic images of aggregates consisting of 2.5×105 MSCs at 10 minutes-72 hours. (D) Histological sections stained with Toluidine Blue at 72 hours. (E) TEM images of aggregates at 72 hours. (F) Histological sections immunostained with lubricin at 72 hours. (G) Real time PCR analysis for MSCs 72 hours after monolayer and pellet culture. RNA was prepared from 500,000 MSCs or 2 aggregates from 5 donors respectively. Gene expression levels in MSCs after monolayer culture were normalized as 1 (n = 5, *p<0.05 by the Wilcoxon’s signed rank test).