Effect of Conditioned Medium From 3d Spheroids of Human Umbilical Cord Mesenchymal Stem Cells on Endothelial Cell Migration in an in Vitro Wound-Healing Model

Three-dimensional (3D) spheroid culture may modulate the paracrine activity of mesenchymal stem cells. This study compared the effects of conditioned media from two-dimensional (2D) cultures and 3D spheroids of human umbilical cord mesenchymal stem cells (hUC-MSCs) on endothelial wound closure in vitro. Passage-3 hUC-MSCs were cultured as adherent monolayers or assembled into spheroids using MicroTissues® agarose micromolds. Spheroid morphology and diameter were assessed at 24, 48, and 72 h. Immunofluorescence analysis included CD73, CD90, CD105, and CD45 in 2D cultures and CD90 and CD105 in intact 3D spheroids. Human umbilical vein endothelial cells (HUVECs) were exposed to 2D-conditioned medium (2D-CM), 3D-conditioned medium (3D-CM), or endothelial growth medium (control). Scratch-assay images were acquired at 0 and 24 h, and wound closure was calculated relative to the initial cell-free area. Spheroids formed in all 81 assessed microwells. Mean diameter increased from 310 ± 20 µm at 24 h to 420 ± 25 µm at 48 h and 500 ± 30 µm at 72 h (mean ± SD). CD90 and CD105 remained detectable following spheroid formation. At 24 h, wound closure estimated from individually selected images (not from independent experiments) was approximately 100%, 66.0%, and 72.7% for 3D-CM, 2D-CM, and control, respectively. These preliminary descriptive findings suggest greater endothelial wound closure with 3D-CM; confirmation requires matched-field analysis across independent experiments with statistical analysis.