Figure 2
From: Efficient scalable production of therapeutic microvesicles derived from human mesenchymal stem cells
Large-scale formation of hMSC-spheroids with precisely controlled size and cell number. (a) Fabrication of a PEG hydrogel microwell array with inverted-pyramidal openings adjoined to cylindrical microwells. (b) Seeded hMSCs, at a density of 5 × 105 cells/array, were evenly entrapped within microwells 20 min after seeding. (c) 12 hours after cell seeding, hMSCs entrapped within microwells were well agglomerated in the shape of a spheroid with a controlled size of approximately 150 μm. The size bar indicates 200 μm. The microwell arrays were inserted in the commercial six-well plates and cultured for 7 days in a CO2 incubator under a 30-rpm orbital-shaking condition. (d) A live (green) and dead (red) assay of the 3D w/shaking group on D5 revealed that most cells in the 3D hMSC-aggregates were highly viable. The size bar indicates 400 μm. (e and f) Histological images after H&E (e) and M&T (f) staining showed that hMSC-spheroids of the 3D w/shaking group on D5 were compactly integrated with cells and secreted ECM, respectively. The size bar indicates 50 μm. (g) Cell growth kinetics was examined by a DNA quantification method. The cell numbers in the 3D w/shaking group were not increased during the culture period from the initial seeding density. Data are presented as the mean ± SEM. Differences among culture days in each group were evaluated by one-way ANOVA at a level of significance of p < 0.05 (*).
