Effect of alginate microencapsulation on the expansion of hESC as aggregates.
hESC aggregates were encapsulated at day 2 and cultured in spinner vessels. (A) Phase contrast and fluorescence images of encapsulated and non-encapsulated cultures at days 3, 7 and 9. Viability of hESC aggregates assessed by staining with fluoresceine diacetate (FDA-live cells, green) and propidium iodide (PI- dead cells, red). Scale bar: 100 µm. (B–C) Cell growth performance of both encapsulated (purple) and non-encapsulated (grey) cultures. (B) Metabolic activity measured by alamarBlue test on the day after microencapsulation (day 3) and at day 15. Error bars denote SD of 3 measurements. ** indicates significant difference (P<0.05) in metabolic activity by one-way ANOVA analysis. (C) Cumulative values of specific rates of LDH release overtime. Error bars denote SD of 3 measurements. (D) Aggregate size of encapsulated cultures at days 2, 4, 7 and 15 of culture. Error bars denote SD of 10 measurements. (E–I) Characterization of encapsulated hESC aggregates expanded in spinner vessels. (E) Percentage of SSEA-4, TRA-1-60 and SSEA-1 positive cells at days 7 (purple bars), 14 (pink stripes bars) and 21 (grey stripes bars). Error bars represent SD of 2 measurements. (F) Flow cytometry analysis of SSEA-4 and TRA-1-60 positive cells at day 7 of culture. (G) Confocal images of aggregates labeled for Oct-4 and TRA-1-60 at day 16 of 3D culture. Scale bar: 50 µm. (F–G) Flow cytometry analysis of the expanded population (H) Immunofluorescence images of Oct-4 and TRA-1-60 labeling and phase contrast pictures of alkaline phosphatase (AP) activity, staining after expansion (2D culture). Nuclei were labeled with DAPI (blue). Scale bars: immunofluorescence images - 200 µm, AP image −1 mm. (I) In vitro pluripotency analysis. Microcapsules were dissolved and hESCs were transferred to a monolayer of inactivated hFF. At confluence, colonies were dissociated and hESCs were able to form embryoid bodies (EBs) in non-adherent conditions and differentiated into cells from all three germ layers. Fluorescence images of differentiated cultures labeled for α–SMA (α smooth muscle actin, mesoderm), FOX-A2 (Forkheadbox A2, endoderm) and βIII-Tub (β tubulin type III, ectoderm). Nuclei were stained with DAPI (blue). Scale bar: 100 µm.