Figure_2.tif (8.43 MB)

Effect of alginate microencapsulation on the expansion of hESC as aggregates.

Download (0 kB)
figure
posted on 05.08.2011 by Margarida Serra, Cláudia Correia, Rita Malpique, Catarina Brito, Janne Jensen, Petter Bjorquist, Manuel J. T. Carrondo, Paula M. Alves

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.

History

Licence

Exports