Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

Figure 1 Cell viability and growth of embryonic stem cells encapsulated in self-assembling three-dimensional scaffolds. Enhanced yellow fluorescent protein-labeled ESCs were encapsulated (2 × 10⁶ cells/mL) in fixed and floating scaffolds prepared using Dex-SH with 7.5% thiol substitution and PEG-4-Acr. Encapsulated cells were cultured in ESC medium and periodically cell growth was visualized using fluorescent microscopy. A and B: Live (green) and dead (yellow) cells in fixed scaffolds stained with PI at day 1 and week 3, respectively. While the viable cells increased with time, the number of dead cells remained constant. Scale bars = 100 μm; C and D: Three-dimensional (3-D) confocal images of floating scaffolds representing ESC growth at day 3 and week 3, respectively; E: Growth of ESCs encapsulated in 3-D scaffolds was assayed by direct count using hemocytometer. Cells seeded in 3-D scaffolds (at 2 × 10⁶ cells/mL) were incubated in ESC medium and counted at various time intervals. Data presented as cell number (x 10⁶ cells/mL) ± SE (n = 3) and plotted against the days of incubation; F: Quantitative determination of ESC proliferation in floating scaffolds at week 0, 1, 2, 3, and 4 of culture after staining with PB as analyzed by microplate reader. The results were expressed as the normalized absorbance ± SE (n = 3), with a significant increase in cell number, compared to initial ESC viability at time 0. ^aP < 0.05 and ^bP < 0.01 using one-way ANOVA with the non-equal variance hypothesis and Games-Howell multiple comparisons test. ESCs: Embryonic stem cells; Dex-SH: Thiol functionalized dextran; PEG-4-Acr: Polyethylene glycol tetra-acrylate.