Schematic of the EB formation process using bioprinting approach. Droplets of cell-medium suspension were bioprinted onto the lid of a Petri dish and were hung up for 24 h to allow for EB aggregation. The formed EBs were transferred to a 96-well plate for additional culture up to 96 h.
EB formation using bioprinting method. (a)–(c) Images of formed EBs with droplet sizes of 1, 4, 10, and at a cell density of . (a) Uniform-sized droplets encapsulating ESCs were generated by bioprinting. (b) Phase contrast images of EBs formed after hanging for 24 h and (c) after culture for 72 h in a 96 multiwell plate. (d) Fluorescent images of GFP positive EBs at stained with ethidium homodimer. (e) Images of EBs formed with printed droplet size of at at different cell concentrations.
The effect of initial cell density, droplet volume, and culture time on the EB size for bioprinting and control with manual pipetting. EBs retrieved from bioprinted droplets after 24 h culture in vitro (a) displayed more uniform size distribution compared to control method (i.e., pipetting based manual hanging-droplet) (b). Statistical analysis of EB size with different initial cell concentrations (, , and ) formed by the bioprinting method [(c), (e), and (g)] and by the control method [(d), (f), and (h)]. The EB sizes formed by bioprinting were well controlled by varying the droplet size (1, 4, 10, and ) and the culture time (24, 48, 72, and 96 h).
Comparison of the uniformity and the size of the EBs formed by control (manual hanging-drop method) and bioprinted hanging-drop methods. The statistical comparison of the EB size uniformity was performed with Levene’s test for equality of variances at the end of the 96 h culture period for different initial cell seeding densities and droplet sizes. The uniformity of the sizes was assessed based on the variance in the data sets. Less variance in the data indicated higher uniformity in the resulting EB sizes. The diameters of the resulting EBs were compared statistically for control and bioprinting methods with Mann–Whitney U test for pairwise comparisons . (a) For initial cell seeding density, bioprinting method resulted in more uniform EB sizes at the end of the 96 h culture period for 1, 4, and droplet sizes. (b) For initial seeding density, bioprinting resulted in significantly higher uniformity in the EB sizes for all droplet sizes compared to control method. (c) For initial cell seeding density, higher uniformity in EB sizes was observed for the droplet size of . Overall, bioprinting method resulted in statistically greater EB sizes compared to control method at the end of the 96 h culture period in all groups.
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