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Generation of co-cultured spheroids consisting of osteogenic HWJSC spheroids seeded with epithelial cells.

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posted on 12.09.2017 by David G. Belair, Cynthia J. Wolf, Carmen Wood, Hongzu Ren, Rachel Grindstaff, William Padgett, Adam Swank, Denise MacMillan, Anna Fisher, Witold Winnik, Barbara D. Abbott

A: Schematic of HWJSC staining with CellTracker Orange, spheroid generation and osteogenic differentiation (7 d), washing in OM, and coating with CellTracker Green-stained HPEKp epithelial cells. B: Representative fluorescent maximum intensity projections (MaxIP) of HPEKp seeded on HWJSC spheroids after one day of incubation at epithelial/mesenchymal ratios (E/M) of 0.1 (i), 0.2 (ii), 0.4 (iii), 0.8 (iv), 1.2 (v), and 1.4 (vi). C: Quantified HPEKp/HWJSC co-localization after one day of incubation at E/M ratios from 0.1–1.4, presented as mean ± 95% confidence interval of fractional epithelial coverage represented by 7 confocal z-stacks per condition and a total of 6 independent experiments. Asterisks represent statistical significance with one-way ANOVA and Tukey’s post-hoc test of each condition relative to E/M = 0.1, 0.2, and 0.4. All conditions were statistically higher than E/M = 0.1 and 0.2. Mean epithelial coverage was statistically indistinguishable between E/M = 0.8, 1.2, and 1.4, and E/M = 1.4 was indistinguishable from E/M = 0.4. D: Quantified HPEKp/HWJSC co-localization versus mean HWJSC spheroid diameter, presented as mean fractional epithelial coverage represented by at least one confocal z-stack per data point. E: Quantified HPEKp/HWJSC co-localization versus mean HPEKp seeding density in cells/cm2 of HWJSC spheroid surface area, calculated from mean HWJSC spheroid diameter in each image, presented as mean fractional epithelial coverage represented by at least one confocal z-stack per data point. F: Regression analysis, presented with a Pearson correlation coefficient and p-value for each variable, of 6 independent variables versus epithelial coverage: HWJSC spheroid diameter, HPEKp passage #, HWJSC passage #, HPEKp/HWJSC seeding ratio, HPEKp seeding density (cells/cm2), and HPEKp seeding density (cells/cm3).

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