Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor

Abstract

To cite this version:Sasitorntime of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3x10 5 cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Slow Turning Lateral Vessel Bioreactor Improves Embryoid Body Formation and Cardiogenic Differentiation of Mouse Embryonic Stem Cells

“…1). We have previously determined that 3 · 10 5 (STLV1) or 5 · 10 5 (STLV2) cells/mL initial seeding density and 10 rpm gives the optimal EB number and yield in STLV conditions (Rungarunlert et al, 2011). By D2, cultures formed spherical EBs with a regular shape and uniform size.…”

“…Therefore, the beating was graded and evaluated ( Fig. S1E; Rungarunlert et al, 2011). EBs plated on D2 and D3 showed the fastest beating over their entire surface area (grade 4).…”

“…Forty-eight EBs derived from each group and each replicate were observed, and the number of beating EBs was presented as the rate of the total plated EBs. The cardiac beatings were further evaluated by grading of cardiac beating (beating rate and the area of cardiac beating) as described earlier (Rungarunlert et al, 2011). The results are presented as the mean -SEM of three independent experiments.…”

“…After investigating the efficiency of cardiac beating the EBs were stained for cardiac troponin-T (cTnT) to measure the area of cTnT as described earlier (Rungarunlert et al, 2011). The selected EB samples were analyzed by a ''doubleblind'' approach to avoid biases (eight EBs per one replication), and the experiments were completed in triplicates.…”

“…To date, the influence of the dynamic culture parameters (i.e., time of EB adherence to gelatin-coated dishes, cell seeding density, etc.) has been reported to affect cardiac differentiation from ESCs (Rungarunlert et al, 2011). However, there is inadequate information concerning the histological characteristics of EBs, which could vary due to differences in cell seeding density and day of culture formed when using the STLV.…”