Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells
Figure 2
Monoculture neuroectodermal differentiation and comparison of differentiation in static and perfusion systems.
(A–C) Schematic of culture conditions and images of 46C mESCs in different culture conditions taken 24 hours after seeding (left), and 6 days after attachment (middle and right) for (A) static differentiating cultures in N2B27 medium, (B) on-chip perfused culture in N2B27 medium, and (C) on-chip perfused culture in N2B27 medium with conditioned medium (CM). D. Fold increase in cell area after 5 days of perfusion culture for two different conditions, N2B27 and N2B27+CM. Data are average ± s.d. of 3 independent experiments. E. Analysis of Sox1 protein level - frequency of Sox1-GFP+ cells after 7 days of differentiation for N2B27+CM (perfusion culture) and N2B27 condition (static culture), assessed via flow cytometry. Data are average ± s.d. of 3 independent experiments. F. Analysis of gene expression for N2B27+CM condition in perfusion culture - relative Sox1 gene expression for N2B27+CM (perfusion culture) on Day 7 of differentiation normalized to GAPDH and gene expression level of Sox1 for N2B27 condition (static culture). Data are average ± s.d. of 2 independent experiments. 46C mESCs in self-renewal condition (N2B27+LIF+BMP4) were used as a negative control for both flow cytometry and qRT-PCR analysis, (* indicates P<0.05; *** indicates P<0.001). G. Representative phase images of mESCs colonies undergoing differentiation for three days in static and perfused cultures.