Figure 1.
A. Image of the microfluidic perfusion device, showing the upper pneumatic control layer (green), the two sets of triplicate culture chambers (red and blue). B. Schematic of the perfusion device. Gray and purple outlines represent fluidic and control layer, respectively. C. Microfluidic perfusion systems use flow to fine-tune the relative significance of convection, diffusion, and reaction.
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.
Figure 3.
FGF signaling in perfusion culture.
A. Phase images and close-ups of representative mESC colonies growing under differentiation and self-renewal conditions. Representative images of time-course changes in mESC morphology under two differentiation conditions in perfusion culture (N2B27+FGF4 and N2B27+CM) (left) or under self-renewal conditions in static culture (serum+LIF) (right). B. FGF4 supplementation in perfused mESC differentiation. Growth curves for cells differentiated in N2B27+CM vs. N2B27+FGF4 (5, 20 ng/mL) in perfusion for 5 days. Data shown are average ± s.d. of 2 independent experiments for each FGF4 concentration (* Indicates P<0.05). (C–D) Inhibition of FGF signaling in perfused mESC differentiation cultures. C. Transmission images of cells cultured in perfusion for 6 days in N2B27+CM and N2B27+CM+FGFRi (300 ng/mL) (left). Growth analysis for cells cultured in the presence of FGFRi at 300 ng/mL. Fold increase in cell area after 5 days of perfusion culture for two different conditions, N2B27+CM and N2B27+CM+FGFRi (right). D. Fluorescence images of cells cultured in perfusion for 6 days in N2B27+CM and N2B27+CM+FGFRi (300 ng/mL) (left). Sox1-GFP+ cell frequency assessed by flow cytometry for cells differentiated in N2B27+CM and N2B27+CM+FGFRi condition on Day 6 of perfusion culture (right). Data are average ± s.d. of 3 independent experiments, (*** Indicates P<0.001).
Figure 4.
Gene expression profiling of committed mESCs in perfusion.
Relative expression of genes expressed in mESCs (Klf4, Rex1) and epiblast (Sox17, Dnmt3b, T, Fgf5) for cells in perfusion culture (N2B27+FGF4 at 5 ng/mL), normalized to GAPDH and to self-renewal static culture (N2B27+LIF+BMP4). Data are shown as average ± s.d. from 4 independent experiments, (* Indicates statistical significance, ° P = 0.06, * P<0.05, ** P<0.01, *** P<0.001 for logarithmic distribution). The large variability in FGF5 gene expression is typical for this gene [26].
Figure 5.
Graphical model of signaling in mESC neuroectodermal differentiation.
Removing cell-secreted factors suppresses growth and differentiation, which can be restored by supplementing N2B27 medium with cell-secreted factors. Supplementation with (cell-secreted) FGF4 does not rescue growth of perfused mESC cultures, while inhibition of FGF signaling downregulates differentiation without compromising mESC growth.