Figure 1.
Microbioreactor array design and validation.
A Microbioreactor array photomask design, with key features marked. B Perspective schematic view showing assembly of glass substrate and PDMS structural layers 1 and 2. Via holes join microchannel structures between layers. C Computational fluid dynamic model of velocity field through centreline planes at nominal operating conditions. Chambers are 1.63 mm in diameter and 250 µm high. D Design normalised concentrations of factors in each column, corresponding to panels E and F. Stock factor and buffer solutions are provided at normalised concentrations of 3 and 0, respectively, to allow for subsequent dilution. E Photograph of microbioreactor array filled with red, yellow and blue food dyes (representing factors A1, B1 & C1, respectively), and mixed with PBS (buffers A0, B0, & C0). F Fluorimetric quantification of soluble factor levels in each column. Stock solution of 40 kDa FITC-dextran was provided at 100 µM, therefore the design concentration levels are 0, 16.7 and 33.3 µM. Bars represent mean ± SD of 2 independently fabricated devices. G Heatmap of number of nuclei in each chamber, with individual numbers marked. Cell numbers do not represent densities used in microbioreactor experiments. H Phase contrast images of hESCs imaged 0 and 2 h after seeding into microbioreactor arrays. Chambers are ∼1.63 mm in diameter.
Figure 2.
Microbioreactor screening of HES3(MIXL1GFP/w) cells undergoing mesendodermal differentiation demonstrates paracrine dependencies.
A Screening panel showing array conditions in each column. Numbers indicate concentrations of BIO (µM), BMP-4 (ng/mL) and Activin A (ng/mL). B Confocal images of HES3(MIXL1GFP/w) hESCs expressing GFP and counterstained with Hoechst at experiment endpoint. Medium flow direction was from top to bottom. C Heat maps of total fluorescence intensities in the array (arbitrary units). D Higher-magnification confocal images of individual wells within the microbioreactor array highlighting position-dependent phenotypes, scale bar: 500 µm. E Main effect magnitudes of exogenous factors (BIO, BMP-4, Activin A) and positional dependency (Position) on expression index IMIXL1-GFP. Units represent global standard deviations relative to global mean. F Interaction effect magnitudes of combinations of 2 factors on expression index IMIXL1-GFP. Full dataset is shown in Figures S2, S3. G Traces of expression index IMIXL1-GFP and DNA-normalised expression index IMIXL1-GFP/DNA versus position coordinate for 0 (Column 6) and 1 µM BIO (Column 15) showing extended expression under BIO treatment.
Figure 3.
Confirmation and screening of paracrine factors involved in hESC mesendodermal differentiation. A
Screening panel showing array conditions, corresponding to B-C. Numbers indicate concentrations of induction-conditioned and factor re-supplemented medium (CM, final % v/v), IWP-4 (µM), and CHIR99021 (CHIR, µM). B Confocal images of HES3(MIXL1GFP/w) hESCs expressing GFP and co-immunostained in situ for NCAM (CD56) at experiment endpoint. Flow direction was from top to bottom. C Heat maps of total fluorescence intensities in the array (arbitrary units). D Selected interaction effect plot showing average effect magnitudes for combinations of CM and Position on expression index IMIXL1-GFP and DNA-normalised expression index IMIXL1-GFP/DNA, highlighting the shift towards earlier rows with CM treatment (legend represents CM concentration in % v/v). Full dataset, Figures S6, S7. E Traces of expression index IMIXL1-GFP and DNA-normalised expression index IMIXL1-GFP/DNA versus position coordinate for 0 (Column 19), 2.5 (Column 20), and 5 µM CHIR (Column 21) showing extended expression under CHIR treatment. F Blocking of MIXL1-GFP induction in static cultures with IWP-4, and improvement and rescue of induction with CHIR. Bars represent mean ± SD of 4 biological replicates over 2 independent experiments. * indicates p<0.05 relative to +None condition.
Figure 4.
Interrogation of FGF stimulation and MEK inhibition on hESC mesendodermal differentiation in HES3(MIXL1GFP/w) hESCs.
A Screening panel showing array conditions. Numbers indicate concentrations of FGF-2 (ng/mL), PD0325901 (PD; µM), and CHIR99021 (CHIR; µM). B Confocal tile scan image of HES3(MIXL1GFP/w) hESCs expressing GFP and and co-immunostained in situ for NCAM (CD56) and Hoechst at 2.5 d experiment endpoint. Flow direction was from top to bottom. C Heatmaps of total fluorescence intensities in the array (arbitrary units). D Higher-magnification images of cells in highest MIXL1-expressing conditions – Column 21, Row 7– demonstrating efficient induction of MIXL1-GFP and NCAM. Scale bar: 500 µm. E Main effect magnitudes of factors on expression index IMIXL1-GFP. Units represent global standard deviations relative to global mean. F Selected interaction effect plots showing average effect magnitudes for combinations of FGF-2 and Position on expression index IMIXL1-GFP and DNA-normalised expression index IMIXL1-GFP/DNA, highlighting the shift towards earlier rows with FGF-2 treatment (legend represents FGF-2 concentration in ng/mL). G Chemical inhibition of MIXL1-GFP induction with MEK cascade inhibitor PD0325901 and FGF receptor tyrosine kinase inhibitor PD173074. Bars represent mean ± s.d., n = 2−3 from one representative experiment. H-J Addition of FGF-2 or FGF-2+ CHIR99021 improved induction efficiency (H) and median fluorescence intensity (I) in static controls, but without significantly increasing total cell count (J). Bars represent mean ± s.d., n = 4 from 2 independent experiments. * p<0.05 compared to +None condition, one-way ANOVA.