Fig 1.
The methodology for PEGDA-based micropatterning on methacrylated glass-bottomed petri dish by photo crosslinking.
(a) Schematic illustration of the fabrication process (i) Glass surface methacrylation; (ii) PEGDA pre-hydrogel solution was added onto the glass surface, and then exposed to UV light (wavelength = 405 μm) with the virtual photomask embedded; (iii) Micropatterned PEGDA gel was fabricated on the glass surface. Uncrosslinked pre-hydrogel solution was removed by multiple PBS washes. (b) Schematic illustration of hESC seeding and culture (i) Laminin coating was applied on the micropatterned surface. Unattached laminin was washed away after 2.5-hr-incubation at 37°C; (ii) hESC suspension was added at desired density on the laminin coated surface. Unattached cells were washed away after 45-min-incubation at 37°C. Following seeding, media with desired reagents was added to the dish for further culture or induction.
Fig 2.
hESC culture and induction on micropatterned PEGDA glass-bottomed dish.
(a) Photographs of a micropatterned glass-bottomed dish with PBS. (b) hESCs attached to micropatterned PEGDA coated glass surface: (i) bright field image of an octagon pattern (ii) hESCs attached well 45 min post cell seeding. Scale bar = 100 μm. (c) hESCs attached to “RICE” pattern were cultured for 3 days. Scale bar = 100 μm. (d) Immunostained hESC patterns for SOX2 and AP2 26 h post BMP treatment at 50 ng/mL. Scale bar = 100 μm. hESCs self-organized to form an outer ring of extra-embryonic cells (ISL1+), and the inner pluripotent cells (SOX2+).
Fig 3.
Characterization of micropatterned PEGDA gel thickness and cell pattern size.
(a) Measurement of thickness of gel bed using different UV exposure time in fabrication. Unit: μm. The linear polynomial curve fitted to the data points has the equation of val(x) = p1*x + p2, where coefficients (with 95% confidence bounds) are p1 = 26.54 (21.93, 31.14), p2 = 0.317 (-33.53, 34.16). (b) Measurement of the sizes of circular cell patterns. All gels used were exposed to UV for 6 s. Major axis length of each cell colony was acquired, which represents the measured size of the cell pattern. Unit: μm. The linear polynomial curve fitted to the data has the same equation of val(x) = p1*x + p2, but with coefficients (with 95% confidence bounds) p1 = 1.042 (0.9707, 1.114) and p2 = -110.5 (-167, -54.1).
Fig 4.
Cell viability and proliferation assessments after 24 hours.
(a) Confocal images of live/dead staining of hESCs on LN521-coated micropatterned (top) or glass (bottom, control) surfaces with calcein-AM (live) and propidium iodide (dead) after 24-hour-culture. Scale bar = 100 μm. Colony size: Diameter of 650 μm. (b) Confocal images of hESCs immunostained for Ki67 on micropatterned (top) or bare glass (bottom, control) surfaces after 24-hour-culture. Scale bar = 100 μm. Colony size: Diameter of 650 μm. (c) The quantities of live and dead cells were quantified from the calcein-AM/PI staining images. Cell viability is calculated as a percentage of living cell (# of living cells/(# of living cells + # of dead cells). The results were reported as the mean ± standard deviation. P<0.05. (d) Cell proliferation assessed by Ki67 assay was quantified and analyzed from Ki67 immunostaining images, and plotted as histograms (micropatterns in (i), and control in (ii)). The red line in each plot indicated the threshold used to calculate the Ki67 positive rate.
Fig 5.
Reproduction of gastrulation and neural ectodermal induction results with the PEGDA-based micropatterning method.
(a) Confocal images of cell colonies immunostained for SOX2, BRA, and ISL1 at 48 h post BMP treatment in different conditions: Control and NODAL −/− cells were treated with 50 ng/ml BMP4. BMP, Bone Morphogenic Protein. Scale bar = 100 μm. Colony size: Diameter of 650 μm. (b) SOX2, BRA, and ISL1 levels of the wild type group (top) and Nodal -/- group (bottom) were quantified as a function of distance from the colony center (n = 3). (c) Confocal images of cell colony immunostained for PAX6, SOX9, and AP2. Patterns were initially treated for 3 days in N2B27 media with 10 ng/mL SB and then subsequently induced for 1 day in N2B27 media with SB, BMP4 and IWP2. Scale bar = 100 μm. Colony size: Diameter of 650 μm. (d) PAX6, SOX9, and AP2 levels of the ectodermally induced group were quantified as a function of distance from colony center (n = 3).