BFC functionality. (a) Large-scale patterning of mESCs, stained with a vital dye on day 1 after seeding. Scale bar, 500 μm. (b) Histogram showing the distribution of cell-cell distances, with mean spacing of 195 ± 18 μm. Inset: high precision patterning, seeding cells within 1 cell diameter of their target. Inset scale bar, 100 μm. (c) Cell patterning of mESCs onto a TCPS dish with patterned fluorescent gelatin (top right), showing the compatibility of these two approaches. Scale bar, 50 μm. (d) Cell patterning of half the cells onto a TCPS dish (left, in-focus) and half the cells on a 100 μm step of polydimethylsiloxane (PDMS) (right, out-of-focus). Scale bar, 50 μm. (e) Cell patterning of mESCs onto a layer of murine embryonic fibroblasts. Scale bar, 100 μm. (f) Fluorescent picture of (e), showing only the mESCs. Scale bar, 100 μm.

Modulating the cell seeding density. (a) By varying the microwell-to-microwell spacing across the chip, we can pattern cells at multiple seeding densities on the same 60×15 mm TCPS dish. Cell patterning with cell-cell spacing of: (b) 200 μm, (c) 400 μm, and (d) 800 μm. Scale bar, 200 μm.

BFC device and operation. (a) BFC packaging, consisting of a BFC, spacer gasket, and cell-culture dish, all held together using binder clips. Scale bar, 15 mm. (b) SEM image of the BFC microwells, spaced 200 μm apart. Scale bar, 1 mm. (c) SEM image of a single microwell, with diameter and height of 30 μm. Scale bar, 15 μm. (d) The cells are pipetted onto the surface of the chip, allowing cells to fall into the microwells. (e) The cells are trapped in the microwells and the other cells are rinsed away. (f) The BFC is flipped upside down onto the dish with a spacer gasket. (g) The cells then fall out of the microwells onto the substrate, where they attach after a few hours. (h) The cells now have room to grow and move.

Multi-day cell tracking. Proliferation and migration of mESCs on (a) day 0, (b) day 1, and (c) day 2. Scale bar, 200 μm.

Precise control of the stem cell microenvironment. (a) By making the well dimensions large enough to trap multiple cells, we can vary the number of cells patterned at each spot. (b) Using a BFC with microwell-to-microwell spacing of 0.75 mm, well diameter of 40 μm, and well height of 50 μm, we patterned clusters of cells with 1-4 cells at each spot. Collage is from images taken randomly from different areas on the substrate. Scale bar, 40 μm. (c) Patterning the cells as clusters allows them to experience cell-cell contact. (d) Using multiple single-cell sized wells, we can pattern small groups of cells without any cell-cell contact. (e) Using a BFC with microwell-to-microwell spacing of 0.75 mm, well diameter of 30 μm, and well height of 30 μm, we patterned small groups of 1-4 single mESCs at each spot. Using microwells with edge-edge distance of ∼20 μm (n = 30), we patterned the cells with a membrane-membrane distance of 45 ± 15 μm (n = 33). Collage is from images taken randomly from different areas on the substrate. Scale bar, 40 μm. (f) Patterning mESCs as small groups of single cells allows us to turn off cell-cell contact. (g) Colony-forming efficiency (2 days after cell seeding) as a function of starting cell number for three cases – model of no cell-cell signaling (solid yellow bars), experiment with cell-cell contact (solid blue bars), experiment without cell-cell contact (solid red bars). Bars on experimental data indicate 1 standard deviation from the mean. (h) Colony-forming efficiency as a function of starting cell number for three cases – cells in contact without exposure to anti-E-cadherin (solid blue bars), cells in contact with exposure to anti-E-cadherin (striped blue bars), and cells without contact with exposure to anti-E-cadherin (striped red bars). Bars on experimental data indicate 1 standard deviation from the mean.