Fig 1.
LOP fabrication of protein patterns on polyacrylamide gels.
(A,B) Photoresist patterns are fabricated by standard contact photolithography on glass coverslips. Inset at right shows array of S1818 photoresist features after development. (C) Unspecific protein adhesion to the resist-patterned coverslip is blocked by incubating with biopassive PLL(20)-g[3.5]-PEG(2) copolymer. (D,E) Following photoresist lift-off, the resulting PLL-g-PEG pattern is backfilled with the ECM protein of interest. Inset at right shows a fluorescence micrograph of labeled gelatin on glass after backfill. (F) To transfer the protein pattern to the PAAm gel, the gel is polymerized between the protein patterned glass coverslip and a silanized coverslip. (G) After gel polymerization, the top coverslip is removed from the PAAm gel. Inset at right shows a fluorescence micrograph of a labeled protein transferred to a PAAm gel. (H) Inset at right shows pairs of epithelial cells on the patterned PAAm gel restricting the geometry of the protein functionalized regions.
Fig 2.
Quantification of protein transfer efficiency to PAAm gels of varying stiffness.
(A,B) Arrays of 45 μm2 square protein patterns on 25 kPa PAAm gels created by LOP and μCP before and after transfer to gel surface. (C) Quantification of protein transfer efficiency from glass coverslips to PAAm gel of varying stiffness. Differences between LOP and μCP for each stiffness are statistically significant (p-value < 2.2E-16, Mann-Whitney-Wilcoxon test). Substantially more protein is transferred from patterns created by photoresist lift-off. Data are represented as box plots. The median, 1st and 3rd quartile, and minimum and maximum values are shown, n = 150 for each method and stiffness shown. (D) Overview of μCP method to pattern proteins on PAAm gels.
Fig 3.
Comparison of pattern accuracy between LOP and μCP methods.
(A) Average images of 150 binarized protein patterns created by LOP and μCP on 25 kPa gels. (B) Difference images calculated by comparing the average images and the theoretical pattern mask. Edges and corners are resolved substantially better in patterns created by LOP. (C) Theoretical pattern shape with a region highlighted corresponding to where profile column average scans were taken. (D) Profile column average scans across 150 binarized patterns show that the variation in protein signal at the pattern edges is strongly reduced in LOP patterns. Plotted are the median (line), 1st / 3rd quartile (box) and 5–95% (whisker) of the probability of protein present across the pattern width.
Fig 4.
LOP yields sharper cell edges with localized actin bundles compared to μCP patterned gels.
Time-lapse acquisitions of MDCK cells transfected with Lifeact-GFP (actin label) grown on 25 kPa PAAm gels showed similar intracellular actin structures on LOP (A,B) and μCP (C,D) protein patterns. Cell doublets rotated around each other on the patterns for both techniques (B,D).
Fig 5.
Lamellipodia are more exploratory for cells on LOP than on μCP patterned substrates.
Phase contrast time lapse imaging of 3 representative MDCK cells on 25 kPa PAAm gels patterned by LOP (A) and μCP (B). Cells on substrates produced by LOP follow the protein pattern border more accurately (dotted line panels A, B) and reveal more pronounced lamellipodia. (C, D) Kymograph analysis of lamellipodia kinetics along pattern edge. Single cell on LOP pattern shows increased lamellipodia protrusions and retractions within a 10 μm wide region of interest outside the protein pattern edge compared to a cell on a μCP pattern (S5 and S6 Movies). Kymographs show cells depicted in bottom row of panel A and B. Regions of interest are straightened and distorted regions at the pattern corners are cleared.