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Figure S1. Representative stacked image and MATLAB aggregate mask used for image analysis. For all image analysis done with MATLAB, cell–microparticle aggregates were first defined by creating masks, which outline the boundaries of the aggregates from a maximum intensity projection of all images from the confocal stack. (A) Maximum intensity projection of all pixels (original image) from which (B) the aggregate mask outlines are created. Each mask (yellow, teal, blue, and red) represents an individual aggregate and defines the boundaries in xy space for pixel calculations from each stacked image. Scale bar represents 100 μm.

Figure S2. Example images used for image analysis of particle content within β-cell aggregates. During image analysis, the original confocal stacked image (A) is split into color channels containing only the (B) green pixels (MIN6 cells), (C) red pixels (laminin-coated microparticles), and (D) blue pixels (fibronectin-coated microparticles). These images are converted to binary representations of each color channel (EG) using a custom threshold to identify the pixels that accurately represent imaged cells and microparticles. From these binary images, the number of (E) green pixels, (F) red pixels, and (G) blue pixels are tabulated using MATLAB. Single, protein-laden microparticle occupied approximately 11 pixels. Scale bars represent 100 μm.

Figure S3. Distribution of microparticles within cell aggregates without pre-incubation with cells. Laminin-coated resin microparticles were seeded in hydrogel microwell arrays with MIN6 cells without overnight pre-incubation. Confocal images of the cell–microparticle aggregates were taken at 1 μm intervals, starting at the bottom of the aggregate, then compressed into 10 μm stacks for image analysis. The number of microparticles incorporated at different z-positions was manually counted for 31 cell–microparticle aggregates. Error bars represent SEM and *denotes P < 0.05 using two-tailed student's t-test with Welch's correction for unequal variances. (A) Percent of microparticles incorporated at various z-positions within the cell–microparticle aggregates. (B) Percent of microparticles incorporated in the bottom, middle, and top third of the cell microparticle aggregates. Data represents n = 31 aggregates analyzed.

Figure S4. z-Distribution of LN- and FN-coated microparticles in β-cell aggregates. The percent of the aggregate cross-sectional area (C.S.A.) comprised of microparticles as a function of z-position within the aggregate was determined using image analysis in MATLAB. For cellmicroparticle aggregates containing both laminin- and fibronectin-coated resin microparticles, the percent of the cross-sectional area containing (A) laminin-coated microparticles, (B) fibronectin-coated microparticles, and (C) both laminin- and fibronectin-coated microparticles is shown for microparticle seeding conditions of 1LN:3FN (solid diamonds), 1LN:1FN (open circles), and 3LN:1FN (solid squares). Error bars represent SEM for n > 30 aggregates (w200) measured for each condition.

Figure S5. Distribution of microparticles throughout different size cell–microparticle aggregates in the z-direction. Average cross-sectional area of aggregates containing microparticles as a function of z-position within the aggregate. No significant differences in percent of aggregate occupied by microparticles were found at any z-position for aggregates formed in 100 μm microwells (open circles) and 200 μm microwells (solid squares). Error bars represent standard error of the mean for n ≥ 35 aggregates measured of each size.

Figure S6. Images of microparticles before and after protein adsorption. (A) Resin microparticles before protein adsorption exist as individual, spherical microparticles approximately 1 μm in diameter. (B) After protein adsorption, laminin-coated microparticles (red) remain mostly as single microparticles with a small percentage forming small microparticle aggregates (white arrow) while fibronectin-coated microparticles (blue) show high levels of agglomeration (yellow arrow). Scale bars represent 100 μm.

bit25153-sm-0002-SupData-S2.docx15KSupplemental Materials and Methods

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