Increased Retention of Cardiac Cells to a Glass Substrate through Streptavidin–Biotin Affinity

Figures

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  Figure 1. Functionalization of a glass substrate to increase cell retention. (A) Epoxy coating of a glass substrate followed by streptavidin conjugation and binding of biotinylated cells. (B) Fluorescent imagining of SA-Cy3 coating on epoxy slides and negative control/unmodified slides. Streptavidin density determined using the Cy3 calibration slide (scale bar = 1 mm).

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  Figure 2. Viability of cardiomyocytes determined by Trypan Blue staining during the first 5 h following retrieval. Black arrows indicate CMs with an intact membrane. (p* < 0.05, p** < 0.01, and scale bar = 100 μm).

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  Figure 3. Retention of H9C2 following PBS rinsing when incubated with laminin and poly-L-lysine cover glass compared to biotinylated H9C2 on an eSA-functionalized cover. A BSA-blocked glass substrate was used as a control. (A) Microscopic images of cell retention at incubation times of 5, 10, 20, and 40 min (scale bar = 50 μm). (B) Summary plot of cell retention versus incubation time. Retention results are an average of three replicates with three areas counted per coverslip (approximately 150 cells per replicate).

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  Figure 4. Retention of primary cardiomyocytes following the introduction of PBS for unmodified cells on a BSA-blocked substrate versus biotinylated cells on a eSA-functionalized substrate (scale bar = 50 μm).

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  Figure 5. Time for dye to reach full concentration through injection with hand pipetting versus perfusion with a 160 mL/min parallel-plate flow chamber. (A) Retention of unmodified H9C2 on a plain glass substrate versus NHS-biotin-modified H9C2 on eSA. (B) Microscopic images of cell retention captured at 1, 4, 6, and 9 s following the introduction of trypan blue.

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  Figure 6. (A) Viability of unmodified H9C2 versus NHS-biotin-modified H9C2 through calcein, ethidium, and caspase assays. (B) Viability of unmodified CM versus NHS-biotin-modified CM determined by trypan blue (p* = 0.29) and calcein assay (p* = 0.60). (C) Contraction of adhered cardiomyocytes following fluidic stimuli. The arrow indicates the position of the deformation wave during cell contraction.

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  Figure 7. Ability to obtain Ca2+ transients following the introduction of a 10 mM caffeine puff. Systems are compared based on CM movement and observed Ca2+ transient. CM movement is directly related to if a transient is useable or not. (n = 5 per sample group; y axis = F340/F380, x axis = time(s)).