Analysis of ex vivo produced platelets morphology and functionality. (A) Silk microtubes are perfused with culture media for 6 hours, and released platelets are collected into gas-permeable bags. Samples are mixed with counting beads to quantify the number of platelets that are identified as CD61⁺CD42b⁺ events. A maximum of 4 different silk microtubes have been perfused concurrently. The graph shows the absolute number of platelets released per microtube embedded in the silk sponge containing 2.5 × 10⁵ Mks. (B) Analysis of platelet morphology. (Bi-Bii) Light microscopy analysis shows preplatelets, dumbbell-shaped platelets, and disc-shaped platelet (scale bar = 10 μm). (Biii) Immunofluorescence staining of β1-tubulin (green) (scale bar = 10 μm). (Biv-Bv) Magnification highlights the microtubule coil typically showed by resting platelets (scale bar = 5 μm). (Bvi) Transmission electron microscopy analysis of ex vivo produced platelets ultrastructure (scale bar = 2 μm). (C) Analysis of platelet adhesion on type I collagen. (Ci) Tetramethylrhodamine isothiocyanate-phalloidin staining of resting platelets (scale bar = 5 μm). (Cii) After adhesion on type I collagen platelets spread and formed filopodia/lamellipodia (scale bar = 5 μm). (Ciii-Civ) Magnification highlights actin cytoskeleton reorganization with evident stress fibers assembly (scale bar = 5 μm). (Cv) CFSE⁺ platelets were suspended into Tyrode’s buffer containing von Willebrand factor and perfused over immobilized type I collagen at shear rate of 1000 s^–1. Image shows a representative filed demonstrating platelet adhesion. Arrows indicate formation of platelet aggregates (scale bar = 10 μm). (D) Aggregation capacity was further measured by flow cytometry after stimulation with thrombin, ADP, and epinephrine. Platelets were separately labeled with CD31 or CD42b (left and right top, respectively), and then mixed 1:1, before being stimulated (bottom right) or not (bottom left) with the cocktail of agonists. (E) Analysis of ex vivo produced platelets participation to clot formation. (Ei) Ex vivo produced CFSE⁺ platelets were mixed with peripheral blood platelet stained with CellTracker Deep Red Dye. Clot formation was favored by addition of thrombin and visualized by confocal microscopy (scale bar = 10 μm). (Eii) Cross-sectional analysis of z-stack of the clot demonstrating that ex vivo produced platelets (green) actively interacted with in vivo derived platelets (red) with appearance of a juxtaposed signal (yellow) (scale bar = 5 μm).