Fig. 5.

Engineered cardiac scaffolds retained cATMSC-EV, as shown by fluorometric scanning, confocal microscopy and SEM imaging. (A) Dotblot of SEC fractions eluting NIR815-labelled porcine cATMSC-EV, scanning fluorescence at 800 nm. EV-containing fractions (squared) were confirmed NIR815⁺, pooled together and concentrated by 100 kDa-ultrafiltration, maintaining NIR815 fluorescence (right). (B–C) NIR815-labelled cATMSC-EV mixed with peptide hydrogel and added to pericardial and myocardial scaffolds were detected after thorough washing of scaffolds. (B) Representative near-infrared fluorescent scanning images of pericardial and myocardial scaffolds (autofluorescence at 700 nm, red) embedded with NIR815-cATMSC-EV (NIR815 fluorescence at 800 nm, green). Scale bar = 1 cm. (C) NIR815-cATMSC-EV fluorescence quantification expressed as relative to hydrogel-alone filled scaffold (background). (D) PKH26-labelled cATMSC-EV (red) were detected inside the scaffolds by confocal imaging, after immunostaining for collagen III (col III; grey). Scale bar = 20 μm. (E) SEM imaging confirms retention of porcine cATMSC-EV within scaffolds after washing and week-long culture, appearing as 100–120 nm protuberances embedded in the hydrogel filling the scaffolds. Controls are scaffolds filled with peptide hydrogel alone. Scale bar = 1 μm.