Confocal imaging system

Gelatin sponges were purchased from Guangzhou Kuaikang Medical Apparatus Co. (Guangzhou, China). According to the previous study (DINH et al., 2018 (link)), the gelatin sponges soaked in dopamine (DA) solution (2 mg/mL in 10 mM Tris-HCl, pH 8.5, Sigma-Aldrich, St. Louis, US) were incubated with shaking at 37°C for 24 h to form the polydopamine (PDA) coating. To remove the non-adherent PDA, the gelatin sponge/polydopamine (GS-PDA) scaffolds were gently shaken in an ultrasonic cleaner with distilled water for 5 times. The GS-PDA scaffolds were dried and sterilized by ethylene oxide before the next step. Then the GS-PDA scaffolds were immersed in PKH67-labeled or non-labeled ADSCs-Exos solution (10¹⁰particles/scaffold) with shaking at 37°C for 12 h. To remove the non-adherent exosomes, and the GS-PDA-Exos scaffolds were gently shaken in an ultrasonic cleaner with distilled water for 5 times. The distribution of PKH67-labeled exosomes on the scaffolds was observed with the confocal imaging system (Nikon, Japan). To measure the ADSCs-Exos release effect of GS-PDA-Exos scaffolds. The amount of ADSCs-Exos released was measured using CD63 ELISA (Beyotime, Shanghai, China) assay. Scanning electron microscopy (SEM, Hitachi, Tokyo, Japan) was used to observe the surface morphology of the scaffolds.

Cells cultured on cover slips were fixed using 4% PFA in PBS for 15 min and then permeabilized with 0.25% Triton X-100. After blocking with 1% BSA and 1% FBS in PBS, cells were incubated with the primary antibody for 1 hr at RT. Cells were then washed and incubated with secondary antibodies (Life Technologies) for 1 hr at RT. DAPI staining was used to visualize nucleus. For three-dimensional culture (3D) organoids, cells were grown in laminin-rich basement membrane growth factor reduced Matrigel (BDBiosciences) (Matrigel) as we previously described (Fessart et al., 2013 (link)). Confocal analysis was performed using Nikon confocal imaging system. Images were generated and converted to Tiff format.