Ethidium homodimer

The freshly isolated fibrin membranes were placed onto ultra-low attachment 24-well plates in 2 mL of stem cell medium, and 25,000 hBM-dMSCs (5 passages) were seeded onto the membranes and cultured on them for 6 days. The medium was refreshed every 2 days. On the 7th day live-dead staining was performed to visualize attaching cells. The membranes were washed 3 times with PBS (phosphate-buffered saline) and stained in PBS containing 1 mM Calcein-AM (Invitrogen, Carlsbad, CA, USA), 4 mg/mL ethidium homodimer (Invitrogen), and 20 mg/mL Hoechst (Invitrogen, Carlsbad, CA, USA) for 30 min. The gels were washed again 3 times for 10 min with FluoroBrite DMEM (Gibco, Paisley, Scotland), and images were taken by an inverse fluorescent Nikon Eclipse Ti2 microscope (Tokyo, Japan).

The effect of marine EVs on proliferation in normal or inflammatory environments was evaluated using the water-soluble tetrazolium salt (WST) assay (EZ-cytox, DoGenBio, Seoul, Korea). Briefly, based on the protein concentration measured by the Bradford assay, mEVs were diluted to 5, 10, 15, and 20 μg/mL in FBS-free RPMI-1640 medium and treated onto RAW 264.7 cells for 24 h. In the inflammatory environment, the same concentration of LPS (50 ng/mL) was used. The WST assay was performed according to the manufacturer’s instructions.
To check the cell viability, highest concentration marine EVs (20 μg/mL) in FBS-free medium were treated on RAW-264.7 for 24 h. Then, the medium was changed using the live and dead assay kit containing calcein-AM and ethidium homodimer (Invitrogen) in Hanks’ balanced salt solution (HBSS, Welgene, Gyeongsan, Korea) for 1 h. After 1 h, live and dead cells were observed using a fluorescence microscope (Carl Zeiss).

Briefly, thin hydrogels encapsulating hCSCs (2 × 10⁶ cells/mL) were prepared by placing a drop of hydrogel formulation (12 µL) at the center of a sterile coverslip. Then, another coverslip was placed immediately on top of the drop to yield a hydrogel sandwiched between the coverslips. The sandwiched hydrogel was then placed in culture media for a few minutes and then the top coverslip was gently released and pushed aside using a pair of sharp pointed forceps without damaging the underlying hydrogel. The hydrogels were cultured for a period of 5 days, following which they were incubated in calcein acetoxymethyl (calcein-AM, 0.2 µg/mL) and ethidium homodimer (2.5 µg/mL) (Invitrogen, Paisley, UK) for 15 min in supplemented DMEM at 37 °C to stain for live cells and dead cells. Live cells were visualized as green and dead cells as red under a fluorescence microscope (EVOS FL Auto2, Thermo Fisher Scientific, Waltham, MA, USA).

Twenty-four hours after trauma or IL-1β treatment, engineered cartilage constructs were bisected transversely and stained with Calcein AM (Invitrogen, Cat# L3224A) and ethidium homodimer (Invitrogen, Cat# L3224B) for 45 min in a 37 °C incubator. Images of the cut, transverse plane through the center of the construct were taken using a confocal microscope (Olympus FluoView FV1200) set for red (527_ex nm/624_em nm) or green (488_ex nm/520_em nm) detection. 40 × and 100 × magnification were used to take pictures. Ten stacks for each construct were taken with a z-stack density of 10 um. Fiji ImageJ software was used to process images and quantify live and dead cells.

After incubation of BMEs in different media, bone chips were washed three times with PBS and stained in PBS containing 1 μM Calcein-AM (Invitrogen, Carlsbad, CA, USA), 5 μg/mL ethidium homodimer (Invitrogen, Carlsbad, CA, USA), and 5 μg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) for 40 minutes. The experiments were performed in case of two patients' four-four BMEs (8 replica) after 5 days' incubation in FCS, HAS, or serum-free media, in total 3 different media. The samples were washed three times with PBS and imaged immediately with a Nikon A1R confocal microscope (Nikon-KOKI Imaging Center, Budapest).

A two-color fluorescence assay (LIVE/DEAD Assay, Molecular Probes, consisting of calcein, as a marker of viable cells, and ethidium homodimer, as a marker of dead cells, Invitrogen) was employed to determine HKs viability within the hybrid constructs immediately after the hybrid construct was produced, and to determine the HCECs viability after the cells incubating on the surface of the construct for 24 h. Each sample was washed in PBS 3 times before staining. Samples were stained for 15 min while avoiding light and washed 3 times in PBS after staining. A confocal microscope (Xcellence, Olympus) was used for image acquisition. Three different fields were counted for each sample.