Kgaf001

A live–dead cell staining kit (KeyGEN, KGAF001) was used to determine the effect of NO on ADSC viability and mortality according to the manufacturer’s protocol [28 (link)]. Images were taken with a fluorescence microscope (Mshot, China) at varying resolutions.

Cell viability of SKPs in fibrin hydrogels was measured through trypan blue stain assay and live/dead staining (KGAF001, KeyGEN BioTECH, China). The SKPs before and cultured in fibrin hydrogels for 3 days were collected and suspended with PBS (Gibco, USA). The cell suspension and 0.4% trypan blue stain solution (Solarbio, China) were mixed at a ratio of 9:1 and the cell viability were automatically calculated in Countstar (Countstar Rigel S2, China). For live/dead staining, the cells were washed with PBS and then incubated with staining solution (PBS: Calcein-AM: PI = 1000:1:1) for 10 min in the dark and then was visualized immediately by a fluorescence microscope (Nikon, Eclipse Ti2-U, Japan).

The samples were sterilized by immersing in 75% ethanol overnight and exposed to UV light for 1 h before the experiment. The direct contact test was evaluated through two biological assays for cell membrane integrity and morphology (KGAF001, KeyGEN BioTECH, China) and cell proliferation (CCK-8, Dojindo, Japan). Cells without samples were used as the control. The cells were seeded onto a 24-well plate at a density of 1.25 × 10⁴ cells/cm₂. After 24 h of incubation, the wells were rinsed twice in phosphate buffer solution (PBS, Thermo Fisher Scientific, USA). The cells were incubated in dark for 30 minutes with a 1 mL working solution (containing 4 μM calcein and 8 μM propidium iodide in 10 mL of PBS). The samples were then examined under a fluorescence microscope (Optiphot-2, Nikon Corporation, Tokyo, Japan). For the determination of proliferative ability, cells were seeded in the samples at a density of 5 × 10⁴ cells/cm₂. After incubation for 24 h and 72 h, the medium was replaced with a medium containing 10% CCK-8 solution and incubated for another 2 h. Next, 100 μL of the medium per well was transferred to a 96-well plate. The absorbance was measured at 450 nm using a microplate reader (ELX808, Bio-Tek, USA).

The effects of 3D bioprinted constructs on cellular viability were assessed using a fluorescent live/dead staining kit based on provided protocols (KeyGEN Bio TECH, KGAF001, China). Briefly, a 10-ml staining solution containing 8 µM of propidium iodide (PI) and 2 µM of calcein-AM was prepared. Scaffolds were stained for 30 min with this solution, washed three times with PBS, and then imaged via fluorescence microscopy, with viable cells staining green (calcein-AM, 490 nm) and dead cells staining red (PI, 535 nm). The sample imaging (n = 3) was performed on Days 1, 4 and 7, with numbers of live and dead cells being quantified using the ImageJ software. The cell viability was quantified by dividing live cell numbers by the total number of cells. A CCK-8 assay (LK815, Japan) was conducted based on provided protocols to assess HGF proliferation within 3D printed constructs on Days 1, 7 and 14. Briefly, samples were incubated with the CCK-8 reagent at these time points, after which the absorbance at 450 nm was assessed via microplate reader (BioTek ELX800, VT, USA).

A batch of three domes (50 μl) of cultured organoids was dissolved by manual disruption to initiate immunostaining.⁴⁴ After centrifugation (100 × g, 4°C, 5 min), sedimentary organoids were fixed in 4% paraformaldehyde solution in PBS for 30–60 min. To block and permeabilize organoids, 1 ml Triton X‐100 and 2 g BSA were added to 1 L PBS to prepare organoid wash buffer (OWB). The fixed organoids were permeabilized using OWB for 20 min. Primary antibodies (anti‐Ki‐67, 11‐5698‐82; anti‐E‐cadherin, 53‐3249‐82; Thermo Fisher; anti‐CK‐20, ab109111; anti‐MUC2, ab272692; anti‐lysozyme, ab108508; anti‐CHGA, ab254322; Abcam, Cambridge, UK) were incubated in OWB (1:100 dilution) at 4°C overnight in rotation (60 rpm).⁴⁵ After extensive washing, second antibodies (GB22301, GB22303; Servicebio) were incubated in OWB (1:200 dilution) at 4°C overnight on rotation (60 rpm). Nuclei (KGA215; KeyGEN BioTECH) and live/dead (KGAF001; KeyGEN BioTECH) staining was performed. Prepared organoids were transported into a glass bottom dish and observed using confocal microscopy (LSM900; Zeiss, Jena, Germany).

HUVECs were added to four groups of bioinks (0%, 0.5%, 1% and 2% ChiNC), with a cell density of 8 × 10⁶ cells/ml. 3D bioprinted grid scaffolds were cultured in ECM, and cell viability within the 3D bioprinted scaffolds was assessed using a fluorescent live/dead activity assay kit (KGAF001, KeyGEN Bio tech, China) on Days 1 and 7 according to the protocol. The scaffolds were visualized under a fluorescence microscope (NIKON Ti-A1, Japan), and images were processed with Image J software.

Cell viability was assessed with a fluorescent live/dead viability assay kit (KeyGEN, KGAF001) following the manufacturer’s instruction. Briefly, samples were immersed in staining solution containing 8 μM propidium and 2 μM Calcein-AM. After incubating for 15 min, the samples were washed three times with phosphate-buffered saline. Fluorescent images were taken with a fluorescence microscope. Live and dead cells were stained green and red by Calcein-AM and propidium. For cell viability calculations, live and dead cells were counted in at least five random sights under 100× magnification.