Crl 1635

A total of 5% CO₂ at 37 °C was used to cultivate human dermal fibroblasts cell line HS68 (ATCC^® CRL-1635™) (ATCC, Manassas, VA, USA) cell at a consistent monolayer culture of Dulbecco’s modified Eagle medium (DMEM) for 24 h. Fetal bovine serum (FBS) (10%), penicillin (100 U/mL), streptomycin (100 mg/mL), amphotericin B (0.25 µg/mL), and amphotericin B (0.25 μg/mL) were the ingredients of DMEM. A. fulica mucus were dissolved in adding dimethyl sulfoxide (DMSO) at different concentrations without any impurity, and the DMSO concentration was less than 1.0% compared to the final working volume. The influences of testing samples on cell development were estimated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 1 × 10⁴ cells/well in 96-well plates and allowed to hatch for 24 h before adding the extracts. After 24 h, the MTT solution was dispensed into each well. After another two hours, the culture medium was discarded, and DMSO was added to each well. The absorbance of the formazan salt was 595 nm, and the cell viability was computed as Equation (3).
$\mathrm{Cell} \mathrm{viability} (\%) = \frac{(A_{sample}-A_{blank})}{(A_{control}-A_{blank})} \times 100\%$

Hepatocytes cell line HepG2 (HB-8065™, ATCC^®, Gaithersburg, MD, USA), fibroblasts cell line HS68 (CRL-1635™ ATCC^®, Gaithersburg, MD, USA), and primary human umbilical vein endothelial cells (HUVEC, C2519A, Lonza, Basel, Switzerland) were utilized in the current study. All the cell types were grown at 37 °C with 5% CO₂ in a humidified incubator. All the cells were cultured for two passages before being utilized in experimental conditions. HepG2 and HS68 were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin. Primary HUVEC cells were grown in endothelial cell media. The experiment for validation of the spheroid culture platform was divided into two phases. Initially, cell number was optimized for spheroid formation in a dynamic culture plate in which only hepatocytes (HepG2 cell line) were used. HepG2 cells were grown until confluency and then seeded in the spheroid plate at different concentrations of 2000, 5000, 10,000, 20,000, 25,000, and 30,000 cells for optimization of the cell number selection. The cells after seeding were progressively monitored for the spheroid formulation. The spheroids were observed in the inverted microscope to monitor their growth every 24 h. For dynamic culture, the spheroid plate was kept on a perfusion rocker (Mimetas B.V., The Netherlands) after the first 24 h of seeding the cells.

Human neuroblastoma cells (SK-N-SH) were purchased from the Korean Cell Line Bank (Seoul, Korea). Non-tumorigenic fibroblast HS 68 (CRL-1635) cells were purchased from the American Type Culture Collection (Manassas, Virginia, USA). Cells were cultured in suitable media (SK-N-SH; RPMI-1640 media, HS 68; DMEM media) with 10% of fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. The cell passage for SK-N-SH and HS 68 subculture were within passage number 3–7.