**4. Materials and Methods**

## *4.1. Cell Cultures and Treatment*

Human fetal right auricle(RA) tissue was received from Pusan National University YangSan Hospital and the study was approved by the Institutional Review Board (IRB) (IRB No. 05-2015-133). C-kit positive hCPCs were isolated and cultured, as previously reported [45,46]. hCPCs were maintained at 37 °C with 5% CO2 in Ham's Nutrient Mixture F-12 (Hyclone, GE Healthcare, Chicago, IL, USA) and supplemented with 10% Fetal bovine serum (FBS; Gibco#16000-044, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin-streptomycin (PS; Welgene, Daegu, Republic of Korea), 0.005 unit/mL of human erythropoietin (hEPO, R&D systems, Minneapolis, MN, USA), 10 ng/mL of recombinant human basic fibroblast growth factor (rb-FGF, Peprotech, Rocky Hill, NJ, USA), and 2 mM of glutathione (Sigma-Aldrich, St. Louis, CA, USA). Passages 4–10 were utilized for the experiments as passage 13 was found to be senescent cells. hCPCs were treated with different concentrations of H2O2 (Sigma-Aldrich, St. Louis, CA, USA) and a final concentration of 600 μM (to investigate the antioxidant effect), 1 mM (to confirm anti-apoptotic effect) was used.

The standardized substance echinochrome A (registration number in Russian Federation is P N002362/01) was isolated from the sea urchin *Scaphechinus mirabilis* by a previously described method [47]. The purity of echinochrome A (99.0%) was confirmed using LC-MS data (Shimadzu LCMS-2020, Kyoto, Japan). Purified echinochrome A appeared as red–brown needles, had a melting point of 221 ◦C, and similar nuclear magnetic resonance (NMR) spectra to that reported previously [47]. We used a solution of echinochrome A sodium salts in ampoules with trade name Histochrome®. Histochrome was generated by combining echinochrome A (1 g) with sodium carbonate (0.4 g) in a water solution heated in inert gas until CO2 was completely removed. This solution at a concentration of 0.2 mg/mL echinochrome A (750 μM) was sealed in ampoules in inert gas. After opening of the ampoule, histochrome was used as a stock solution to be diluted with appropriate solvents or culture media.

#### *4.2. Cell Cytotoxicity Assay*

To determine cell cytotoxicity, hCPCs were seeded at 5000 cells/well in a 96-well plate. The following day, cells were pretreated with different concentrations of histochrome and incubated for 24 h. Cell viability was evaluated using a cell-counting kit (CCK) cell viability assay kit (#CCK-3000, DonginLS, Seoul, Republic of Korea), following the manufacturer's instructions [48,49]. The cell viability was measured by incubating cells with the CCK solution. The plates were incubated for 1 h, and the absorbance of each well was measured. Absorbance was measured at 450 nm using a microplate reader (TECAN, Mannedorf, Switzerland ). Cell viability of the experimental group was represented as percentage to 0 μM.
