Poly 2 hydroxyethyl methacrylate solution

Low-adherent well plates were prepared
by coating them with 5 mg/mL poly(2-hydroxyethyl methacrylate) solution
(Sigma) and stored sealed at 4 °C until use. BC and BC-Ppy materials
were sterilized with UV irradiation for 15 min on each side and secured
by polytetrafluoroethylene rings at the bottom of each well.
The scaffolds were immersed in DMEM containing 4.5 g/L glucose, 50%
FBS (Gibco, Invitrogen), and incubated overnight inside a cell culture
incubator at 37 °C with 5% CO₂. Scaffolds were coated
with collagen (100 mg/mL, VitroCol, Advanced Biomatrix) prior to myoblast
seeding.
Thirty thousand cardiomyoblasts/cm² or fifteen
thousand cardiac fibroblasts/cm² were seeded on top of
each scaffold. Culture plastic wells (without scaffolds) were used
as controls. Adult human cardiac fibroblasts (p5-p6, PromoCell) were
cultured in fibroblast growth medium (PromoCell), whereas H9c2 rat
cardiac myoblasts (p2-p9, ATCC) were cultured with DMEM containing
4.5 g/L glucose, 10% FBS, 1% l-glutamine (Gibco, Thermo Fisher),
and 1% penicillin/streptomycin (Gibco, Invitrogen). Cultures were
maintained in a humidified atmosphere with 5% CO₂ at 37
°C, and culture medium was changed every 2 days.

Sphere formation assays were performed to confirm the self-renewal capacity of colon CSCs as previously described [23 (link)]. Briefly, sphere medium was prepared with DMEM-F12 (1:1; Welgene) containing 20 ng/mL epidermal growth factor (EGF, Pepro Tech, London, UK), 40 ng/mL basic fibroblast growth factor 2 (bFGF, Pepro Tech), and 2% B27 (Invitrogen) and 6-well plates were coated with a 10% poly-(2-hydroxy- ethyl methacrylate) solution (Sigma Aldrich). After CD133⁺CD44⁺ HCT116 cells were plated in the 6-well plates (5 × 10⁴ cells/well), they were treated with WPE (0, 10, 20, and 40 μg/mL) or doses of (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid that were comparable to 40 μg/mL WPE. After 6 days, the number of spheres containing more than 50 cells were counted and photographed.