Matrigel matrix solution

Patient-derived retinoblastoma cells (RB170 cells) were established using a previously reported protocol¹⁷ (link) as suspension and organoid cultures. Briefly, tumor tissue was dissociated, and the resulting cells were cultured in the growth medium developed previously.¹⁷ (link) A final concentration of 1% Matrigel matrix solution (Corning, Inc., Corning, NY, USA) was added in suspension culture, and that of 65% Matrigel matrix (growth factor reduced) was used to embed cells for organoid culture. RB170 was manually dissociated and passaged at a 1:2 or 1:4 ratio once a week for suspension culture or every 3 weeks for organoids. Cold freezing medium (culture medium containing 10% dimethyl sulfoxide) was used to freeze cells at −80°C for 24 hours before long-term storage in liquid nitrogen. A human retinoblastoma cell line (Y79; American Type Culture Collection, Manassas, VA, USA) was maintained in RPMI-1640 (HyClone Laboratories, Inc., Logan, UT, USA) containing 15% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. Medium was changed every 3 days for both RB170 and Y79 cells.

Note that 1 × 10⁴ indicated cells were suspended in a 500‐μl DMEM‐F12 serum‐free medium (Invitrogen, USA) and mixed with a 500‐μl Matrigel matrix solution (Corning Life Science, USA). Then, the mixture was gently added on a 6‐well ultra‐low attachment culture plate and placed at 37°C for 4 h to solidify the gel. Four hours later, 2‐ml DMEM‐F12 medium (comprising basic fibroblast growth factor [b‐FGF, 20 ng/ml], epidermal growth factor [20 ng/ml], insulin [5 μg/ml], 4% BSA and 2% B27) was added to the top of gels. Cells were cultured in a CO₂ incubator for 14 days, and the spheroids were imaged using a 10× microscope (Carl Zeiss, Germany). Measurements were performed in ImageJ software, where the minimized spheroid size was 40 cells per case. Mean values of triplicate replicates were used for data analysis.