70 μm nylon mesh cell strainer

Cell suspension from the mouse liver and spleen were prepared by dispersing the tissue in 8 mL phosphate-buffered saline (PBS) containing 5% FBS using a syringe needle. 8 mL red blood cell (RBC) lysis buffer (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA; pH 7.2–7.4) were added to lysed the erythrocytes. After centrifugation at 250× g for 5 min, cells were resuspended in 5% FBS-containing PBS and were passed through a 70 μm nylon mesh cell strainer (Corning, Inc., Corning, NY, USA) [89 (link),90 (link)].

Human iPSC-derived cardiomyocytes were purified according to the method described previously [23 (link)]. The differentiated iPSCs containing cardiomyocytes harvested from the bioreactor were dissociated with 0.05% trypsin/EDTA treatment and seeded on culture dishes at 1.0–1.7 × 10⁵ cells/cm² (Day 15). The cells were cultured in Medium A (defined as DMEM (D6429; Sigma-Aldrich) containing 10% FBS and Penicillin-Streptomycin (Sigma-Aldrich)) in a humidified incubator with 5% CO₂ at 37°C (Panasonic Healthcare, Tokyo, Japan). On Day 20, 1.5 μg/ml puromycin (Sigma-Aldrich) was added for 24–36 hours to eliminate non-cardiomyocytes which did not express the puromycin-resistant gene. On the next day (Day 21), the cells were passaged with 0.05% trypsin/EDTA treatment and seeded on culture dishes at 1.0–1.7 × 10⁵ cells/cm². The above process was repeated again. That is, 1.5 μg/ml puromycin was added again on Day 26, and on the next day (Day 27) the purified cardiomyocytes were harvested with 0.05% trypsin/EDTA treatment. The dissociated cells were suspended in a fresh medium, passed through a 70-μm nylon mesh cell strainer (Corning, Corning, NY, USA) to remove the cell aggregates, and used for cardiac cell sheet engineering. Through the purification process, the medium was changed to fresh medium on the day after cell seeding, and then every other day.