Production process of iPSC-PLTs from the imMKCL MCB. (A) Dox-ON condition for imMKCL proliferation: On day 1, the MCB cryopreserved in vials was thawed and cultured in a 6-well culture plate at 37°C and 5% CO₂. On days 4, 8, 11, 14, 17, and 20, the cells were transferred and cultured in a 90-mm culture dish, 250-mL culture flask, 2 500-mL culture flasks (both with 100 revolutions per minute [rpm] shaking), or 2-L, 10-L, and 50-L WAVE bags (each with rocking motion). On day 23, the imMKCLs were pelleted and washed twice using 2 ACP215 centrifugation systems and resuspended in Dox-OFF medium. (B) Dox-OFF condition for platelet production: the processed imMKCL solution was applied to 4 vessels of 10 L-scale VerMES bioreactors with 8 L medium solution in each and cultured with vertical stirring motion at 37°C for 6 days. (Ci) On day 29, the culture solution was added with 1/5 volume of ACD-A solution and concentrated to about 1/10 volume using a hollow fiber membrane filter. (Cii) imMKCLs and iPSC-PLTs were separated by continuous centrifugation using an ACP215 at 2500 rpm. With extruding solution, the platelet supernatant fraction volume became 4 L. (Ciii) The iPSC-PLT suspension was concentrated and extruded out to 700 mL using a hollow fiber membrane filter and then filtered through a leukocyte removal filter to remove residual megakaryocytes. (Civ) The iPSC-PLT suspension filtrate was centrifugated in an ACP215 at 200 mL per minute and 6000 rpm. Then, the pellets in the centrifuge pod were resuspended in 250 mL bicarbonate Ringer's solution (BRS) with 10% anticoagulant citrate dextrose solution (ACD)-A and 2.5% human serum albumin (HSA). From the 250 mL solution, 50 mL was drawn for evaluation.