*2.3. Manufacture of Kartigen®*

The manufacture of Kartigen*®* was described previously [34]. In brief, cell culture was performed under standard operative procedures at the cell processing unit (CPU) of Kartigen Biomedical Inc. (Taipei, Taiwan), following the Good Tissue Practice regulations. Fifteen mL of heparinized blood was aspirated from the iliac crest, collected in sterile 50-mL tubes. The MSCs were isolated using the density-gradient medium Ficoll (Cat. No. 17-5446-52, GE Healthcare, Little Chalfont, UK). The nucleated cells were collected from the interface, washed twice in PBS, and then suspended in Dulbecco's Modified Eagle's Medium (DMEM-LG, Cat. No. 31600, Gibco, Carlsbad, CA, USA) supplemented with the patient's serum. BMSC were expanded to a sufficient number, enough to fill the cartilage defects of patients. The required cell number was estimated according to the size of the defects. Before the BMSCs were induced into CPs, a small portion of BMSCs were used to assess the number, viability, and the immunophenotype of BMSCs, and to undergo sterility testing. The cell number and viability were quantified using trypan blue staining and an automated cell counter. BMSC's immunophenotype must be fulfilled by the minimal ISCT criteria: More than 95% of BMSCs are CD90-positive, and less than 2% of BMSCs are CD34-negative. Sterility testing must be negative for aerobic and anaerobic bacteria, mycoplasma, and <0.5 EU/mL endotoxin. The expanded cells were then seeded in excipient (Kartigen®) and cultured in CPs' induction medium.
