Cs 900

Peripheral blood samples were collected from donors’ peripheral veins and stored in blood bags with CPDA (citrate phosphate dextrose adenine), carried out in the Asia University Hospital (CMUH 109-REC 1-012), Show Chwan Memorial Hospital (1090803), and Taichung Tzu Chi Hospital (REC 109-45). A 100 ml peripheral blood sample was mixed with 80 ml normal saline (Nang Kuang Pharmaceutical, Tainan, Taiwan) or good manufacturing practice (GMP)-grade MoFi medium (comprising a chemical-defined medium for ex vivo human cells; classified as a medical device; Duogenic StemCells Corporation, Taichung, Taiwan) for 30 min. The mixed blood was then carefully loaded in a set of cell isolation (CS. 900.2, Cytiva, Marlborough, MA, USA) with the Sepax II machine (Cytiva). PBMNCs were separated by density-gradient centrifugation with Ficoll-Paque premium (Cytiva). The isolated PBMNCs were automatically washed with normal saline three times to remove the residue of the MoFi medium and Ficoll-Paque before the cell transplantation. The final concentration of the PBMNCs was adjusted to be 3.5 to 4 ml for one knee/person, containing 7–12 × 10⁷ PBMNCs from 100 ml blood. The cell numbers of engrafted cells were estimated by manual counting using a hemocytometer.

For leukocyte-poor PRP preparation, we collected 10 ml blood from peripheral veins and the PRP was concentrated to 2 ml using an authorized centrifuge tube with separating gel (class II medical device; CPKit, Aeon Biotherapeutics, Taipei, Taiwan). For the PBMC preparation (peripheral blood cell purification [PCP]), 100 ml peripheral blood samples were taken and stored in blood bags with citrate phosphate dextrose adenine (CPDA; Japan Medical System, JMS, Singapore). We mixed 100 ml peripheral blood and 80 ml good manufacturing practice (GMP)-grade MoFi medium (comprising a chemical-defined medium for ex vivo human cells; class II medical device; Duogenic StemCells, Taichung, Taiwan) for 30 min. The mononuclear cells separation from mixed blood sample was automatically processed in a closed-tubing set (CS. 900.2, Cytiva, Marlborough, MA, USA) with the Sepax II machine (Cytiva) and Ficoll-Paque premium solution (Cytiva). The MoFi medium and Ficoll-Paque were removed by three times washing with normal saline. The final PBMC solution was adjusted to 2 ml, consisting of 6–9 × 10⁷ PBMCs. The PRP and PCP processes were carried out at the point of care. The cell numbers of engrafted cells were analyzed by manual counting using a hemocytometer.