Umbilical cord-derived mononuclear cells were isolated from umbilical cord (UC) blood (100~ 150 mL) that was collected from healthy unrelated donors.
Each blood sample was tested for communicable diseases. Cord blood was diluted (2:1) with saline and 30 ml of the diluted blood was then added to 15 ml of Ficoll in every 50 ml centrifuge tube and then centrifuged (750 g × 22 minutes). Mononuclear cells were collected and washed twice in saline. Contaminating erythrocytes were lysed with lysis buffer comprising of injection-grade water. Cells were then cultured at a cell density adjusted to 2 ~ 6 × 106/ml, in DMEM/F12 culture medium with bFGF and EGF at a concentration of 20 ng/ml. Culture media was mixed with 2% v/v B-27 Stem Cell Culture Supplements. Cells were harvested for clinical application after one week of cultivation at a cell quantity ≥1 × 107 and viability ≥95%. Finished product control: final cell count (≥1 × 107, containing 1.0-2.0% CD34+ cells, as determined by flow cytometry), cell viability (≥95%) and sterility test.