Abstract: BACKGROUND: To obtain a large number of high purity trophoblast-free, animal-derived natural killer (NK) cells from deep cryopreserved cord blood for better experimental studies on tumor killing by NK cells, a safe and efficient method for expanding and culturing NK cells in vitro was obtained by using NK expansion culture kits and serum substitutes.
OBJECTIVE: To establish a technical system for the expansion of NK cells from frozen cord blood using pure cytokines, and demonstrate their tumor-killing ability in vitro.
METHODS:  The frozen cord blood stored in liquid nitrogen was thawed to optimize the recovery system. The individual nucleated cells were collected by Ficoll density gradient centrifugation and inoculated in pre-plated T175 culture flasks. Cells were cultured using the cytokine + serum replacement method for 21 days and counted. The ratio of CD3-CD56+ in cells was detected by flow cytometry. The data obtained were compared with the data of K562 trophoblast cells + fetal bovine serum culture method. Meanwhile, myeloid leukemia cells K562 were used as target cells, and CCK-8 kit was used to perform different potent target ratios of NK cells to kill K562 cells. Later, simulated storage transport experiments were performed for 12 hours. The changes in the ratio of CD3-CD56+ were detected by flow cytometry and the apoptosis ratio was determined using Annexin V/PI double-staining method. 
RESULTS AND CONCLUSION: (1) The cells were expanded and cultured for 14 days using K562 trophoblast cells + fetal bovine serum, and the cells expanded (51.47±4.28) fold. After 21 days of expansion culture using cytokine + serum substitute method, the number of cells was more than 5×109; the cells expanded (328.32±116.36) times; the cell expansion ploidy was significantly higher; the CD3-CD56+ purity ratio obtained by both methods was above 80%; no significant difference was seen. (2) The killing effect of NK cells cultured by pure cytokine method on K562 cells (effective target ratio=10:1) was more than 80%. (3) The ratio of CD3-CD56+ did not change significantly during the 12-hour simulated storage experiment until the 12 hours, when the apoptosis rate of NK cells was < 7%. (4) The results showed that the optimized recovery system and pure factor expansion method can efficiently culture high-purity NK cells with the ability to effectively kill K562 tumor cells, and still have good cell purity and low apoptosis rate after 12-hour storage and transportation.

Key words: cryopreserved cord blood, NK cells, cytokine, expansion, cytotoxicity, apoptosis rate, matching, clinical application