OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.