[The experimental study on inducing and expanding T/NK cells from mononuclear cells of human umbilical cord blood].
OBJECTIVE
To explore the most efficient culture system which can induce cord blood (CB)-mononuclear cells (MNC) to differentiate into mature T/NK cells in vitro.


METHODS
The CB MNCs were cultured in six culture systems respectively for 4 weeks. The T/NK cell surface phenotypes were analyzed by flow cytometry and the absolute numbers of nucleated cells (NCs) were counted at each time point. Moreover, cell morphology was identified by Giemsa-Wright staining, and cytotoxicity of the cultured cells to K562 and Raji tumor cells was also evaluated by MTT method.


RESULTS
Cultured in the cytokine cocktail of SCF + FLT-3L + IL-7 + IL-15 + TNF-alpha + IL-2, the NCs were (20 approximately 26) x 10(6)/ml in numbers at day 22. The percentage of lymphocytes in the NCs and that of CD(3)(+) T cells in the lymphocytes both exceeded 90% at the same time. Most of the CD(3)(+) T cells were CD(3)(+)CD(8)(+) and the percentage of CD(3)(+)CD(4)(+) T cells declined gradually. The percentage of CD(3)(+)CD(56)(+) NKT cells and gamma delta(+)T cells in the lymphocytes arised from lower than 2% to 30% approximately 40% and 10% approximately 15%, respectively. CD(3)(-)CD(56)(+) NK cells were not expanded. The cytotoxic activity of the cultured cells to K562 and Raji cells at an effector:target (E:T) ratio of 50:1 was over 75% and about 32% approximately 65%, respectively.


CONCLUSIONS
The most efficient culture system which can induce CB MNC to differentiate into mature T/NK cells in vitro is the cytokines cocktail of SCF + FLT-3L + IL-7 + IL-15 + TNF-alpha + IL-2, and the optimum culture time is 22 days.
Chen
Ge-yu
GY
The Second Affiliated Hospital of Zhongshan University, Guangzhou 510120, China.
Huang
Shao-liang
SL
Zhou
Dun-hua
DH
Wu
Yan-feng
YF
Wei
Jing
J
Chen
Qin
Q
D002454:Cell Differentiation; D002455:Cell Division; D003602:Cytotoxicity, Immunologic; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D007231:Infant, Newborn; D007376:Interleukin-2 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000166:cytology*; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000032:analysis; D013601:T-Lymphocytes / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007376:Interleukin-2; D016692:Receptors, Antigen, T-Cell, gamma-delta; D014409:Tumor Necrosis Factor-alpha