[Experimental study on cryopreservation of immature dendritic cells derived from cord blood].
OBJECTIVE
To investigate the biological properties of immature dendritic cells( imDC) derived from cord blood before and after cryopreservation, so as to provide a method for preservation of imDC.


METHODS
Immature dendritic cells were generated from human cord blood (CB) monocytes and cultured with rhGM-CSF and rhIL-4, and 10% DMSO was added into culture medium as cryopreservation reagent. After freezing in - 80 degrees C refrigerator, the cells were finally cryopreserved in - 196 degrees C liquid nitrogen, and then thawed with 40 'C water, and they were finally named frozen-thawed imDC. The morphology of imDC were observed with light microscope, and TBR were calculated. Cellular surface markers for DC maturation were determined with flow cytometry, and the ability of the cells to stimulate proliferation of non-sensitized T lymphocyte was determined with allogeneic mixed lymphocyte reaction.


RESULTS
Monocyte (MNC) from cord blood could differentiate into DC after GM-CSF and rhIL-4 induction. Under light microscope, the cells showed irregular morphology, with branch-like prominence on the cell surface. Similar changes were also observed with scan electron microscope. The cryopreserved imDC were resistant to trypan blue staining, and TBR was (86. 8 +/- 1. 3) % . There was no obvious difference in the cell morphology between cryopreserved and fresh imDCs. The expression of cell surface markers and maturation markers in imDCs before cryopreservation were as follows: CDla(62 +/-8)% , CD14 (18 +/- 7)% , HLA-DR (67 +/- 5)% , CD80 (13+/-7)%, CD 86 (12+/- 5) % . Though the expression of CD80, CD86 and CD83 of cryopreserved imDC increased to (15 +/-5)% , (17 +/-5)% and (7.4 +/-3. 3)% , respectively( P <0.05), they still possessed the phenotype of imDC. There exhibited no obvious difference in cmp value between fresh imDC[ (463 +/- 104) min(-1) ] and cryopreserved imDC[ (512 +/-78 )min(-1) ] , ( P > 0. 05 ). The cpm in control group was (488 +/- 197 ) min'. The stimulation index in all groups was lower than 2, and both fresh imDC and cryopreserved imDC could not stimulate the proliferation of non-sensitized T lymphocyte.


CONCLUSIONS
The cryopreserved imDC exhibits immature characteristic in cell phenotypes, function and good cell activity, indicating that the method of cryopreservation of imDC is feasible.
Wang
Yi-tao
YT
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chonqing, PR China.
Peng
Yi-zhi
YZ
Tang
Jin
J
Wang
Qiang
Q
Wang
Yong-quan
YQ
You
Bo
B
D002454:Cell Differentiation; D002469:Cell Separation; D015925:Cryopreservation / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D009000:Monocytes / Q000166:cytology