In vitro culture and characterization of human umbilical cord blood-derived plasmacytoid dendritic cell subsets

Objective To establish a method for in vitro culture of plasmacytoid dendritic cell( pDC). Methods Umbilical cord blood( 40 ml) was collected from healthy parturients in the First Affiliated Hospital of Hunan University of Chinese Medicine,and cord blood mononuclear cell( CBMC) were isolated. The CBMC were cultured for 7 days with RPMI 1640 complete medium containing rh Flt3-ligand( Flt3-L)( 100 ng/ml) and rh interleukin( IL)-3( 10 ng/ml),and the medium was half changed every 2 days. On the eighth day,CpG ODN( 2 [mu]g / ml) was added to the cells,and the attached cells and supernatant were collected 24 h later for flow cytometry and interferon( IFN) [alpha] measurement,respectively. On days 1,3,5,7,and 8 of cell culture,the morphological changes of pDC were observed. Results After 2 h of culture,the CBMC showed circular,flat morphology. Twenty-four hours later,the cells began to adhere to the wall,with extended cytoplasm and increased volumes,and they became round and translucent,with scattered small colonies. On days 3-4 of culture,the cell volume continued increasing; most cells were round,and some had small protrusions; few cells were spindle-,tadpole-,star-or irregularly shaped; the number and volumes of colonies increased substantially. On days 5-8 of culture,the number of colonies and the number of cells in colonies gradually decreased,and suspended cells that were round or had small protrusions gradually increased in the medium. The cells expressing CD123,BDCA-2,and BDCA-4,which were considered pDC,were detected by flow cytometry. Flow cytometry revealed that the proportion of pDC in CBMC increased during the culture: increasing from 1. 08% at the beginning of culture to 5. 32%on day 4,and finally reaching a peak of 19. 8% on day 8. On day 8,the level of IFN[alpha] in pDC culture supernatant was( 11 302. 61 + or - 1745.31) pg / ml. Conclusion pDC can be successfully induced in vitro by rh Flt3-L combined with IL-3 from human umbilical CBMC.