Establishment of in vitro stable culture of human peripheral blood dendritic cells and its comparation with magnetic activated cell sorting / 中国地方病学杂志

ABSTRACT

Objective To establish a economic and stable method to induce and culture dendritic cells (DCs) from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] than that at day of 3rd[(68.667 ± 3.215)%, all P ＜ 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant (F = 0.737,P＞ 0.05) at days of 3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P ＜ 0.05). Before and after using the magnetic activated cell sorting, the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. The difference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]was higher than those of culturing for 1, 5 h[(15.033 ± 6.189)%, (21.233 ± 4.895)%, all P ＜ 0.05]. Compared with the 1st day[(32.328 ± 14.517)%], the CD14 expression level[(2.200 ± 1.356)%] on surface of DCs was significantly reduced(t = 5.467, P ＜ 0.05) at the 6th day of culturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased than that of the 1st day[(12.300 ± 6.223)%, t = 2.545, P ＜ 0.05];while the expressions of CD86, CD83, HLA-DR[(16.857 ± 5.686)%,(9.343 ± 5.230)%,(72.800 ± 17.881)%] were similar(t = 0.652,1.137,0.907, all P ＞ 0.05) compared with that of the 1st day[(12.550 ± 16.758)%, (6.250 ±1.323)%, (64.671 ± 15.588)%]. In mixed lymphocytes reactions, with increasing of lymphocytes, T lymphocytes proliferating activities were reduced. In the magnetic activated cell sorting, when the ratio of DCs and lymphocytes were 1 50, 1 100, cells proliferation ability(1.502 ± 0.055,1.507 ± 0.029) were lower than that of ratio of 1 10(1.859 ± 0.049, all P ＜ 0.05);in adherent culture method, the ratio of DCs and lymphocytes was 1 100, the cells proliferation ability(1.545 ± 0.066) was decreased than that of ratio 1 10(2.015 ± 0.301, P ＜ 0.05). When the proportion of DCs and lymphocytes remained the same, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Comparied with the magnetic activated cell sorting, after culture of PBMC for 2 h the induction of DCs can produce better formed and functional cells, and this method is stable, simple,economic, and is a suitable method for basic and clinical research of DCs in vitro.