Establishment of in vitrostable culture of human peripheral blood dendritic cells and its comparation with magneticactivated cell sorting

Abstract

：Objective To establish aeconomic and stable method to induce and culture dendritic cells (DCs) from peripheralblood of human being, and compare with the magnetic activated cell sorting. MethodsMonocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) bydensity gradient separation,cultured and compared with that of cells isolated by themagnetic activated cell sorting or adherent culture,respectively. PBMC were cultured withrecombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) andrecombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs.Morphological changes was observed under inverted microscope. Meanwhile, cell viabilitywas tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DRwere analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. Afteradding 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. Tcells proliferating activity was determined by allogeneic mixed lymphocyte reaction invitro. 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)%] thanthat at day of 3rd[(68.667 ± 3.215)%, all P ＜0.05] with themagnetic activated cell sorting, but with adherent culture method, the difference was notstatistically significant (F = 0.737,P＞0.05) at days of3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,comparedwith the magnetic activated cell sorting, there were differences in cell viability ofadherent 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. Thedifference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]washigher 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 wassignificantly reduced(t = 5.467, P ＜ 0.05) at the 6th day ofculturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased thanthat 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 oflymphocytes, T lymphocytes proliferating activities were reduced. In the magneticactivated cell sorting, when the ratio of DCs and lymphocytes were 1: 50, 1: 100, cellsproliferation 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 culturemethod, 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 thesame, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Compariedwith the magnetic activated cell sorting, after culture of PBMC for 2 h the induction ofDCs 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. Key words: Dendritic cells; Cell culture techniques; Cytokines; Flowcytometry; Lymphocyte culture test,mixed