Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction

Abstract

Abstract: Objective To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)-α during their induction of dendritic cell differentiation.Methods PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-α, given before or 30 min after pre-treatment with 0.5 μg/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells.Results PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-α for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-α.Conclusion s Under serum-free conditions, both CI and TNF-α are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-α.

CLC Number:

R392.3

WU Jun¹, WANG Xiao-huai², YANG De-mao², YANG Tai-cheng², WANG Jie², CHEN Zheng-liang¹. Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction[J]. Journal of Southern Medical University, 2004, 24(11): 1263-1266.

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