Cultivation and idenification of decidual dendritic cell in URSA

Objective To develop a method for the cultivation of human decidual dendritic cells from patients with an unexplained recurrent spontaneous abortion(URSA).Methods Between July 2008 and April 2009,decidual tissues from URSA patients at week-7 to 14 of gestation were collected.The tissues were dispersed mechanically.Mononuclear cells were released by Ⅳ collagenase/DNA-Ⅰ digestion and the cells were subsequently isolated using density gradient centrifugation.DCs were then treated with GM-CSF and IL-4.The cell suspension was gently collected at day-10 after incubation.The cells were then stained with FITC-HLADR and PE-Lin,and analyzed by flow cytometric method.Results Decidual dendritic cells were obtained from the freshly collected decidual tissues..Microscopic study indicated that the adherent cells gradually form small colony in the suspension.At day-9,most of the cells were in single cell suspension.The cells were still in small and round morphology.The formation of dendritic processes was not apparent.The culture consisted of a mixture of small slender spindle-shaped macrophages and DCs.At day-10,the purity of DCs was above 80%.Conclusion The method using enzyme digestion and gradient centrifugation can successfully separate decidual dendritic cells from the tissues obtained from URSA patients.GM-CSF and IL-4 can promote the growth of the immature dendritic cells.The dendritic cells may be used the study the maternal immunity and immunological tolerance of the fetus in URSA patients.