Objective: Using adipose-derived stem cells(ADSCs)to halt stress urinary incontinence(SUI)progression and regenerate urethral sphincter(US)has emerged as a promising method to restore normal voiding function.However,traditional two-dimensional(2D)cell culture systems have limitations in mimicking in vivo conditions losing some important properties.This study aims to examine the potential and mechanism of three-dimensional(3D)cultures of ADSCs in the treatment of SUI in a rat model simulating childbirth injury.Methods: ADSCs were used to generate micro-tissues(MTs)with a hanging drop method.A total of 48 postpartum Sprague-Dawley(SD)rats were developed SUI models by 4 h vagina dilation(VD)followed by bilateral ovariectomy(OV).Ten rats underwent sham OV without VD as control group.The SUI rats were divided into three groups and received urethral injection of PBS,ADSCs and MTs.specimens were harvested for histology and tracking CMDil-Labelled ADSCs at day 1,3,7(n=3),and 28(n=7)post-injection.At day 28,the remaining rats in each group were examined for voiding function.Western blot,immunofluorescence(IF)and immunohistochemistry(IHC)staining were performed to examine the histological change and cytokines.Results: Compared with ADSCs,MTs expressed increased vascular endothelia growth factor(VEGF),TNFα stimulated gene/protein 6(TSG-6)in vitro and represented a higher retention rate in vivo.The voiding function and histopathological recovery were significantly better in MTs group than ADSCs group.Conclusions: Urethral injection of MTs better restored the voiding function than ADSCs.The up-regulation of some bioactive factors and the improved cellular retention in the urethra may be responsible for the underlying mechanisms.