Application Of Microfluidic Technology To Establish Endometriosis In Vitro Cell Coculture Model And The Investigation Of Endometrial Stromal Cell Migration And Invasion

BackgroundEndometriosis is a common disease of the reproductive aged women, the incidence of it is rising recently, the disease performance devisity, the invasion, distant metastasis, recurrence characters such as malignant tumor behavior, so far the pathogenesis of it is unclear and treatment measures for it is limited.To establish a good Mode is very important in studying endometriosis, although many models have been successfully constructed, but there are still some insufficiency. Such as rabbit and rat models are regarded not suitable for lack of menstrual cycle. The ideal models of macaque or baboon are rare and too expensive. In the cell model, one kind of cell culture can not simulate the in vivo interaction of multiple cell method, In vitro methods for investigating interaction between multiple types of cells, however, are very limit, Boyden chambers (or called Transwell) are commercial available produce for researching interactions between only two types of cells, but it is hard to get real time observation of interactions between two types of cellsIn this study, we are in cooperation with the Chinese Academy of Nano-Science Center, they have used micro-nano technology to establish various methods for patterning multiple types of cells which have a large potentiality for application to cell-cell interaction models. However, the model currently applied in cell biology rarely reported. Using this model, we can observe the procession of endometrial stromal cells in reflux of blood attaching and invasng peritoneal mesothelial cells.Objective1,To establish endometriosis endometrial stromal cells, peritoneal mesothelial cells in vitro co-culture model. 2,Using this model, to observe the endometrial stromal cells and peritoneal mesothelial cell movement, migration and invasion from endometriosis and non-endometriosis patients, to compare the cell cocultured behavior of endometriosis and controls.3,Time-lapse phase-contrast micrographs for cells interaction.Methods1,Isolated and cultured primary endometrial stromal cells (ESC) and human peritoneal mesothelial cells (HPMC)2,Designed and prepared PDMS stamps3,Using stamps in healing test for endometrial stromal cells and peritoneal mesothelial cells from endometriosis and non-endometriosis4,Crossed coculture and immunofluorescence of endometrial stromal cells and peritoneal mesothelial cells from endometriosis and non-endometriosis, photographed every 24 hours then compared their features.5,Time-lapse phase-contrast micrographs for coculture ESC and HPMC interaction.6,Try to use this model to investigate the motility of ESC and HPMC after 17βestradiol, progesterone or TriptorelinResults1,When the channels width and height of 300μm, the channel spacing of 500μm, endometrial stromal cell density 5×106/l, peritoneal mesothelial cells in 3×105/l, cells grew well in the channels and adhered to solid plastic within 2 hours.2,Peritoneal mesothelial cells moved faster than endometrial stromal cells. When the channel width of 600μm, endometrial stromal cells completely healed in 93 hours,while peritoneal mesothelial cells healed in 24 hours, there was no significant difference between endometriosis and non-endometriosis group3,Interaction between ESC cells (ESC em) and HPMC cells (HPMC em) from endometriosis, ESC em cells invaded into areas of HPMC em cells which died and disappeared.4,Interaction between ESC cells (ESC em) from endometriosis and HPMC cells (HPMC con) from controls, and HPMC con cells invaded into areas of ESC em cells which died and almost disappeared5,Interaction between ESC cells (ESC con) from control and HPMC cells (HPMC em) from endometiosis, ESC con cells also invaded into areas of HPMC em cells and. HPMC em cells almost disappeared.6,Interaction between ESC cells (ESC con) f and HPMC cells (HPMC con) from control, cells grew in the areas of themselves, invasion was not be observed.7,Distance of ESC movement between study and control group were significantly different when 5nM, 10nM 17βestradiol,5 x 10-7g/ml Triptorelin. Progesterone has no effect an it.17βestradiol when 10nM, may accelerate ESC and cocultur cells movement.Conclusion1,The subject of applications of microfluidic technology successfully established endometriosis in vitro cell coculture model. Use this model we can get real time observation of interactions between two or more types of cells.2,Endometrial stromal cells have invasive capacity to peritoneal mesothelial cells, which is in accordance with clinical manifestations and histological observation.3,The peritoneal state has a certain correlation in the pathogenesis of endometriosis4,Estrogen has effect on endometrial stromal cells movement, but need advanced test.