Gelatin/polycaprolactone Bioscaffold For Uterine Cell Culture And Its Potential Application Valu

Research backgroundThe uterus is divided into endometrial layer,basal layer and serous layer from inside to outside.The endometrium is mainly composed of the functional layer that changes periodically with the hormone and the basal layer that does not change with the hormone periodically.It mainly contains two cell components:epithelial and stromal cellsSecond paragraph.The muscular layer is thick and can be roughly divided into submucosa,intermediate layer and serosa from the inside to the outside.It is mainly composed of smooth muscle cells.The isolation and culture of primary uterine cells in vitro can establish a model to study uterus-related diseases,which is the basis of many in vitro experiments.The traditional two-dimensional(2D)cell culture method has great limitations.Although a large number of cells can be obtained in a short time,it is difficult to simulate the microenvironment of cell growth in vivo and the spatial interaction between cells.The research can only be limited to the cell level,which has become increasingly difficult to meet the needs of in vitro research.In recent years,three-dimensional(3D)cell culture has developed rapidly and has wide application value in tumor microenvironment,organoid,drug research and development and other fields.Three-dimensional model culture of cells in vitro can effectively simulate the migration,growth and information exchange of cells in space,which is closer to the environment of the uterus in the body,and can study the pathophysiology or pharmacological mechanism of related diseases by changing relevant factors.Three-dimensional culture of uterine cells has broad application prospects in the study of uterus-related diseases such as intrauterine adhesions(IUA),peritoneal disseminated leiomyoma(LPD).endometriosis(EMT).adenomyosis(AM)and other fields.It is a common method for three-dimensional cell culture to culture cells on scaffolds with specific spatial structure.Gelatin/polycaprolactone(GT/PCL)gradient biofilm has sufficient mechanical strength,good biocompatibility,no cytotoxicity,and the advantages of preventing cell invasion and infiltration.It has carried out in vitro fine cell culture in heart,cartilage,skin and other fields and applied to tissue engineering technology research.Therefore,we believe that the scaffold material can also play a role in the culture of uterine cells.Research objectiveTo isolate endometrial cells and uterine smooth muscle cells in vitro and co culture them with scaffolds,explore the feasibility of three-dimensional culture of uterine cells using GT/PCL gradient biofilm scaffolds as carriers.Research methodIsolation and culture of Human uterine smooth muscle cells(HSMC),endometrial cells including endometrial stromal cells(HESC)and endometrial glandular epithelial cells(HEEC)in vitro.The second generation cells were selected for cell identification and cell growth curve determination.The cells were inoculated on the GT/PCL gradient biofilm scaffold for culture,and the cell adhesion rate was estimated by counting with a cell counter at 4 hours after culture,and the cell adhesion was observed by two-photon microscopy at 12 hours after culture;Cck-8 kit was used to determine the activity of cells on the scaffold;The cells were cultured on the scaffold materials on the first day and the the fourth day day respectively,and the cell scaffold complex was photographed by SEM Immunofluorescence assay for cells cultured on scaffold for four days a The expression of actin,vimentin,and cytokerotin 18 protein was used to observe the adhesion and growth of cells on the scaffold,and to evaluate the feasibility of GT/PCL gradient layer biofilm as the scaffold for three-dimensional culture of uterine cells in vitro.Research resultsThis experiment successfully isolated,cultured and identified the primary endometrial glandular epithelial cells,endometrial stromal cells,and uterine smooth muscle cells in vitro.After being inoculated on the gelatin/polycaprolactone gradient layer biofilm,the cells can remain active and proliferate for a long time,and complex network structure can be seen between cells,which may be the establishment of spatial information exchange between cells;Cells are not limited to the surface of the scaffold,but also can grow into the interior of the scaffold.Therefore,scaffold materials can provide three-dimensional space for cell growth and better simulate the growth environment of cells in vivo.Immunofluorescence assay for the determination of marker proteins α-actin,vimentin and cytokerotin-18 of cells cultured on scaffolds for four daysto observe cell adhesion and growth on the scaffold,and evaluate the feasibility of using GT/PCL gradient layer biofilm as a three-dimensional culture scaffold for uterine cells in vitro.ConclusionGT/PCL gradient biofilm scaffolds can simulate the spatial microenvironment of cells within the body in vitro through electrospinning technology.Endometrial cells and uterine smooth muscle cells can adhere to and grow well on the GT/PCL gradient biofilm scaffold material.A possible information exchange network has been established between cells,and their own characteristics have not changed.Using GT/PCL biofilm scaffold as a carrier to three-dimensional culture uterine cells in vitro and design related disease models is feasible.