The Protective Effect Of Umbilical Cord Mesenchymal Stem Cells In Three-dimensional Microcapsule Culture On Endometrial Injury

Objective: 1.To develop a three dimensional(3D)microcapsule culture system,explore the effects of various parameters on the size and morphology of the microcapsules,and further explore the conditions for stable and efficient 3D microcapsule h UC-MSCs culture.2.To explore the effect of different culture methods of h UC-MSCs on the migration and apoptosis of the endometrial stromal cells(ESCs).Methods: 1.The 3D microencapsulation culture system was used to conduct experiments on parameters such as voltage and sodium alginate and summarized the most suitable parameters for the formation of the microcapsules.Based on the above results,h UCMSCs were encapsulated to choose the optimal h UC-MSCs microcapsules formation parameters and to detect the growth status of the cells in the microcapsules.2.ESCs were isolated and cultured,and the morphology of endometrial cells was observed.The damaged ESCs model was established by the use of the mifepristone.The ESCs were treated with 60 ul/ml mifepristone for 48 hours to observe the changes in cell morphology.CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of the damaged ESCs.3.The non-contact co-culture of the damaged ESCs with h UC-MSCs under traditional adherent culture(2D-h UC-MSCs)and 3D culture(3D-h UC-MSCs)were performed through the transwell chamber.After 48 hours of co-culture,the migration of the damaged ESCs was detected by the scratch test,and the survival and apoptosis of the damaged ESCs were detected by flow cytometry.Results: 1.The diameter of the microcapsules decreased with increasing voltage,and the concentration of sodium alginate mainly affected the microcapsule morphology and uniformity.When encapsulating stem cells,the microcapsule morphology and encapsulation rate were mainly related to cell concentration.When the concentration was too large,it was easy to block the needle,which affected the uniformity and diameter of the microcapsules.If the concentration was too small,more cell-free encapsulated microcapsules will be produced,which will affect the encapsulation efficiency of the microcapsules.The proliferation capacity of h UC-MSCs cultured by uniaxial microencapsulation was restricted.HUC-MSCs cultured in the coaxial microencapsulated culture system had better proliferation ability and survival status,and the cell growth in the capsule showed a tissue-like structure.By optimizing the conditions,we established a microencapsulated culture system with an encapsulation rate of about 95%,a regular microcapsule morphology,a suitable size,and a suitable amount of encapsulated cells.Through the in vitro culture of microencapsulated stem cells,we found that the proliferation of stem cells was significant.Cell proliferation increased significantly after 4 days of culture,and cells within the microcapsule showed a similar tissue-like structure after 6 days of culture.2.After treating mifepristone 48 hours,the morphology of ESCs changed significantly,the proliferation of the ESCs decreased,and apoptosis increased(P < 0.05).The injury of the ESCs induced by mifepristone can be reduced to a certain extent by co-culture with h UC-MSCs under different culture methods.After 48 hours of co-culture,the migration of the damaged ESCs was increased(P < 0.05),and the effect of 3D-h UC-MSCs was better than that of 2D-h UC-MSCs(P = 0.002).2D-h UC-MSCs could increase the survival of the damaged ESCs,but the difference was not statistically significant(P = 0.051).3Dh UC-MSCs could increase the survival of the damaged ESCs,and the difference was statistically significant(P < 0.001).The apoptosis of the damaged ESCs was decreased by co-culture with h UC-MSCs under both culture methods,and the effect of 3D-h UCMSCs was better than that of 2D-h UC-MSCs(P < 0.001).Conclusions: 1.The coaxially microencapsulated culture system is a great choice for the stem cell therapy,which has the advantages of fast reaction speed,stable process,high encapsulation rate,regular morphology and appropriate size of microcapsules.2.A suitable damaged ESCs model can be established by isolating the ESCs and treating ESCs with the mifepristone.The protective effect of 3D-h UC-MSCs on damaged ESCs is more significant than that of 2D-h UC-MSCs,and 3D-h UC-MSC can be a great choice for stem cell therapy.