Preparation Of BDM Scaffold "Artificial Amniotic Membrane" And Preliminary Experimental Study Of Three-dimensional Cultivation

Objective:Objective to explore the use of human amniotic mesenchymal stem cells(hAMSCs)and biological matrix in vitro prefabrication of"artificial amnion",and"artificial amnion"in vitro three-dimensional culture.Methods:The method of double enzyme digestion adpots separate and extract hmamscs,which were cultured and purified to P3 generation,and identified by flow cytometry.The method adpots prepare"artificial amnion"by mixing with BD matrix(BDM).The stem cells were combined with BD matrix glue in a volume ratio of 1:1 to make a circular membrane with a bottom area of 2cm~2,a thickness of about 3-5mm and a concentration of 3×10~5/ml,i.e."artificial amniotic membrane",which was cultured in a three-dimensional culture system(experimental group),at the same time,the stem cells with a concentration of 3×10~5/ml and no BD matrix glue were placed in the same culture system(control group).On the 1st,4th and 7th day of stem cell culture,the growth and morphological changes of the cells were observed under ordinary light microscope.The AnnexinV-FITC/PI apoptotic kit was adopted to stain hAMSCs in the experimental group and the control group.The flow cytometer(FCM)was applied to detect the hAMSCs.The proportion of AnnexinV-FITC/PI positive cells was calculated Apoptosis rate.The ability of the two groups of cells to secrete vascular endothelial growth factor(VEGF)was detected by ELISA kit.Results:Cell morphological observations revealed that the mesenchymal stem cells in the control group began to grow completely adherent about 48 hours after primary culture,and the degree of fusion reached more than 90%at 7 days,which was uniformly spindle-shaped to the third generation;mesenchymal stem cells in the experimental group showed aggregated spheroid growth;most of the two groups of cells showed good growth,and floating cells were rare.After Annexin-V staining,the results of flow cytometry showed that the apoptosis rates of the experimental group and the control group were 4.91%and 2.91%,respectively,and the difference was statistically significant(P<0.05).ELISA results showed that the secretory quantity of VEGF in mesenchymal stem cells in the experimental group was not statistically different from that in the control group(P>0.05).Conclusion:Human amniotic mesenchymal stem cells can survive in the"artificial amnion"with BDM as the scaffold,and have a good ability to secrete VEGF.