The Primary Culture And 3-Dimensional Culture On Collagen â…  Scaffold Of Human Amnion Cells For Fetal Membrane Healing

Objective: To establish the technique for isolation and culture of pure amniotic epithelial cells and mesenchymal cells; To study the most potent proliferation-inducing medium for amniotic cells.; To establish amniotic cells/collagenⅠcomplex for the purpose of fetal membrane healing.Methods:1. Amniotic epithelial cells and mesenchymal cells were separated and purified enzymatically.Cell types are characterized immuocytochemistry. 2. Proliferation of amniotic cells in four different media was measured photometrically.(MediumⅠ:DMEM /F12+10%FBS+50.0ng/mlEGF+2.5ug/ml insuline+5ug/ml transferrin+0.1ng/ml T3;mediunⅡ:DMEM /F12+10%FBS;mediumⅢ:DMEM /F12+50.0ng/mlEGF+2.5ug/ml insuline+5ug/ml transferrin+0.1ng/ml T3;mediumⅣ:DMEM /F12+50.0ng/mlEGF). 3. Amnion mesenchymal cells were embedded in three-dimensional(3D) collagenⅠ, and epithelial cells were placed on top of it. The amniotic cells/collagenⅠcomplex were created to mimic the architecture of native amnion, 4. As judged by DAPI staining, the number of amniotic cells cultured in 3D collagen I was counted after 2 and 8 days culture. Cell morphology cultured on 3D collagen I was visualized by F-actin filaments incubated with rhodamine-labelled phalloidin. Cell ultrastructure was visualized by SEM and TEM. 5. Collagen I with different quantities of amniotic mesenchymal cells was measured of the tensile strength after 15 days culture.Results: 1. Human amniotic cells can be successfully isolated by trypsin and collagenase respectively, and the cells can proliferate and passaged in vitro. 2. Amniotic mesenchymal cells proliferated only with fetal bovine serum (FBS). Proliferation of amnion cells in medium containing FBS, epithelial growth factor (EGF), insulin, transferring and triidothyronine(T3) was significantly increased compared with the other three media(p < 0.001). (AMEs: mediumⅠ:1.130±0.203;mediumⅡ:0.487±0.141;mediumⅢ:0.466±0.094;mediumⅣ:0.451±0.144;AMCs: mediumⅠ: 1.356±0.173 ; mediumⅡ: 0.930±0.112 ; mediumⅢ:0.200±0.053;mediumⅣ:0.190±0.029;). 3. Preterm amnion epithelial and mesenchymal cells cultured in collagen I matrices assume cell morphologies similar to those observed in vivo. Amniotic cells was not significantly increased with increasing three-dimensional culture time. Mesenchymal cells were capable of remodelling collagen I, as seen by extensive volume contraction, and could not be inhibited by GM6001 and/or aprotinin. 4. The tensile strength of amniotic cells/collagenⅠscaffold complex was strengthened with the increase of amnion mesenchymal cells (P<0.001).Conclusion: 1. Human amniotic epithelial and mesenchymal cells can be isolated and proliferated in vitro. These findings suggest that amnion cells may be a practical cell source for cell therapy and tissue engineering. 2. CollagenⅠmight be useful in amnion cell tissue engineering, Three-dimensional cell-matrix system might be useful in the study and be a therapeutic strategy for preterm premature rupture of the membrane.