The Research Of Human Amniotic Cells In Three-Dimensional Fibrin Matrices For Fetal Membrane Healing And Repairing After Membrane Rupture

Objective: Human amniotic cells were cultured in 3D fibrin matrix and observed whether it can generate well in three-dimensional fibrin matrices. Biological characteristics of human amniotic cells were investigated to pursue the new methods that the compounds of amniotic cells and fibrin scaffold are created to repair preterm rupture of the membrane.Methods: 1.The epithelial cells and mesenchymal cells were isolated from human amniotic membrane through primary culture. 2. Before fibrinogen was conglomerated, mesenchymal cells were mixed with fibrin solution .After that, epithelial cells were plated onto the surface of fibrin scaffold to simulate a three-dimensional culture in vivo. Then the amniotic cells and fibrin complex were created to mimic the architecture of native amnion. 3. The morphology and connection of epithelial cells on the fibrin scaffold were observed with scanning electron microscope. Cell ultrastructure and bundles of fibrin fibrils was visualized by transmission electron microscope. Cell morphology cultured on 3D fibrin gel was visualized by F-actin filaments incubated with rhodamine-labelled phalloidin. The reconstruction of the 3D system was performed using a stacked series of optical slices obtained by confocal laser scanning microscopy. 4. The growth curves of human amniotic cells which cultured in monolayer and 3D were drawn. The proliferation of amniotic cells under different culture conditions was measured. 5. The migration of the amniotic cells in fibrin gel was observed, and the construction of fibrin matrices were measured over a 15-days period and expressed as percentage of original gel thickness.Results: 1.The human amniotic cells were highly viable on the three-dimensional fibrin matrices. Cells are expanded along the scaffold and pseudopodia spread out with secretion functions well. The epithelial cells on the top of the 3D matrices grow on the same plane. While the mesenchymal cells embedded in 3D matrices were distributed in the different layer of the matrices. This model assumed cellular and structural composition is similar to those observed in vivo. 2. The human amniotic cells in monolayer culture demonstrated higher cell proliferation than in fibrin gel. 3. The mesenchymal cells could migrate and form cell groups in the fibrin clot. 4. In the 3D culture of amniotic mesenchymal cells, fibrin glue gradually shrank and gel thickness gradually decreased.Conclusion: 1.Human amniotic cells are highly viable on the fibrin scaffold and the amniotic cells interacted with the scaffold, which are similar to those observed in vivo.2. The compounds of amniotic cells and fibrin scaffold can be the biomaterials of fetal membrane defects healing and be a therapeutic strategy for preterm premature rupture of the membrane.