The Study Of Seeding Methods For Human Umbilical Cord Derived Mesenchymal Stem Cells Tissue Development In 3-Dimentional Matrix

In recent years, the promising prospect of human umbilical cord derived mesenchymal stem cells (hUCMSCs) for bone tissue engineering is indicated by numerous studies. The cell seeding method is an important factor which influences the bone tissue construct in 3-dimentional scaffold.To investigate the use of cycling perfusion seeding method for hUCMSCs bone tissue engineering.Primary hUCMSCs were cultured and passaged. The cell morphology was observed with an inverted light microscope. The fifth passage of cells were characterized by determining their surface antigen and then seeded onto 3-dimentional (3D) non-woven Polyethylene terephthalate (PET) disc scaffolds by static seeding method and cycling perfusion seeding method. The 3D disc seeded with cells were cultured for 18 days followed by 20 days of induced osteogenic differentiation. Cell damage were determined by measuring the LDH level during seeding process, cell number were estimated by CCK-8 assay to calculate the seeding efficiency and draw the growth curve. Alkaline phosphatase (ALP) activities of induced cells were measured using microplate reader. The experimental data were analyzed and compared between different seeding methods.The hUCMSCs of fifth passage were steady in the cell shapes, with good viability and highly expressed CD90, CD105 while were negative for CD14, CD45. The seeding efficiency, maximum cell number and ALP activity achieved by cycling perfusion seeding method is significantly higher than that achieved by static seeding method(P<0.05). LDH leakage and lag phase of cell growth kinetic were higher and longer in cycling perfusion seeding group compared with static seeding group, respectively. The data suggested that cycling perfusion seeding method is more suitable for hUCMSCs bone tissue engineering and further optimization of this method is needed.