Various seeding methods for tissue development of human umbilical-cord-derived mesenchymal stem cells in 3-dimensional PET matrix

Mesenchymal stem cells derived from human umbilical cords (hUCMSCs) are attractive as a new cell source for tissue engineering. It is essential to investigate and optimize the seeding process of these cells for the success of cell culture and tissue regeneration in vitro. In this study, a static seeding method (SSM), a centrifugal seeding method (CSM), and a novel method-cycling filtration seeding method (CFSM) are evaluated in terms of seeding efficiency, cell damage, and distribution inside the scaffolds, cell proliferation, and osteogenic differentiation. Cells were seeded on three-dimensional (3-D) nonwoven PET discs at a density of 1×10⁴ cells/disc, followed by 21 days of cell culture and 20 days of osteogenic differentiation. Cells grown in 3-D conditions exhibited higher metabolic activity than those grown on a 2-D control surface. The CSM and CFSM groups showed higher seeding efficiency, proliferation capacity, and differentiation potential. H&E staining indicated a more uniform spatial distribution of cells in CFSM groups. LDH level measurements suggested that more cell damage was caused by the CFSM process. Above all, the results showed that the cells maintained their proliferation ability and differentiation potential ex vivo during approximately 7 weeks of culture. The CSM and CFSM are recommended for hUCMSC tissue engineering, although the seeding parameters still require further investigation and optimization.