Cell culture design for homogeneous proliferation of cells in three-dimensional nonwoven polymer scaffolds

The objective of this study is to establish strategies to uniformly proliferate cells in a three-dimensional nonwoven polyethylene terephthalate (PET)/ethylene vinyl alcohol (EVOH) scaffold by simple adjustments in seeding and culture methods and the scaffold design. The combined dynamic and static seeding (intermittent agitations at 300 rpm with 1 h interval) resulted in the highest seeding efficiency (71%) comparing to the static and continuous agitating seeding methods. Cell-attached scaffolds were cultivated under different conditions. The stirring culture permitted cells to proliferate to a significantly greater extent than the static or agitating cultures, although faster cell proliferation in the outer region of the scaffold was observed. Next, based on this observation, scaffolds were opened with holes to alleviate the cell aggregation. The effect of hole size and number of scaffolds on the distribution of cells proliferated in the scaffold was evaluated. Two of 1-mm holes showed to be an optimal adjustment to allow cells to proliferate in a homogeneous manner. After 14 days culture, both of the holes were filled by cells proliferated with a fourfold increase in the cell number. The cell viability in the scaffolds was also high upon evaluating the live/dead and 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) staining examinations. Different cell types of 3T3-L1, C3H/10T1/2, and KUM6 cells showed similar behavior of cell proliferation and distribution in the scaffold, indicating the applicability of the established procedure. It is concluded that the nonwoven PET/EVOH scaffold serves as a potential cell culture substrate for an efficient cell proliferation.