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However, the lactic acid present in the conditioned medium could inhibit ESC growth and induce spontaneous differentiation when its concentration exceeded 1.5 g/l. In addition, the 3D static culture could be limited by oxygen, which was depleted in the long-term culture when cell density in the matrix was high. However, these problems can be alleviated in dynamic culture with improved oxygen transfer and continuous media perfusion. The matrix pore size also had profound effects on ESCs. The smaller-pore (30 -60 m) matrix gave a higher proliferation rate and Oct-4 and stage specific embryonic antigen-1 expressions. Overall, the 3D culturing method is superior to the 2D culture method and can provide an economical way to mass-produce undifferentiated ESCs in uncoated matrices and conditioned media. STEM CELLS 2007;25:447-454

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Three-dimensional (3D) cell cultures have many advantages over two-dimensional cultures. However, seeding cells in 3D scaffolds such as nonwoven fibrous polyethylene terephthalate (PET) matrices has been a challenge task in tissue engineering and cell culture bioprocessing. In this study, a centrifugal seeding method was investigated to improve the cell seeding efficiency in PET matrices with two different porosities (93% and 88%). Both the centrifugal force and centrifugation time were found to affect the seeding efficiency. With an appropriate centrifugation speed, a high 80-90% cell seeding efficiency was achieved and the time to reach this high seeding efficiency was less than 5 min. The seeding efficiency was similar for matrices with different porosities, although the optimal seeding time was significantly shorter for the low-porosity scaffold. Post seeding cell viability was demonstrated by culturing colon cancer cells seeded in PET matrices for over 5 days. The centrifugal seeding method developed in this work can be used to efficiently and uniformly seed small fibrous scaffolds for applications in 3D cell-based assays for high-throughput screening.

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