Human embryonic stem cells derived from human blastocysts have the capability to proliferate extensively and differentiate into any cell type. The cells differentiated from hES cells can be transplanted to restore tissue function in a variety of human diseases such as cardiac infarcts, leukemia, Parkinson's disease, etc. To exploit the clinical applications of hES cells, their mass production is a necessary and important step. Traditionally hES cells are cultured on Matrigel pre-coated surface and frequent subcultures are required in order to maintain their undifferentiated state. These culture methods are expensive, labor intensive and inappropriate for the mass production of hES cells. In order to overcome these limitations, the expansion of undifferentiated hES cells by culturing them in a three-dimensional (3D) polyethylene-terephthalate scaffold was studied. It was found that hES cells grown in the 3-D scaffold did not require Matrigel or any other type of ECM coating in a feeder-free culture system using a conditioned medium. In addition, the conditioned medium from human foreskin fibroblast was used to replace the conditioned medium from MEF, which increases the risk of transferring murine pathogens to hES cells. Compared to conventional 2-D culturing systems such as tissue flasks, higher cell growth rate and expansion fold and better maintenance of pluripotency of hES cells were obtained in the fibrous bed bioreactor, demonstrating that the FBB is advantageous for mass production of undifferentiated hES cells. Repeated batch culture and perfusion culture systems were also developed to further improve the long-term expansion of hES cells in the FBB.