AbstractsEngineering

Characterized by the unlimited self-renewal and the capability of differentiating into all cell types, embryonic stem (ES) cell is an attractive cell source for applications ranging from cell therapy, drug discovery to tissue engineering. The realization of these great applications such as the treatment of a variety of degenerative diseases including Parkinson’s disease, Alzheimer’s disease, heart failure and diabetes requires access to a large quantity of quality-controlled cells. However, the current culture techniques for ES cell expansion and differentiation are expensive, labor-intensive and can not meet the high demand for large numbers of stem cells needed in clinical applications. To overcome these limitations, several different culture systems, including suspension culture in spinner flasks, immobilized culture in fibrous bed bioreactors (FBB) and fibrous microcarrier culture in stirred-tank bioreactors were developed for expansion and differentiation of ES cells. By controlling the culture conditions such as the agitation rate, a suspension culture system in spinner flasks was established for ES cell expansion as aggregates. Four serial passages were studied over a culture period of 16 days in suspension culture and the system was confirmed to be reproducible. However, frequent subculturing is required to maintain the pluripotency of ES cell. Three-dimensional (3-D) culture of ES cells was studied within non-woven fibrous polyethylene terephthalate (PET) scaffolds, which not only provide a large specific surface area for cells to grow onto but also mimic more in vivo-like 3-D microenvironment, and protect cells from detrimental shear stress in bioreactors. Two different 3-D immobilized culture systems, FBB and fibrous microcarrier stirred cultures were developed using PET matrices as cell supports for ES cell cultures. It was demonstrated that the FBB culture can be extended to a longer period of 15 days without subculturing and a final high cell density of above 400 million cells/mL matrix volume was achieved for the purpose of ES cell expansion. In addition, neural differentiation of ES cells was investigated in the FBB, and the results showed that the 3-D culture can improve the differentiation efficiency as compared to the 2-D culture in tissue culture plates. The results suggested that the FBB culture system represents a simple, economical, and scalable process for mass production of ES and their derivative cells.Furthermore, PET fibrous matrices were first introduced as microcarriers into stirred-tank bioreactors for ES cell expansion and differentiation. The results suggested that the culture system was also capable of supporting high density ES cell culture. In addition, long-term integrated expansion and neural differentiation of ES cells with an improved differentiation efficiency was accomplished in the system. The culture system can be further improved for ES cell expansion and differentiation by optimizing the operating parameters, such as the microcarrier concentration, medium renewal…