Expansion of embryonic stem cells in suspension and fibrous bed bioreactors

• Embryonic stem (ES) cells were cultured in suspension as aggregates or in a fibrous matrix.
• Suspension cultures with cell aggregates require frequent passaging to avoid mass transfer limitation.
• The fibrous matrix system could expand ES cells to a high density with minimal passaging.
• The process is scalable and promising for mass production of ES cells.

Applications of embryonic stem (ES) cells in cellular transplantation and tissue engineering require scalable processes for mass production of these cells with controlled qualities. The main objective of this work was to evaluate two cell culture processes for long-term expansion of murine embryonic stem (mES) cells. With serial passaging, suspension cultures in spinner flasks were able to expand mES cells as aggregates for 12.5-fold in each passage of 4 days. However, extending the culturing time to 6 days in each passage caused significant loss in cell viability and induced differentiation as indicated by the reduced expression levels of SSEA-1 and Oct-4. Long-term expansion of mES cells in a fibrous bed bioreactor (FBB) was also studied for 30 days in 2 passages, 15 days in each passage. With periodically refreshing the culture medium, a high expansion fold of 60–77 was achieved in each passage. Flow cytometry and RT-PCR were used to analyze key pluripotency and differentiation markers. The results showed that the expanded cells in both suspension and FBB cultures remained in a highly pluripotent state, which was also confirmed with the embryoid body (EB) forming efficiency test. It is concluded that both the suspension and FBB cultures are suitable to support long-term expansion of undifferentiated mES cells. However, the FBB culture can sustain cell growth for a longer period without frequent passaging, requires less media and labor, and is thus more economical to use for mass production of ES cells.