Foundation Of Suspension Culture System For Large-Scale Human Embryonic Stem Cell Production

In recent years,development of cell therapy has given hope for treating top diseases such as cancer,age-related diseases,organic damages and degenerative diseases.Human embryonic stem cells(h ESCs)hold great promise in the field of regenerative medicine due to their potential of differentiating into any functional cell type of our body.It is estimated that to recover organ function for organs like liver,kidney,pancreas,and heart etc.,109-1010 functional cells per patient will be needed.Therefore,large numbers of high-quality h ESCs as well as their derivations require to be produced.However,conventional 2D adherent cultures occupy large space to scale up h ESC production for example,about 100 T225 flasks can generate a yield of ~109 h ESCs,and that takes 3incubators..And it costs lots of money and people to culture massive high-quality h ESCs.Moreover,functional cells derived in 2D differentiation systems have shown lack of maturity and functioncal defects.Consequently,2D culture platform is not suitable for large-scale expansion and standard production of h ESC,while 3D suspension culture systems expansion and differentiation bring hope for cell therapyThough scientists have made many attempts to establish robust and economic h ESC suspension culture system,there are existing limitations including suboptimal passage methods and shear force caused by dynamic stirring.Here,we report an efficient and simple static 3D culture system that employs defined xeno-free E8 medium and single-cell inoculation.Our system enables long-term,serial expansion of h ESCs with a yield of ~1.5 × 109 cells per 1.5-L culture,while maintaining good pluripotency.This system can be used in large-scale h ESC production for cell therapy and pharmaceutics screening in the future.