Expansion of Human Neural Precursor Cells in Large-Scale Bioreactors for the Treatment of Neurodegenerative Disorders

摘要

The transplantation of in vitro expanded human neural precursor cells(hNPCs)represents a potential new treatment alternative for individuals suffering from incurable neurodegenerative disorders such as Parkinson's disease(PD)and Huntington's disease(HD).However,in order for cell restorative therapy to have widespread therapeutic significance,it will be necessary to generate unlimited quantities of clinical grade hNPCs in a standardized method.We report here that we have developed a serum-free medium and scale-up protocols that allow for the generation of clinical quantities of human telencephalon-derived hNPCs in 500-mL computer-controlled suspension bioreactors.The average KNPC aggregate diameter in the bioreactors was maintained below a target value of 500 mu m by controlling the liquid shear field.The human cells,which were inoculated at 10~5 cells/mL,exhibited a doubling time of 84 h,underwent a 36-fold expansion over the course of 18 days,and maintained an average viability of over 90%.The bioreactor-derived hNPCs retained their nestin expression following expansion and were able to differentiate into glial and neuronal pheno-types under defined conditions.It has also been demonstrated that these hNPCs differentiated to a GABAergic phenotype that has recently been shown to be able to restore functional behavior in rat models of HD and neuropathic pain(Mukhida,K.et al.Stem Cells 2007;DOI 10.1634/stemcells.2007-0326).This study demonstrates that clinical quantities of hNPCs can be successfully and reproducibly generated under standardized conditions in computer-controlled suspension bioreactors.