TY - JOUR
T1 - Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors
T2 - Bioprocess intensification and scaling-up approaches
AU - Aguiar, Tiago
AU - Costa, Rita
AU - Alves, Paula M.
AU - Aspegren,, Anders
AU - Alves, Patrícia Isabel
AU - Abecassis, Bernardo
AU - Arnault, Émilie
AU - Serra, Margarida
PY - 2017/3/20
Y1 - 2017/3/20
N2 - Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3 day−1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7 × 106 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells’ pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.
AB - Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3 day−1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7 × 106 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells’ pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.
KW - Aggregate dissociation
KW - Continuous expansion
KW - Human induced pluripotent stem cells
KW - Scale-up
KW - Stirred tank bioreactors
KW - Whole proteome
UR - http://www.scopus.com/inward/record.url?scp=85013073444&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2017.01.004
DO - 10.1016/j.jbiotec.2017.01.004
M3 - Article
C2 - 28131858
AN - SCOPUS:85013073444
SN - 0168-1656
VL - 246
SP - 81
EP - 93
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -