Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

Abstract

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 10 6 hiPSCs were expanded into 2 × 10 9 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

“…Expanding the LIF-dependent piPSCs line under dynamic conditions did not affect their pluripotency potential, as clearly confirmed by the sustained expression of OCT4, SOX2, cMYC and SSEA-4, which is consistent with previous reports on SSB culture of miPSCs (13) and hiPSCs (12). These piPSCs resemble hESCs in the expression of SSEA-4 and SSEA-1 (23), presumably due to the presence of LIF.…”

“…The purpose of this study was to establish an alternative, scalable culture method for the expansion of piPSCs using SSBs, based upon previous work involving human and murine PSCs (8; 9; 10; 11; 12; 13). Here we demonstrate that piPSCs from an established line and a newly generated line grown in static and SSB culture exhibit similar characteristics in terms of apparent doubling time, gene expression and differentiation potential, illustrating the utility of SSBs as an alternative culture method for the expansion of piPSCs.…”

Online measurement of oxygen enables continuous noninvasive evaluation of human‐induced pluripotent stem cell ( hiPSC ) culture in a perfused 3D hollow‐fiber bioreactor

“…In bioreactors inoculated with a low initial cell number, a more than 100‐fold expansion of hiPSCs was achieved during bioreactor cultures, which has rarely been reported by others; mostly, 3.5‐ to 12‐fold expansion rates have been described (Haraguchi, Matsuura, Shimizu, Yamato, & Okano, ; Meng, Liu, Poon, & Rancourt, ; Olmer et al, ; Rodrigues et al, ; Wang et al, ). To date, such high expansion yields as presented in this study have only been realized by Abecasis et al (), obtaining 10 10 hiPSCs and an 1100‐fold expansion rate within an 11‐day culture period in stirred‐tank bioreactors, and Kwok et al (), achieving a 125‐fold expansion rate and a cell number of 2 × 10 9 hiPSCs after 14 days of stirred suspension culture.…”

Large‐scale expansion of feeder‐free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation

“…Our future studies will focus on translating these protocols to hESC and hiPSC lines. Although human pluripotent cells are more challenging to work with, numerous authors have shown expansion and chromosomal normality following single‐cell inoculation (Abbasalizadeh, Larijani, Samadian, & Baharvand, 2012; Abecasis et al, 2017; Haraguchi, Matsuura, Shimizu, Yamato, & Okano, 2015; Kwok et al, 2018) and aggregate preformation (Hunt, Meng, Rancourt, Gates, & Kallos, 2014; Meng, Liu, Poon, & Rancourt, 2017) into stirred suspension bioreactors. As our lab has had success with translating bioprocess protocols from mouse to human stem cells in the past (Baghbaderani et al, 2008; Gilbertson, Sen, Behie, & Kallos, 2006) and are hopeful this can be replicated with our PSC work.…”

“…(c) Representative CFU plate (seeded with 500 cells taken from the end of the bioreactor serial passage) and magnified mESC colony stained with alkaline phosphatase 5 days after seeding. CFU, colony forming unit; ESC, embryonic stem cell; mESC, mouse ESC [Color figure can be viewed at wileyonlinelibrary.com] et al, 2017; Haraguchi, Matsuura, Shimizu, Yamato, & Okano, 2015;Kwok et al, 2018) and aggregate preformation (Hunt, Meng, Rancourt, Gates, & Kallos, 2014;Meng, Liu, Poon, & Rancourt, 2017) into stirred suspension bioreactors. As our lab has had success with translating bioprocess protocols from mouse to human stem cells in the past (Baghbaderani et al, 2008;Gilbertson, Sen, Behie, & Kallos, 2006) and are hopeful this can be replicated with our PSC work.…”