Scalable expansion of human pluripotent stem cells in suspension culture

Zweigerdt, Robert; Olmer, Ruth; Singh, Harmeet; Haverich, Axel; Martin, Ulrich

doi:10.1038/nprot.2011.318

Cited by 253 publications

(205 citation statements)

References 22 publications

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“…Industrializing these therapies will require the development of scalable and controlled suspension bioreactors for PSC‐based cell therapy manufacturing. The main culture methods used to translate laboratory adherent culture (Figure 1a‐i) to suspension‐based expansion of PSC are as cell aggregates in stirred suspension (Figure 1a‐ii; Amit et al, 2011; Kehoe, Jing, Lock, & Tzanakakis, 2010; Zweigerdt, Olmer, Singh, Haverich, & Martin, 2011) or using microcarriers as a substrate for cell attachment (Figure 1a‐iii; Chen, Chen, Choo, Reuveny, & Oh, 2011; Oh et al, 2009; Phillips et al, 2008). Aggregate‐based expansion methods provide simplicity and a reduction in the number of processing steps required.…”

Section: Introductionmentioning

confidence: 99%

Chemically controlled aggregation of pluripotent stem cells

Lipsitz

Tonge

Zandstra

2018

Biotech & Bioengineering

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Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical‐based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension‐based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.

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Section: Introductionmentioning

confidence: 99%

Chemically controlled aggregation of pluripotent stem cells

Lipsitz

Tonge

Zandstra

2018

Biotech & Bioengineering

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“…The stirred suspension culture technique is one promising method for the large-scale bioprocessing of pluripotent stem cells and maintaining their pluripotency (Amit et al, 2011;Badenes et al, 2015;Chen et al, 2015;Elanzew, Sommer, Pusch-Klein, Brüstle, & Haupt, 2015;Zweigerdt, Olmer, Singh, Haverich, & Martin, 2011). The aim of this study was to demonstrate the scalability of the technique in a singleuse stirred bioreactor (Kaiser, Eibl, & Eibl, 2011), and to develop a protocol for the expansion of billions of hiPSCs using stirred suspension culture vessels, while maintaining the undifferentiated, self-renewing state and pluripotency of the hiPSCs.…”

mentioning

confidence: 99%

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

Kwok

Ueda

Kadari

et al. 2017

J Tissue Eng Regen Med

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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.

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“…[22][23][24][25] These organizations have been shown to affect the secretion of ECM proteins and autocrine factors, producing an ECM microenvironment of distinct characteristics. 20,26,27 For example, deposition of endogenous transforming growth factor-beta inhibitor, Lefty, into ECMs has been reported for undifferentiated ESCs but not EBs, whereas the EBs displayed a unique ECM-shell structure and different cytokine secretion profiles compared to undifferentiated ESCs.…”

Section: Introductionmentioning

confidence: 99%

Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity

Sart

2014

Tissue Engineering Part A

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Embryonic stem cells (ESCs) emerge as a promising tool for tissue engineering and regenerative medicines due to their extensive self-renewal ability and the capacity to give rise to cells from all three-germ layers. ESCs also secrete a large amount of endogenous extracellular matrices (ECMs), which play an important role in regulating ESC self-renewal, lineage commitment, and tissue morphogenesis. ECMs derived from ESCs have a broader signaling capacity compared to somatic ECMs and are predicted to have a lower risk of tumor formation associated with ESCs. In this study, ECMs from undifferentiated ESC monolayers, undifferentiated aggregates, or differentiated embryoid bodies at different developmental stages and lineage specifications were decellularized and their capacities to direct ESC proliferation and differentiation were characterized. The results demonstrate that the ESC-derived ECMs were able to influence ESC proliferation and differentiation by direct interactions with the cells and by influencing the signaling functions of the regulatory macromolecules such as retinoic acid. Such matrices have the potential to present regulatory signals to direct lineage-and developmentspecific cellular responses for in vitro applications or cell delivery.

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Scalable expansion of human pluripotent stem cells in suspension culture

Cited by 253 publications

References 22 publications

Chemically controlled aggregation of pluripotent stem cells

Chemically controlled aggregation of pluripotent stem cells

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

Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity

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