Process engineering of human pluripotent stem cells for clinical application

Serra, Margarida; Brito, Catarina; Correia, Catarina; Alves, Paula M.

doi:10.1016/j.tibtech.2012.03.003

Cited by 268 publications

(271 citation statements)

References 79 publications

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“…Fold increase, FIPD and volumetric yield are parameters that have been used as indicators of growth rates of cells across different culture platforms (reviewed in Chen et al, 2014 andSerra, Brito, Correia, &Alves, 2012). Our workflow has an FIPD of 2.28 in a 125 ml spinner and an FIPD of 1.43 in the bioreactor when analyzed independently of each other.…”

Section: Quantification Of Spinner and Bioreactor Aggregatesmentioning

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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Section: Quantification Of Spinner and Bioreactor Aggregatesmentioning

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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“…However, these applications require large numbers of cells of high quality (4,(6)(7)(8). For instance, ∼10 5 surviving dopaminergic (DA) neurons, ∼10 9 cardiomyocytes, or ∼10 9 beta cells are likely required to treat a patient with Parkinson disease (PD), myocardial infarction (MI), or type I diabetes, respectively (9). Additionally, far more cells are needed initially because both in vitro cell culture yields and subsequent in vivo survival of transplanted cells are typically very low.…”

mentioning

confidence: 99%

“…As examples of the latter, only ∼6% of transplanted dopaminergic neurons or ∼1% of injected cardiomyocytes reportedly survive in rodent models several months after transplantation (10,11). Furthermore, there are large patient populations with degenerative diseases or organ failure (9), including over 1 million people with PD, 1-2.5 million with type I diabetes, and ∼8 million with MI in the United States alone (12). Large numbers of cells are also necessary for applications such as tissue engineering, where for example ∼10 10 hepatocytes or cardiomyocytes would be required for an artificial human liver or heart, respectively (6).…”

mentioning

confidence: 99%

“…Current 2D-based cell culture systems-which suffer from inherent heterogeneity and limited scalability and reproducibility-are emerging as a bottleneck for producing sufficient numbers of high-quality cells for downstream applications (9,16). An attractive approach for scaling up production is to move cell culture from 2D to 3D (9,17), and accordingly several 3D suspension systems have been probed for hPSCs production: cell aggregates (18)(19)(20)(21), cells on microcarriers (22,23), and cells in alginate microencapsulates (24) (SI Appendix, Table S1).…”

mentioning

confidence: 99%

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A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Lei

Schaffer

2013

Proc. Natl. Acad. Sci. U.S.A.

284

309

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Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human-or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 10 72 -fold expansion over 280 d), yield (∼2.0 × 10 7 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 10 7 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.H uman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) (1) and induced pluripotent stem cells (iPSCs) (2), have the capacities for indefinite in vitro expansion and differentiation into all cell types within adults (3). They therefore represent highly promising cell sources for numerous biomedical applications, such as cell replacement therapies (4, 5), tissue and organ engineering (6), and pharmacology and toxicology screens (7,8). However, these applications require large numbers of cells of high quality (4, 6-8). For instance, ∼10 5 surviving dopaminergic (DA) neurons, ∼10 9 cardiomyocytes, or ∼10 9 beta cells are likely required to treat a patient with Parkinson disease (PD), myocardial infarction (MI), or type I diabetes, respectively (9). Additionally, far more cells are needed initially because both in vitro cell culture yields and subsequent in vivo survival of transplanted cells are typically very low. As examples of the latter, only ∼6% of transplanted dopaminergic neurons or ∼1% of injected cardiomyocytes reportedly survive in rodent models several months after transplantation (10, 11). Furthermore, there are large patient populations with degenerative diseases or organ failure (9), including over 1 million people with PD, 1-2.5 million with type I diabetes, and ∼8 million with MI in the United States alone (12). Large numbers of cells are also necessary for applications such as tissue engineering, where for ex...

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“…6,7 Due to their capacity for long-term self-renewal and multipotent differentiation, human iPSCs are highly promising cell sources for a wide range of biomedical applications, including disease mechanism research, tissue/organ engineering, cellular replacement therapies, and pharmacology and toxicology screening. [8][9][10] The capacity of hiPSCs to differentiate into particular cellular lineages is undergoing intensive study. Recently, hiPSCs have been differentiated into cardiomyocytes, pancreatic b-like cells, endothelial cells, and other cell types.…”

Section: Introductionmentioning

confidence: 99%

Human induced pluripotent stem cells derived endothelial cells mimicking vascular inflammatory response under flow

et al. 2016

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Endothelial cells (ECs) have great potential in vascular diseases research and regenerative medicine. Autologous human ECs are difficult to acquire in sufficient numbers in vitro, and human induced pluripotent stem cells (iPSCs) offer unique opportunity to generate ECs for these purposes. In this work, we present a new and efficient method to simply differentiate human iPSCs into functional ECs, which can respond to physiological level of flow and inflammatory stimulation on a fabricated microdevice. The endothelial-like cells were differentiated from human iPSCs within only one week, according to the inducing development principle. The expression of endothelial progenitor and endothelial marker genes (GATA2, RUNX1, CD34, and CD31) increased on the second and fourth days after the initial inducing process. The differentiated ECs exhibited strong expression of cellsspecific markers (CD31 and von Willebrand factor antibody), similar to that present in human umbilical vein endothelial cells. In addition, the hiPSC derived ECs were able to form tubular structure and respond to vascular-like flow generated on a microdevice. Furthermore, the human induced pluripotent stem cell-endothelial cells (hiPSC-ECs) pretreated with tumor necrosis factor (TNF-a) were susceptible to adhesion to human monocyte line U937 under flow condition, indicating the feasibility of this hiPSCs derived microsystem for mimicking the inflammatory response of endothelial cells under physiological and pathological process. V C 2016 AIP Publishing LLC. [http://dx

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Process engineering of human pluripotent stem cells for clinical application

Cited by 268 publications

References 79 publications

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

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

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Human induced pluripotent stem cells derived endothelial cells mimicking vascular inflammatory response under flow

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