Progress and challenges in large-scale expansion of human pluripotent stem cells
TL;DR: Recent progress in the large-scale expansion of hPSCs with particular focus on suspension culture, which represents a universal strategy for controlled mass cell production, and bioreactor-based approaches, including technical aspects ofBioreactor technologies and operation modes are reviewed.
About: This article is published in Process Biochemistry.The article was published on 2017-08-01 and is currently open access. It has received 123 citations till now.
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01 Apr 2008
TL;DR: In this paper, the authors discuss applications of CFD for tissue engineering-related bioreactors and conclude that CFD should be seen as an invaluable tool for analyzing and visualizing the impact of fluidic forces and stresses on cells and TECs.
Abstract: The complex relationship between the hydrodynamic environment and surrounding tissues directly impacts on the design and production of clinically useful grafts and implants. Tissue engineers have generally seen bioreactors as 'black boxes' within which tissue engineering constructs (TECs) are cultured. It is accepted that a more detailed description of fluid mechanics and nutrient transport within process equipment can be achieved by using computational fluid dynamics (CFD) technology. This review discusses applications of CFD for tissue engineering-related bioreactors -- fluid flow processes have direct implications on cellular responses such as attachment, migration and proliferation. We conclude that CFD should be seen as an invaluable tool for analyzing and visualizing the impact of fluidic forces and stresses on cells and TECs.
159 citations
TL;DR: It is shown that phagocytes similar to primitive macrophages can be generated from human induced pluripotent stem cells, by the use of industry-compatible, stirred-tank bioreactors, and applied as a cell-based therapy to treat acute bacterial infections in mice.
Abstract: The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies. Pulmonary infections constitute a substantial health problem worldwide. Here the authors show that phagocytes similar to primitive macrophages can be generated from human induced pluripotent stem cells, by the use of industry-compatible, stirred-tank bioreactors, and applied as a cell-based therapy to treat acute bacterial infections in mice.
95 citations
TL;DR: Different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs are discussed, to enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of h PSCs thereby increasing their potential applications.
Abstract: Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.
92 citations
Cites background from "Progress and challenges in large-sc..."
...The key parameters such as pH, dissolved oxygen (DO) levels, fluid shear stress, growth factors, nutrients, and metabolite concentrations in hPSC scale-up culture are precisely and carefully controlled to ensure a uniform environment with adequate nutrient levels and oxygenation [129]....
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TL;DR: Based on their success and other significant research accomplishments, platforms of innovation were identified and will enable tissue engineering inspired therapies to meet the requirements necessary for large-scale manufacturing and Food and Drug Administration approval for a diverse range of indications.
Abstract: In the past 25 years, the tissue engineering field has made incredible strides in developing tangible therapies for patients in need. Applications of the tissue engineering paradigm, involving vary...
77 citations
TL;DR: The factors driving concerted research endeavors in each of the main classes of bioreactors are briefly examined and the major advancements that have been reported in the last three years are described.
Abstract: Bioreactors have become indispensable tools in the cell-based therapy industry. Various forms of bioreactors are used to maintain well-controlled microenvironments to regulate cell growth, differentiation, and tissue development. They are essential for providing standardized, reproducible cell-based products for regenerative medicine applications or to establish physiologically relevant in vitro models for testing of pharmacologic agents. In this review, we discuss three main classes of bioreactors: cell expansion bioreactors, tissue engineering bioreactors, and lab-on-a-chip systems. We briefly examine the factors driving concerted research endeavors in each of these areas and describe the major advancements that have been reported in the last three years. Emerging issues that impact the commercialization and clinical use of bioreactors include (i) the need to scale up to greater cell quantities and larger graft sizes, (ii) simplification of in vivo systems to function without exogenous stem cells or growth factors or both, and (iii) increased control in the manufacture and monitoring of miniaturized systems to better capture complex tissue and organ physiology.
57 citations
References
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TL;DR: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages.
Abstract: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.
15,555 citations
"Progress and challenges in large-sc..." refers background in this paper
...The cumbersome co-culture of hPSC colonies grown on mitotically inactivated fibroblasts (“feeder cells”) – as initially described for their routine maintenance [26,27] – has been largely replaced by semi- or fully-defined matrices such as Matrigel [26], recombinant proteins (such as laminins [28]), or synthetic polymers [29]....
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Stanford University1, Harvard University2, University of Florida3, University of Washington4, University of Texas Medical Branch5, University of Colorado Denver6, University of Texas Southwestern Medical Center7, University of Rochester8, University of Pittsburgh9, University of Toronto10, University of California, San Francisco11, Loyola University Chicago12, Washington University in St. Louis13, Rutgers University14
TL;DR: This study shows that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another.
Abstract: A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R2 between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.
2,438 citations
TL;DR: It is believed that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.
Abstract: Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.
2,413 citations
"Progress and challenges in large-sc..." refers background in this paper
...ent state while overcoming the necessity of external matrix upplementation [38,39] (Fig....
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TL;DR: Application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency and facilitates subcloning after gene transfer, and enables SFEB-cultured hES Cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors.
Abstract: Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor1,2, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from ∼1% to ∼27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture3 and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.
2,094 citations
"Progress and challenges in large-sc..." refers background in this paper
...However, following the finding that the small molecule Y27632 (a Rho-associated coiled-coil kinase (ROCK) inhibitor) temporarily permits survival of single cell-dissociated hESCs [62], we and other researchers showed the robust multipassage expansion of undifferentiated hPSCs rigorously propagated as single cells in suspension [44,56,57]....
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TL;DR: A cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined is formed, and improved derivation efficiencies of vector-free human iPSCs with an episomal approach are demonstrated.
Abstract: We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
1,330 citations
"Progress and challenges in large-sc..." refers background in this paper
...reported “essential 8” (E8), a xeno-free and ompletely defined culture medium, for hPSC cultivation [99]....
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