BACKGROUND AIMS:The appearance of genetically variant populations in human pluripotent stem cell (hPSC) cultures represents a concern for research and clinical applications. Genetic variations may alter hPSC differentiation potential or cause phenotype variation in differentiated cells. Further, variants may have properties such as proliferative rate, or response to the culture environment, that differ from wild-type cells. As such, understanding the behavior of these variants in culture, and any potential operational impact on manufacturing processes, will be necessary to control quality of putative hPSC-based products that include a proportion of variant threshold in their quality specification.METHODS:Here we show a computational model that mathematically describes the growth dynamics between commonly occurring genetically variant hPSCs and their counterpart wild-type cells in culture.RESULTS:We show that our model is capable of representing the growth behaviors of both wild-type and variant hPSCs in individual and co-culture systems.CONCLUSIONS:This representation allows us to identify three critical process parameters that drive critical quality attributes when genetically variant cells are present within the system: total culture density, proportion of variant cells within the culture system and variant cell overgrowth. Lastly, we used our model to predict how the variability of these parameters affects the prevalence of both populations in culture.

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation‐based model for prediction of T‐cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T‐cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 106 cell h−1) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost‐effective T‐cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T‐cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.

Cell therapy manufacturing is limited by lack of online tools capable of realtime in-process monitoring, particularly of simultaneous changes in multiple orthogonal (mutually independent) parameters. Here, we studied changes in CD36 expression, number density and size (area) of erythroblasts through different stages of erythropoiesis in vitro using a quartz crystal resonator (QCR), integrated with a microscope, and flow cytometry in parallel. An analytical model was developed extending the Kanazawa-Gordon theory. Based on this model, independent correlations were established between changes in each QCR parameter, dissipation (ΔΓ) and resonance frequency (−Δf0), and CD36 expression (from flow cytometry) and cell area (from microscope). The correlation functions were used to derive an acoustic signature (−ΔΓ/Δf0) of the differentiation process that uniquely mapped the relative changes in CD36 expression and late-stage enucleation-related deviations. A method to quantify relative changes in cell area purely from the acoustic parameters was also proposed. This work demonstrated for the first time the potential of an electromechanical tool for online monitoring of concurrently varying orthogonal phenotypic parameters in cell therapy manufacturing.

Manufacture of red blood cells (RBCs) from progenitors has been proposed as a method to reduce reliance on donors. Such a process would need to be extremely efficient for economic viability given a relatively low value product and high (2x10(12)) cell dose. Therefore, the aim of these studies was to define the productivity of an industry standard stirred-tank bioreactor and determine engineering limitations of commercial red blood cells production. Cord blood derived CD34+ cells were cultured under erythroid differentiation conditions in a stirred micro-bioreactor (Ambr (TM)). Enucleated cells of 80% purity could be created under optimal physical conditions: pH7.5, 50% oxygen, without gas-sparging (which damaged cells) and with mechanical agitation (which directly increased enucleation). O-2 consumption was low (similar to 5x10(-8) mu g/cell.h) theoretically enabling erythroblast densities in excess of 5x10(8)/ml in commercial bioreactors and sub-10 l/unit production volumes. The bioreactor process achieved a 24% and 42% reduction in media volume and culture time, respectively, relative to unoptimized flask processing. However, media exchange limited productivity to 1 unit of erythroblasts per 500 l of media. Systematic replacement of media constituents, as well as screening for inhibitory levels of ammonia, lactate and key cytokines did not identify a reason for this limitation. We conclude that the properties of erythroblasts are such that the conventional constraints on cell manufacturing efficiency, such as mass transfer and metabolic demand, should not prevent high intensity production; furthermore, this could be achieved in industry standard equipment. However, identification and removal of an inhibitory mediator is required to enable these economies to be realized. Copyright (c) 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Manufacture of Red Blood Cell based products in vitro requires highly efficient erythroblast culture for economic viability. It has previously been shown that efficiency of erythroblast culture in scalable bioreactors is not primarily limited by mass transfer, availability of medium components, or commonly recognised inhibitory metabolites or cytokines. We have developed a dynamic mechanistic model that describes an autocrine feedback loop in which a cell-derived factor accumulates in culture medium resulting in reversible erythroblast growth inhibition. Cells exhibited two phases of growth: a relatively uninhibited followed by an inhibited phase. Cell cycle analysis during inhibition identified slight accumulation of cells in S phase, distinct from the G1 accumulation anticipated in growth factor or nutrient deprivation. Substantial donor to donor growth rate variability (mean 0.047 h(-1), standard deviation 0.008 h(-1)) required the growth rate parameter to be refitted for different donors. The model could then be used to predict growth behaviour with full medium exchange, but showed some reduced predictive ability after partial medium exchange. The model could predict the growth inflexion point over a range of phenotypic maturities from early to late maturity erythroblasts: however the secondary phase of growth differed substantially with less inhibition observed in more mature cells. The model provided a framework to optimise culture economics based on cost of production time and input consumables. It also provided a framework to evaluate the benefits of biological process engineering in medium design or cell modification vs. operational optimisation depending on the specific cost scenario of a process developer. (C) 2018 The Author(s). Published by Elsevier B.V.

Dynamic mechanistic modelling of cell culture is a key tool in bioprocess development to support optimisation and risk assessment. However, the approach is underutilised in the bioprocess industry due to challenges including lack of accessible tools to support a structured approach, the difficulty of realising computationally tractable (low parameter) yet realistic models, and the specialised skill sets required. We have proposed that these issues could be partly addressed by developing a parsimonious framework comprising a set of model building blocks, based on the ordinary differential equation modelling paradigm, representing common cell culture dynamics and modulation thereof. The framework is designed to avoid obvious pathological behaviours. Further, specific model instances within the framework can be simply visualised as a directed graph with vertices representing system species, dynamics and modulations, and arcs representing the interactions between them. The directed graph can be extended to describe the timing and nature of experimental interventions. A visual and intuitive route to describing models with an associated mathematical framework enables realisation in a software interface and integration with standard mathematical tools such as those for sensitivity analysis and parameter estimation. Such a framework is sufficient to represent some of the simple mechanisms underpinning bioprocesses that nonetheless lead to highly non-linear and counterintuitive outcomes. It also has a relatively low learning burden for users without formal mathematical training. The concept could be extended to include, for example, partial differential equation-based approaches to stochastic or spatially complex systems built up from a small number of parametrically parsimonious and well-behaved building blocks. (C) 2018 The Authors. Published by Elsevier B.V.

Background: Umbilical cord blood provides a source of hematopoietic stem cells for transplantation with immunological and availability advantages over conventional bone marrow sources. Limited cell numbers and slower engraftment from umbilical cord blood units has led to the clinical development of immobilised Notch ligand Delta-Like 1 to promote ex vivo expansion of a rapidly engrafting cell population. However, current immobilisation methods are not simple to scale in a controlled manner.Results: Delta-Like 1 was immobilised onto streptavidin coated magnetic particles via a heterobifunctionalised polyethylene glycol linker molecule to provide an easily manipulated format of surface protein presentation. CD34(+) enriched cord blood cells were treated with Delta-Like 1 immobilised particles, and immunophenotypic markers measured to monitor population distributions using cluster identification, characterization, and regression software. The amenability of the approach to scalability was evaluated in a micro-scale stirred tank bioreactor. Surface concentration of Delta-Like 1 was well controlled used differing stoichiometric reagent ratios. Protein immobilisation was a cost effective process and particles were efficiently removed from the final cell product. Immobilised Delta-Like 1 is functional and stimulates qualitatively similar CD34(hi), CD38(lo), CD90(lo), CD133(hi), CD135(hi) progenitor expansion in both static culture and scalable stirred culture platforms.Conclusions: Immobilised Delta-Like 1 in this form has the potential to improve the manufacturing efficiency and control of final ex vivo expanded cell product through compatibility with highly controlled and characterised suspension culture systems.

Hematopoietic therapies require high cell dosages and precise phenotype control for clinical success; scalable manufacturing processes therefore need to be economic and controllable, in particular with respect to culture medium and growth factor (GF) strategy. The aim of this work was to demonstrate the biological function, and integration within scalable systems, of a highly controllable immobilized growth factor (iGF) approach. GFs were biotinylated and attached to streptavidin coated magnetic particles. GF concentration during biotinylation, GF-biotin ratio, and GF lysine content were shown to control iGF surface concentration and enable predictable co-presentation of multiple GF on a single bead. Function was demonstrated for immobilized GMCSF, SCF, TPO and IL-3 in GF dependent cell lines TF-1 and M-07e. Immobilized GMCSF (iGMCSF) was analyzed to show sustained activity over 8 days of culture, a 2-3 order of magnitude potency increase relative to soluble factor, and retained functionality under agitation in a micro-scale stirred tank bioreactor. Further, short exposure to iGMCSF demonstrated prolonged growth response relative to soluble factor. This immobilization approach has the potential to reduce the manufacturing costs of scaled cell therapy products by reducing GF quantities and offers important process control opportunities through separation of GF treatments from the bulk media.

Human umbilical cord blood (CB) has long been established as a source of stem and progenitor cells for bone marrow (BM) transplantation. Although CB transplants have a number of advantages over other stem cell sources, the relatively low cell dose in a CB collection has limited their therapeutic potential. In order to overcome this disadvantage a number of novel technologies have been reported for proliferating haematopoietic progenitors whilst maintaining a cell phenotype and population balance that is therapeutically useful for rapid repopulation. To date the most promising of these technologies have sought to recreate the BM niche in order for progenitor cells to retain their repopulating ability during ex vivo expansion. Of the molecular components identified within the BM niche, Notch family members have been shown to play a key role in hematopoiesis, influencing cell fate decisions in vivo. The induction of Notch signalling in vitro using immobilised Delta1 ligand enhanced the generation of CD34+ cells and cells capable of NOD/SCID. This technology has demonstrated powerful proof of principle, but as of yet has not been developed into a well-controlled scalable system.

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored, using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony “edge,” “core periphery” or “core” cells. Conventional biomarkers, such as Oct3/4, Nanog, and Sox-2, were shown to correspond to specific morphologies using immunostaining and flow cytometry techniques. Quantitative monitoring of these morphological attributes in-process using the reference image libraries showed rapid sensitivity to changes induced by different media exchange regimes or the addition of mesoderm lineage inducing cytokine BMP4. The imaging sample size to precision relationship was defined for each morphological attribute to show that this sensitivity could be achieved with a relatively low imaging sample. Further, the morphological state of single colonies could be correlated to individual colony outcomes; smaller colonies were identified as optimum for homogenous early mesoderm differentiation, while larger colonies maintained a morphologically pluripotent core. Finally, we show the potential of the same image libraries to assess cell number in culture with accuracy comparable to sacrificial digestion and counting. The data supports a potentially powerful role for quantitative image analysis in the setting of in-process specifications, and also for screening the effects of process actions during development, which is highly complementary to current analysis in optimization and manufacture. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:215–223, 2016

SummaryConditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2hours, and shear to 3hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer- term cultures, reduction in expression of β1-integrin (but not β3-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β1-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β1-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

BACKGROUND AIMS:Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control.METHODS:CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers.RESULTS:The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state.CONCLUSIONS:Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps.

Background: Apart from haematopoietic stem cell transplantation for haematological disorders many stem cell-based therapies are experimental. However, with only 12 years between human embryonic stem cell isolation and the first clinical trial, development of stem cell products for regenerative medicine has been rapid and numerous clinical trials have begun to investigate their therapeutic potential.Source of data: This review summarizes key clinical trial data, current and future perspectives on stem cell-based products undergoing clinical trials, based on literature search and author research.Areas of agreement: It is widely recognized that the ability to stimulate stem cell differentiation into specialized cells for use as cellular therapies will revolutionize health care and offer major hope for numerous diseases for which there are limited or no therapeutic options.Areas of controversy: Stem cell-based products are unique and cover a large range of disorders to be treated; therefore, there is significant potential for variation in cell source, type, processing manipulation, the bioprocessing approach and scalability, the cost and purity of manufacture, final product quality and mode of action. As such there are gaps in regulatory and manufacturing frameworks and technologies, only a small number of products are currently within late phase clinical trials and few products have achieved commercialization.Growing points: Recent developments are encouraging acceleration through the difficulties encountered en route to clinical trials and commercialization of stem cell therapies.Areas timely for developing research: The field is growing year on year with the first clinical trial using induced pluripotent stem cells anticipated by end 2013.

Umbilical cord blood provides a source of hematopoietic stem cells for transplantation with immunological and availability advantages over conventional bone marrow sources. Limited cell numbers and slower engraftment from umbilical cord blood units has led to the clinical development of immobilised Notch ligand Delta-Like 1 to promote ex vivo expansion of a rapidly engrafting cell population. Further, bone marrow stromal cells also promote ex vivo proliferation and differentiation of hematopoietic stem cells, supporting the concept that bone marrow niche cues may influence their fate. Surface receptors for a number of proteins released from the bone marrow niche have been identified on hematopoietic stem cells. The immobilisation of these niche proteins in a controllable manner presents an exciting opportunity for the improved expansion of cells with high clinical potential.

Using an automated stirred tank micro-bioreactor (ambr®, Sartorius Stedim, UK) we have shown that initially erythroblasts rapidly proliferate but then enter an inhibited growth phase. Experimentally we have confirmed that the conventional constraints on cell manufacturing efficiency, such as mass transfer, common metabolic limitations, or previously reported paracrine signals were not responsible for this inhibition.