RESUMEN
Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags. In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg-1 ), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 106 viable cells ml-1 obtained at the highest P/M of 74 × 10-4 W kg-1 in comparison with 0.91 ± 0.07 × 106 viable cells ml-1 at the lowest P/M of 3.1 × 10-4 W kg-1 . We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications.
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Reactores Biológicos , Técnicas de Cultivo de Célula , Linfocitos T/metabolismo , Células Cultivadas , Humanos , Linfocitos T/citologíaRESUMEN
Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS . The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06-0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253-2266. © 2017 Wiley Periodicals, Inc.
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Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Microfluídica/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero/metabolismo , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Microfluídica/métodos , Miniaturización , Proyectos Piloto , Robótica/instrumentaciónRESUMEN
BACKGROUND AIMS: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. METHODS: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. RESULTS: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. CONCLUSIONS: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.
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Plaquetas/citología , Técnicas de Cultivo de Célula/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Microtecnología/métodos , Andamios del Tejido , Animales , Bovinos , Proliferación Celular , Células Cultivadas , Humanos , Investigación Biomédica TraslacionalRESUMEN
BACKGROUND AIMS: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. METHODS: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. RESULTS: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R(2) = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. CONCLUSIONS: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.
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Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Proliferación Celular , Tamaño de la Célula , Medios de Cultivo/química , Expresión Génica , Humanos , Células Madre Mesenquimatosas/fisiología , OsteogénesisRESUMEN
Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.
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Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Criopreservación/métodos , Medio de Cultivo Libre de Suero/química , Células Madre Mesenquimatosas/fisiología , Microesferas , Preservación Biológica/métodos , Supervivencia Celular , Humanos , Proyectos Piloto , Células MadreRESUMEN
Extracellular surface proteins are used to identify fully-functional human mesenchymal stem cells (hMSCs) in a mixed population. Here, a multiparameter flow cytometry assay was developed to examine the expression of several bone marrow-derived hMSC markers simultaneously at the single cell level. The multiparameter approach demonstrates a depth of analysis that goes far beyond the conventional single or dual staining methods. CD73, CD90 and CD105 were chosen as positive markers as they are expressed on multipotent hMSCs, whilst CD34 and HLA-DR were chosen as negative indicators. Single colour analysis suggested a population purity of 100 %; in contrast, when analysed via the multiparameter method, the CD73(+ve)/CD105(+ve)/CD90(+ve)/HLA-DR(-ve)/CD34(-ve) phenotypes represented 94.5 ± 1.3 % of the total cell population. Also, although CD271 has been posited as a definite early stage hMSC marker, here we show it is not present on pre-passage cells, highlighting the need for careful marker selection.
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Biomarcadores/análisis , Citometría de Flujo/métodos , Proteínas de la Membrana/análisis , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/clasificación , Técnicas Citológicas/métodos , HumanosRESUMEN
When considering the development pathway for a genetically modified cell therapy product, it is critically important that the product is engineered consistent with its intended human use. For scientists looking to develop and commercialize a new technology, the decision to select a genetic modification method depends on several practical considerations. Whichever path is chosen, the developer must understand the key risks and potential mitigations of the cell engineering approach. The developer should also understand the clinical implications: permanent/memory establishment versus transient expression, and clinical manufacturing considerations when dealing with transplantation of genetically engineered cells. This review covers important topics for mapping out a strategy for developers of new cell-based therapeutics. Biological, technological, manufacturing, and clinical considerations are all presented to map out development lanes for the initiation and risk management of new gene-based cell therapeutic products for human use.
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Ingeniería Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , HumanosRESUMEN
Chimeric antigen receptor T-cell (CAR-T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR-T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR-T cells from multiple donors in an automated stirred-tank bioreactor. The authors successfully produced functional human CAR-T cells from multiple donors under dynamic conditions in a stirred-tank bioreactor, resulting in overall cell yields which were significantly better than in static T-flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR-T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 cells ml-1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T-flasks and gas-permeable culture bags). Importantly, engineered T-cells post-expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR-T cell manufacture in stirred-tank bioreactors and the findings present significant implications and opportunities for larger-scale allogeneic CAR-T production.
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Reactores Biológicos , Técnicas de Cultivo de Célula , Recuento de Células , Humanos , Inmunoterapia Adoptiva , Linfocitos TRESUMEN
PURPOSE OF REVIEW: Many cell therapy products are beginning to reach the commercial finish line and a rapidly escalating pipeline of products are in clinical development. The need to develop manufacturing capability that will support a successful commercial business model has become a top priority as many cell therapy developers look to secure long-term visions to enable both funding and treatment success. RECENT FINDINGS: Manufacturing automation is both highly compelling and very challenging at the same time as a key tactic to address quality, cost of goods, scale, and sustainability that are fundamental drivers for commercially viable manufacturing. This paper presents an overview and strategic drivers for application of automation to cell therapy manufacturing. It also explores unique automation considerations for patient-specific cell therapy (PSCT) where each full-scale lot is for one patient vs off-the-shelf cell therapy (OTSCT) where a full-scale lot will treat many patients, and finally some practical considerations for implementing automation.
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Automatización , Ingeniería Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ingeniería Genética , Animales , Automatización/economía , Automatización/métodos , Automatización/normas , Automatización de Laboratorios , Ingeniería Celular/economía , Ingeniería Celular/métodos , Ingeniería Celular/normas , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Tratamiento Basado en Trasplante de Células y Tejidos/economía , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Ingeniería Genética/economía , Ingeniería Genética/métodos , Ingeniería Genética/normas , Humanos , Control de CalidadRESUMEN
Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.
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Técnicas de Cultivo de Célula/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Investigación Biomédica Traslacional/métodos , Ensayos Clínicos como Asunto , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citologíaRESUMEN
Nonunion fractures and large bone defects are significant targets for osteochondral tissue engineering strategies. A major hurdle in the use of these therapies is the foreign body response of the host. Herein, we report the development of a bone tissue engineering scaffold with the ability to release anti-inflammatory drugs, in the hope of evading this response. Porous, sintered scaffolds composed of poly(D,L-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were prepared with and without the anti-inflammatory drug diclofenac sodium. Analysis of drug release over time demonstrated a profile suitable for the treatment of acute inflammation with â¼80% of drug released over the first 4 days and a subsequent release of around 0.2% per day. Effect of drug release was monitored using an in vitro osteoblast inflammation model, comprised of mouse primary calvarial osteoblasts stimulated with proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Levels of inflammation were monitored by cell viability and cellular production of nitric oxide (NO) and prostaglandin E2 (PGE2). The osteoblast inflammation model revealed that proinflammatory cytokine addition to the medium reduced cell viability to 33%, but the release of diclofenac sodium from scaffolds inhibited this effect with a final cell viability of â¼70%. However, releasing diclofenac sodium at high concentrations had a toxic effect on the cells. Proinflammatory cytokine addition led to increased NO and PGE2 production; diclofenac-sodium-releasing scaffolds inhibited NO release by â¼64% and PGE2 production by â¼52%, when the scaffold was loaded with the optimal concentration of drug. These observations demonstrate the potential use of PLGA/PEG scaffolds for localized delivery of anti-inflammatory drugs in bone tissue engineering applications.
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Diclofenaco/uso terapéutico , Sistemas de Liberación de Medicamentos , Inflamación/tratamiento farmacológico , Osteoblastos/patología , Polietilenglicoles/química , Poliglactina 910/química , Andamios del Tejido/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diclofenaco/administración & dosificación , Diclofenaco/farmacología , Dinoprostona/biosíntesis , Humanos , Inflamación/patología , Interferón gamma/farmacología , Interleucina-1beta , Ratones , Modelos Biológicos , Óxido Nítrico/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Cráneo/patología , Factor de Necrosis Tumoral alfaRESUMEN
The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of -10 to -40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haperzine-A-loaded NPs. Furthermore, a range of drug encapsulates NPs were produced and showed a sustained release of the encapsulated drug. This study demonstrates the ability to control the size of drug-loaded particles by manipulation of the production variables, which will allow targeted and controlled drug release to fit a variety of applications.