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Understanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, differences between assays and the body are common, indicating the importance of in vitro-in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fibre (Plasmaphan P1LX, 3M) that serves as a porous scaffold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant flow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2. MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fluorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown significant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fivefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efficiency compared to Transwell cell cultures while efflux of the P-gp-specific substrates Hoechst and Rhodamine 123 was decreased. These results further support the effect of the microenvironment and fluidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.
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Técnicas de Cultura de Células , Túbulos Renais Proximais , Animais , Transporte Biológico , Transporte Biológico Ativo , Túbulos Renais Proximais/metabolismo , Ratos , Estresse MecânicoRESUMO
BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION: The prospect of a temporary microcarrier that can be used to expand cells and then 'disappear' for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality.
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Mesenchymal stem cells (MSCs) represent an invaluable asset for the field of cell therapy. Human Bone marrow-derived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its effect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not affect BM-MSCs morphology, surface marker expression, differentiation or proliferation potential. However, mixed results exist regarding the effect on colony forming ability and the effects on viability, attachment and migration, genomic stability and paracrine function are undefined mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assays.
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Criopreservação , Células-Tronco Mesenquimais/citologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Forma Celular , Sobrevivência Celular , Senescência Celular , Congelamento , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/metabolismo , Especificidade da EspécieRESUMO
The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non-invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi-illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12-day culture noted, prior to the onset of aggregation. The developed image acquisition system and post-processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032-2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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Rastreamento de Células/instrumentação , Aumento da Imagem/instrumentação , Transplante de Células-Tronco Mesenquimais/instrumentação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Microscopia/instrumentação , Dispositivos Ópticos , Crescimento Celular , Células Cultivadas , Humanos , Aumento da Imagem/métodos , Miniaturização , Reconhecimento Automatizado de Padrão/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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 Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Microfluídica/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura Livres de Soro/metabolismo , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Microfluídica/métodos , Miniaturização , Projetos Piloto , Robótica/instrumentaçãoRESUMO
BACKGROUND: Human mesenchymal stem/stromal cells (hMSCs) are at the forefront of regenerative medicine applications due to their relatively easy isolation and availability in adults, potential to differentiate and to secrete a range of trophic factors that could determine specialised tissue regeneration. To date, hMSCs have been successfully cultured in vitro on substrates such as polystyrene dishes (TCPS) or microcarriers. However, hMSC sub-cultivation and harvest typically employs proteolytic enzymes that act by cleaving important cell membrane proteins resulting in long-term cell damage. In a process where the cells themselves are the product, a non-enzymatic and non-damaging harvesting approach is desirable. RESULTS: An alternative system for hMSC expansion and subsequent non-enzymatic harvest was investigated here. A liquid/liquid two-phase system was proposed, comprising a selected perfluorocarbon (FC40) and growth medium (DMEM). The cells exhibited similar cell morphologies compared with TCPS. Moreover, they retained their identity and differentiation potential post-expansion and post-harvest. Further, no significant difference was found when culturing hMSCs in the culture systems prepared with either fresh or recycled FC40 perfluorocarbon. CONCLUSIONS: These findings make the FC40/DMEM system an attractive alternative for traditional cell culture substrates due to their ease of cell recovery and recyclability, the latter impacting on overall process costs. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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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/citologia , Técnicas de Cultura de Células/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Microtecnologia/métodos , Alicerces Teciduais , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Humanos , Pesquisa Translacional BiomédicaRESUMO
With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to â¼70% and only â¼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.
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Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Congelamento , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
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 Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Tamanho Celular , Meios de Cultura/química , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/fisiologia , OsteogêneseRESUMO
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 Cultura de Células/métodos , Proliferação de Células , Criopreservação/métodos , Meios de Cultura Livres de Soro/química , Células-Tronco Mesenquimais/fisiologia , Microesferas , Preservação Biológica/métodos , Sobrevivência Celular , Humanos , Projetos Piloto , Células-TroncoRESUMO
Encouraging advances in cell therapies have produced a requirement for an effective short-term cell preservation method, enabling time for quality assurance testing and transport to their clinical destination. Low temperature pausing of cells offers many advantages over cryopreservation, including the ability to store cells at scale, reduced cost and a simplified procedure with increased reliability. This review will focus on the importance of developing a short-term cell preservation platform as well highlighting the major successes of cell pausing and the key challenges which need addressing, to enable application of the process to therapeutically relevant cells.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Células/efeitos da radiação , Preservação Biológica/métodos , Sobrevivência Celular/efeitos da radiação , Temperatura Baixa , Fatores de TempoRESUMO
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/análise , Citometria de Fluxo/métodos , Proteínas de Membrana/análise , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/classificação , Técnicas Citológicas/métodos , HumanosRESUMO
Cell encapsulation into spherical microparticles is a promising bioengineering tool in many fields, including 3D cancer modelling and pre-clinical drug discovery. Cancer microencapsulation models can more accurately reflect the complex solid tumour microenvironment than 2D cell culture and therefore would improve drug discovery efforts. However, these microcapsules, typically in the range of 1 - 5000 µm in diameter, must be carefully designed and amenable to high-throughput production. This review therefore aims to outline important considerations in the design of cancer cell microencapsulation models for drug discovery applications and examine current techniques to produce these. Extrusion (dripping) droplet generation and emulsion-based techniques are highlighted and their suitability to high-throughput drug screening in terms of tumour physiology and ease of scale up is evaluated.
3D microencapsulation models of cancer offer a customisable platform to mimic key aspects of solid tumour physiology in vitro. However, many 3D models do not recapitulate the hypoxic conditions and altered tissue stiffness established in many tumour types and stages. Furthermore, microparticles for cancer cell encapsulation are commonly produced using methods that are not necessarily suitable for scale up to high-throughput manufacturing. This review aims to evaluate current technologies for cancer cell-laden microparticle production with a focus on physiological relevance and scalability. Emerging techniques will then be touched on, for production of uniform microparticles suitable for high-throughput drug discovery applications.
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Descoberta de Drogas , Neoplasias , Humanos , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Descoberta de Drogas/métodos , Encapsulamento de Células/métodos , Modelos Biológicos , Cápsulas , Animais , Composição de Medicamentos/métodos , Microambiente Tumoral/efeitos dos fármacosRESUMO
For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me2SO). Me2SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me2SO. Although reduced (by 22 ± 2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me2SO, where the doubling time was significantly reduced (by 41 ± 4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.
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Permeabilidade da Membrana Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/metabolismo , Polímeros/metabolismo , Trealose/metabolismo , Linhagem Celular , Sobrevivência Celular , Humanos , Polímeros/químicaRESUMO
Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis.
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Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Citometria de Fluxo/métodos , Antígenos de Superfície/análise , Antígenos de Superfície/química , Biomarcadores/análise , Biomarcadores/química , Humanos , Antígenos CD15/análise , Antígenos CD15/química , Proteoglicanas/análise , Proteoglicanas/química , Antígenos Embrionários Estágio-Específicos/análise , Antígenos Embrionários Estágio-Específicos/químicaRESUMO
For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10(5) cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.
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Reatores Biológicos , Células-Tronco Mesenquimais/fisiologia , Microesferas , Contagem de Células , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Humanos , Concentração de Íons de Hidrogênio , Oxigênio/análise , Fatores de TempoRESUMO
Introduction: For stem cell therapies to be adopted in mainstream health care, robust, reliable, and cost-effective storage and transport processes must be developed. Cryopreservation remains the best current platform for this purpose, and freezing cells at high concentration may have many benefits, including savings on cost and storage space, facilitating transport logistics, and reducing cryoprotectant volume. Cells, such as mesenchymal stem cells (MSCs), are typically frozen at 1 million cells per milliliter (mL), but the aim of this study is to examine the post-thaw attributes of human bone marrow derived MSCs (hBM-MSCs) frozen at 1, 5, and 10 million cells per mL. Methods: Thawed cells were assessed for their morphology, phenotypic marker expression, viability, apoptosis level, metabolic activity, proliferation, and osteogenic and adipogenic differentiation. Results: In this study, for the first time, it is shown that all assessed cells expressed the typical MSC markers (CD90, CD105, and CD73) and lacked the expression of CD14, CD20, CD34, CD45, and HLA-DR. In addition, all cells showed elongated fibroblastic morphology. Post-thaw viability was retained with no difference among the three concentrations. Moreover, no significant statistical difference was observed in the post-thaw apoptosis level, metabolic activity, proliferation, and osteogenic potential, indicating that these cells are amenable to cryopreservation at higher concentrations. Conclusion: The results of this study are of paramount importance to the development of manufacturing processes around a useful freezing concentration when cells are targeted to be stored for at least 6 months.
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Measured variability of product within Cell and Gene Therapy (CGT) manufacturing arises from numerous sources across pre-analytical to post-analytical phases of testing. Operators are a function of the manufacturing process and are an important source of variability as a result of personal differences impacted by numerous factors. This research uses measurement uncertainty in comparison to Coefficient of Variation to quantify variation of participants when they complete Flow Cytometry data analysis through a 5-step gating sequence. Two study stages captured participants applying gates using their own judgement, and then following a diagrammatical protocol, respectively. Measurement uncertainty was quantified for each participant (and analysis phase) by following Guide to the Expression of Uncertainty in Measurement protocols, combining their standard deviations in quadrature from each gating step in the respective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57%, increasing confidence in a more uniform reported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytometry gating process is from the very first gate, where isolating target cells from dead or dying cells is required. This work has demonstrated the potential for greater usage of measurement uncertainty within CGT manufacturing scenarios, due to the resolution it provides for root cause analysis and continuous improvement.
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Flow cytometry is a complex measurement characterization technique, utilized within the manufacture, measurement, and release of cell and gene therapy products for rapid, high-content, and multiplexed discriminatory cell analysis. A number of factors influence the variability in the measurement reported including, but not limited to, biological variation, reagent variation, laser and optical configurations, and data analysis methods. This research focused on understanding the contribution of manual operator variability within the data analysis phase. Thirty-eight participants completed a questionnaire, providing information about experience and motivational factors, before completing a simple gating study. The results were analyzed using gauge repeatability and reproducibility techniques to quantify participant uncertainty. The various stages of the gating sequence were combined through summation in quadrature and expanded to give each participant a representative uncertainty value. Of the participants surveyed, 85% preferred manual gating to automated data analysis, with the primary reasons being legacy ("it's always been done that way") and accuracy, not in the metrological sense but in the clear definition of the correct target population. The median expanded uncertainty was calculated as 3.6% for the population studied, with no significant difference among more or less experienced users. Operator subjectivity can be quantified to include within measurement uncertainty budgets, required for various standards and qualifications. An emphasis on biomanufacturing measurement terminology is needed to help understand future and potential solutions, possibly looking at translational clinical models to engage and enhance better training and protocols within industrial and research settings.
Assuntos
Análise de Dados , Citometria de Fluxo , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , IncertezaRESUMO
Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.