Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions
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ABSTRACT:
SUBMITTER: Zhang B
PROVIDER: S-EPMC10992254 | biostudies-literature | 2023 Dec
REPOSITORIES: biostudies-literature
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Project description:The traditional breeding industry has been increasingly saturated and caused environmental pollution, disease transmission, excessive resource use, and methane emission; however, it still cannot meet the needs of the growing population. To explore other alternatives, researchers focused on cell agriculture and cell-based meat, especially large-scale cell culture. As a prerequisite for production, large-scale culture technology has become an important bottleneck restricting cell-based meat industrialization. In this study, the single-factor variable method was adopted to examine the influence of Cytodex1 microcarrier pretreatment, spinner flask reaction vessel, cell culture medium, serum and cell incubation, and other influencing factors on large-scale cell cultures to identify the optimization parameters suitable for 3D culture environment. Collagen and 3D culture were also prospectively explored to promote myogenesis and cultivate tissue-like muscle fibers that contract spontaneously. This research lays a theoretical foundation and an exploratory practice for large-scale cell cultures and provides a study reference for the microenvironment of myoblast culture in vitro, a feasible direction for the cell therapy of muscular dystrophy, and prerequisites for the industrialized manufacturing of cell-based meat. Graphical summary: Research on large-scale myoblast culture using spinner flasks and microcarriers. For cell culture, the microcarriers were pretreated with UV and collagen. Cell seeding condition, spinner flask speed, resting time, and spinner flask culture microenvironment were then optimized. Finally, two culture systems were prepared: a culture system based on large-scale cell expansion and a culture system for myogenesis promotion and differentiation.
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Project description:Rapidly evolving cell-based therapies towards clinical trials demand alternative approaches for efficient expansion of adherent cell types such as human mesenchymal stem cells (hMSCs). Using microcarriers (100-300?µm) in a stirred tank bioreactor offers considerably enhanced surface to volume ratio of culture environment. However, downstream purification of the harvested cell product needs to be addressed carefully due to distinctive features and fragility of these cell products. This work demonstrates a novel alternative approach which utilizes inertial focusing to separate microcarriers (MCs) from the final cell suspension. First, we systematically investigated MC focusing dynamics inside scaled-up curved channels with trapezoidal and rectangular cross-sections. A trapezoidal spiral channel with ultra-low-slope (Tan(?)?=?0.0375) was found to contribute to strong MC focusing (~300?<?Re?<?~400) while managing high MC volume fractions up to ~1.68%. Accordingly, the high-throughput trapezoidal spiral channel successfully separated MCs from hMSC suspension with total cell yield~94% (after two passes) at a high volumetric flow rate of ~30?mL/min (Re~326.5).
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Project description:In the tissue engineering field dynamic culture systems, such as spinner flasks, are widely used due to their ability to improve mass transfer in suspension cell cultures. However, this culture system is often unsuitable to culture cells in three-dimensional (3D) scaffolds. To address this drawback, we designed a multicompartment holder for 3D cell culture, easily adaptable to spinner flasks. Here, the device was tested with human mesenchymal stem cells (MSCs) seeded in 3D porous chitosan scaffolds that were maintained in spinner flasks under dynamic conditions (50 rpm). Standard static culture conditions were used as control. The dynamic conditions were shown to significantly increase MSCs proliferation over 1 week (approximately 6-fold) and to improve cell distribution within the scaffold. Moreover, they also promoted osteogenic differentiation of MSCs, inducing an earlier peak in alkaline phosphatase (ALP) activity, and a more homogenous ALP staining and matrix mineralization in the whole scaffolds, but particularly in the center. Overall, this study shows a new multicompartment holder to culture 3D scaffolds that can broaden the application of spinner flasks.
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Project description:A method of growing rat Pneumocystis carinii with human embryonic lung fibroblasts (HEL-299 cells) sheeted onto microcarrier beads has been developed. This method allows production of large quantities of P. carinii organisms with very little contamination of host cells. A fivefold increase in the numbers of organisms was achieved, as determined by organism count, antigen detection, and DNA quantification. The majority of organisms produced by this method are trophozoites.
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Project description:In this methodological paper, lyophilized fibroin-coated alginate microcarriers (LFAMs) proposed as mesenchymal stem cells (MSCs) delivery systems and optimal MSCs seeding conditions for cell adhesion rate and cell arrangement, was defined by a Design of Experiment (DoE) approach. Cells were co-incubated with microcarriers in a bioreactor for different time intervals and conditions: variable stirring speed, dynamic culture intermittent or continuous, and different volumes of cells-LFAMs loaded in the bioreactor. Intermittent dynamic culture resulted as the most determinant parameter; the volume of LFAMs/cells suspension and the speed used for the dynamic culture contributed as well, whereas time was a less influencing parameter. The optimized seeding conditions were: 98 min of incubation time, 12.3 RPM of speed, and 401.5 µL volume of cells-LFAMs suspension cultured with the intermittent dynamic condition. This DoE predicted protocol was then validated on both human Adipose-derived Stem Cells (hASCs) and human Bone Marrow Stem Cells (hBMSCs), revealing a good cell adhesion rate on the surface of the carriers. In conclusion, microcarriers can be used as cell delivery systems at the target site (by injection or arthroscopic technique), to maintain MSCs and their activity at the injured site for regenerative medicine.
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Project description:Chinese Hamster Ovary cell lines are currently the primary host for production of therapeutic glycoproteins. Fast process development resulting in robust and scalable processes is a critical success factor in the highly competitive market for biosimilars. In process development screening of hundreds of clones and selection of process conditions are routinely performed in uncontrolled cultivation systems like shake flasks. A handful of potential candidate clones is nominated to be evaluated more intensively in well controlled small-scale bioreactors. Cell performance in the uncontrolled systems and to a lesser extent in the small-scale bioreactors may, however, be different from that in the final production reactor, which may result in failures during scale-up and thus extra development time. In this work, the focus is on better understanding the differences in cell performance between controlled and uncontrolled systems, which can be used to make process development faster and more robust in terms of scale-up. For this, we evaluated differences in gene expression profiles between shake flask and bioreactor cultures at three different time points during the exponential and stationary phase of a batch culture using commercially available Affymetrix GeneChip CHO Gene 2.1 ST arrays and multivariate data analysis on the outcomes. The outcomes were correlated with differences in glycosylation patterns and other culture parameters. Results showed large differences in gene expression over time and much smaller differences between the two cultivation systems. Furthermore, our study identified differentially expressed genes and corresponding metabolic and mechanistic pathways between the two systems, which were directly related to the degree of the control of the systems.
2023-11-04 | GSE104787 | GEO
Project description:This paper presents the preliminary steps required for conducting experiments to obtain the optimal operating conditions of a hybrid impeller mixer and to determine the residence time distribution (RTD) using computational fluid dynamics (CFD). In this paper, impeller speed and clearance parameters are examined. The hybrid impeller mixer consists of a single Rushton turbine mounted above a single pitched blade turbine (PBT). Four impeller speeds, 50, 100, 150, and 200 rpm, and four impeller clearances, 25, 50, 75, and 100 mm, were the operation variables used in this study. CFD was utilized to initially screen the parameter ranges to reduce the number of actual experiments needed. Afterward, the residence time distribution (RTD) was determined using the respective parameters. Finally, the Fluent-predicted RTD and the experimentally measured RTD were compared. The CFD investigations revealed that an impeller speed of 50 rpm and an impeller clearance of 25 mm were not viable for experimental investigations and were thus eliminated from further analyses. The determination of RTD using a k-ε turbulence model was performed using CFD techniques. The multiple reference frame (MRF) was implemented and a steady state was initially achieved followed by a transient condition for RTD determination.
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Project description:ObjectivesCellular aggregates are readily applicable in cell-based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated.Materials and methodsThe aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow-derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion-related molecules was analysed.ResultsIt was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin β1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery.ConclusionsCellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.
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Project description:Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell's metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells.
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Project description:This study focusses on the design and scale-up of industrial lactic acid production by fermentation of dairy cheese whey permeate based on standard methodological parameters. The aim was to address the shortcomings of standard scale-up methodologies and provide a framework for fermenter scale-up that enables the accurate estimation of energy consumption by suitable selection of turbine and speed for industrial deployment. Moreover, life cycle assessment (LCA) was carried out to identify the potential impacts and possibilities to reduce the operation associated emissions at an early stage. The findings showed that a 3000 times scale-up strategy assuming constant geometric dimensions and specific energy consumption (P/V w ) resulted in lower impeller speed and energy demand. The Rushton turbine blade (RTB) and LightninA315 four-blade hydrofoil (LA315) were found to have the highest and lowest torque output, respectively, at a similar P/V w of 2.8 kWm-3, with agitation speeds of 1.33 and 2.5 s-1, respectively. RTB demonstrating lower shear damage towards cells (up to 1.33 s-1) was selected because it permits high torque, low-power and acceptable turbulence. The LCA results showed a strong relation between the number of impellers installed and associated emissions suggesting a trade-off between mixing performance and environmental impacts.Supplementary informationThe online version contains supplementary material available at 10.1007/s13197-021-05239-6.
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