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:Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. KEY POINTS: • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics.
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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:Introduction Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.
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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:The turbocharger, a pivotal technology for energy conservation and emission reduction, offers substantial academic significance, particularly in the in-depth study of its core component: the centrifugal compressor impeller. This research aims to enhance the centrifugal compressor's overall efficiency by optimizing its impeller meridional profile. By modifying the impeller meridional profile of a certain centrifugal compressor impeller to address the issue of discontinuous curvature, this paper aims to optimize the comprehensive average efficiency under variable speed and variable flow conditions. The optimization goal is to improve the overall average efficiency while maintaining the high pressure ratio based on the prototype scheme. The NSGA-III optimization algorithm is employed for multi-condition optimization, aiming to achieve comprehensive efficiency improvements under multiple operating speeds. The following conclusions are drawn: under multi-speed conditions, the optimized scheme exhibits comprehensive efficiency improvements over the prototype scheme, with an expanded stable operating flow range and maintained high-pressure ratio based on efficiency improvement, without sacrificing the operating flow range. Comparisons of internal flow conditions indicate that the optimized impeller features a smoother meridional passage and a reduced high-entropy region at the outlet, leading to lower entropy values. Additionally, pressure increases on both sides of the impeller blades while pressure differentials diminish, signifying enhanced internal flow conditions.
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