Considerations for the bioprocessing, manufacture and translation of extracellular vesicles for therapeutic applications

There is growing interest in the potential and use of extracellular vesicles (EVs) for a range of diagnostic and therapeutic applications. EVs have been shown, in some instances, to mediate the regenerative effects elicited by stem cell therapies. As such, they are being studied to identify the extent to which these extracellular bodies can be employed as a therapeutic entity, and significant R&D activity is underway to further understand their clinical and commercial potential. However, successful translation will first require further characterization and standardization of EV production, as well as addressing some of the major challenges associated with their reproducible manufacture. This includes the capacity to produce EVs at a scale that is both clinically and commercially effective. This article will highlight some of the bioprocessing and manufacturing considerations and challenges associated with the standardized production of EVs

Understanding the impact of bioactive coating materials for human mesenchymal stromal cells and implications for manufacturing

Bioactive materials interact with cells and modulate their characteristics which enable the generation of cell-based products with desired specifications. However, their evaluation and impact are often overlooked when establishing a cell therapy manufacturing process. In this study, we investigated the role of different surfaces for tissue culture including, untreated polystyrene surface, uncoated Cyclic Olefin Polymer (COP) and COP coated with collagen and recombinant fibronectin. It was observed that human mesenchymal stromal cells (hMSCs) expanded on COP-coated plates with different bioactive materials resulted in improved cell growth kinetics compared to traditional polystyrene plates and non-coated COP plates. The doubling time obtained was 2.78 and 3.02 days for hMSC seeded in COP plates coated with collagen type I and recombinant fibronectin respectively, and 4.64 days for cells plated in standard polystyrene treated plates. Metabolite analysis reinforced the findings of the growth kinetic studies, specifically that cells cultured on COP plates coated with collagen I and fibronectin exhibited improved growth as evidenced by a higher lactate production rate (9.38 × 105 and 9.67 × 105 pmol/cell/day, respectively) compared to cells from the polystyrene group (5.86 × 105 pmol/cell/day). This study demonstrated that COP is an effective alternative to polystyrene-treated plates when coated with bioactive materials such as collagen and fibronectin, however COP-treated plates without additional coatings were found not to be sufficient to support cell growth. These findings demonstrate the key role biomaterials play in the cell manufacturing process and the importance of optimising this selection

The role of biopreservation in cell and gene therapy bioprocessing

Cell and gene-based therapies represent a novel therapeutic modality that has the potential to provide a treatment option for a range of medical conditions. There are, however, numerous processing and manufacturing challenges that must be addressed before such therapies are considered commercially and clinically viable. A significant challenge associated with the manufacture of such therapies is ensuring cell quality and the product’s critical quality attributes are retained throughout the entire bioprocess. Biopreservation is an important aspect of cell and gene-based therapy bioprocessing, which enables the development of cell banks. It increases process flexibility by providing a shelf-life to the product, enables the storage of intermediates and provides breakpoints within the process. In this article, we summarize the advances and challenges associated with biopreservation of cell and gene therapies

Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: lessons learnt and what the future holds

Human mesenchymal stem/stromal cells (hMSCs) present a key therapeutic cellular intervention for use in cell and gene therapy (CGT) applications due to their immunomodulatory properties and multi-differentiation capability. Some of the indications where hMSCs have demonstrated pre-clinical or clinical efficacy to improve outcomes are cartilage repair, acute myocardial infarction, graft versus host disease, Crohn’s disease and arthritis. The current engineering challenge is to produce hMSCs at an affordable price and at a commercially-relevant scale whilst minimising process variability and manual, human operations. By employing bioreactors and microcarriers (due to the adherent nature of hMSCs), it is expected that production costs would decrease due to improved process monitoring and control leading to better consistency and process efficiency, and enabling economies of scale. This approach will result in off the shelf (allogeneic) hMSC-based products becoming more accessible and affordable. Importantly, cell quality, including potency, must be maintained during the bioreactor manufacturing process. This review aims to examine the various factors to be considered when developing a hMSC manufacturing process using microcarriers and bioreactors and their potential impact on the final product. As concluding remarks, gaps in the current literature and potential future areas of research are also discussed

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (NJS) and associated specific power input (P/V)JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

This article was published in the journal, Biotechnology Letters [© Springer Science+Business Media] and the definitive version is available at: http://dx.doi.org/10.1007/s10529-013-1211-9For 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 × 105 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

Numerical methods for the design and description of in vitro expansion processes of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called “Digital Twin” of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks. In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown

Scientific, sustainability and regulatory challenges of cultured meat

Producing meat without the drawbacks of conventional animal agriculture would greatly contribute to future food and nutrition security. This Review Article covers biological, technological, regulatory and consumer acceptance challenges in this developing field of biotechnology. Cellular agriculture is an emerging branch of biotechnology that aims to address issues associated with the environmental impact, animal welfare and sustainability challenges of conventional animal farming for meat production. Cultured meat can be produced by applying current cell culture practices and biomanufacturing methods and utilizing mammalian cell lines and cell and gene therapy products to generate tissue or nutritional proteins for human consumption. However, significant improvements and modifications are needed for the process to be cost efficient and robust enough to be brought to production at scale for food supply. Here, we review the scientific and social challenges in transforming cultured meat into a viable commercial option, covering aspects from cell selection and medium optimization to biomaterials, tissue engineering, regulation and consumer acceptance