Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

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 100mL stirred spinner flasks at a density of 3×10⁵cells.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 3h 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.

Enhancing the performance of intelligent control systems in the face of higher levels of complexity and uncertainty

Modern advances in technology have led to more complex manufacturing processes whose success centres on the ability to control these processes with a very high level of accuracy. Plant complexity inevitably leads to poor models that exhibit a high degree of parametric or functional uncertainty. The situation becomes even more complex if the plant to be controlled is characterised by a multivalued function or even if it exhibits a number of modes of behaviour during its operation. Since an intelligent controller is expected to operate and guarantee the best performance where complexity and uncertainty coexist and interact, control engineers and theorists have recently developed new control techniques under the framework of intelligent control to enhance the performance of the controller for more complex and uncertain plants. These techniques are based on incorporating model uncertainty. The newly developed control algorithms for incorporating model uncertainty are proven to give more accurate control results under uncertain conditions. In this paper, we survey some approaches that appear to be promising for enhancing the performance of intelligent control systems in the face of higher levels of complexity and uncertainty.

Green operations strategy of a luxury car manufacturer

This paper investigates the strategic environmental decisions of a luxury car manufacturer. Through case study research, the investigation sheds light on why and how the company is adopting green technologies. Being pressured by different stakeholders to become greener, luxury car manufacturers carry significant opportunities for environmental improvement given the nature of their manufacturing processes and products. Because of their low-volume production, manufacturers may be able to increase output and still reduce overall emissions when compared to high-volume manufacturers. In the case study company this was found to be possible only because of new ideas brought by a change in ownership. Luxury manufacturers may also be a test-bed for the development and experimentation of green technologies as part of a strategic approach to environmental initiatives. This paper contributes to the fields of green technology adoption and operations strategy in automotive manufacturing groups.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

An exploration into measurement consistency on coordinate measuring machines

In high precision industry, the measurement of geometry is often performed using coordinate measuring machines (CMMs). Measurements on CMMs can occur at many places within a long and global supply chain. In this context it is a challenge to control consistency, so that measurements are applied with appropriate levels of rigour and achieve comparable results, wherever and whenever they are performed. In this paper, a framework is outlined in which consistency is controlled through measurement strategy, such as the number and location of measurement points. The framework is put to action in a case study, demonstrating the usefulness of the approach and highlighting the dangers of imposing rigid measurement strategies across the supply chain, even if linked to standardised manufacturing processes. Potential mitigations, and the requirements for future research, are outlined.

High accuracy mobile robot positioning using external large volume metrology instruments

A method of accurately controlling the position of a mobile robot using an external large volume metrology (LVM) instrument is presented in this article. By utilising an LVM instrument such as a laser tracker or indoor GPS (iGPS) in mobile robot navigation, many of the most difficult problems in mobile robot navigation can be simplified or avoided. Using the real-time position information from the laser tracker, a very simple navigation algorithm, and a low cost robot, 5mm repeatability was achieved over a volume of 30m radius. A surface digitisation scan of a wind turbine blade section was also demonstrated, illustrating possible applications of the method for manufacturing processes. Further, iGPS guidance of a small KUKA omni-directional robot has been demonstrated, and a full scale prototype system is being developed in cooperation with KUKA Robotics, UK. © 2011 Taylor & Francis.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

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 (R² = 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.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.

High accuracy mobile robot positioning using an external large volume metrology instrument

A method of accurately controlling the position of a mobile robot using an external Large Volume Metrology (LVM) instrument is presented in this paper. Utilizing a LVM instrument such as the laser tracker in mobile robot navigation, many of the most difficult problems in mobile robot navigation can be simplified or avoided. Using the real- Time position information from the laser tracker, a very simple navigation algorithm, and a low cost robot, 5mm repeatability was achieved over a volume of 30m radius. A surface digitization scan of a wind turbine blade section was also demonstrated, illustrating possible applications of the method for manufacturing processes. © Springer-Verlag Berlin Heidelberg 2010.

Optical fiber sensors embedded in flexible polymer foils

In traditional electrical sensing applications, multiplexing and interconnecting the different sensing elements is a major challenge. Recently, many optical alternatives have been investigated including optical fiber sensors of which the sensing elements consist of fiber Bragg gratings. Different sensing points can be integrated in one optical fiber solving the interconnection problem and avoiding any electromagnetical interference (EMI). Many new sensing applications also require flexible or stretchable sensing foils which can be attached to or wrapped around irregularly shaped objects such as robot fingers and car bumpers or which can even be applied in biomedical applications where a sensor is fixed on a human body. The use of these optical sensors however always implies the use of a light-source, detectors and electronic circuitry to be coupled and integrated with these sensors. The coupling of these fibers with these light sources and detectors is a critical packaging problem and as it is well-known the costs for packaging, especially with optoelectronic components and fiber alignment issues are huge. The end goal of this embedded sensor is to create a flexible optical sensor integrated with (opto)electronic modules and control circuitry. To obtain this flexibility, one can embed the optical sensors and the driving optoelectronics in a stretchable polymer host material. In this article different embedding techniques for optical fiber sensors are described and characterized. Initial tests based on standard manufacturing processes such as molding and laser structuring are reported as well as a more advanced embedding technique based on soft lithography processing.

Large volume metrology process model:measurability analysis with integration of metrology classification model and feature-based selection model

Aircraft manufacturing industries are looking for solutions in order to increase their productivity. One of the solutions is to apply the metrology systems during the production and assembly processes. Metrology Process Model (MPM) (Maropoulos et al, 2007) has been introduced which emphasises metrology applications with assembly planning, manufacturing processes and product designing. Measurability analysis is part of the MPM and the aim of this analysis is to check the feasibility for measuring the designed large scale components. Measurability Analysis has been integrated in order to provide an efficient matching system. Metrology database is structured by developing the Metrology Classification Model. Furthermore, the feature-based selection model is also explained. By combining two classification models, a novel approach and selection processes for integrated measurability analysis system (MAS) are introduced and such integrated MAS could provide much more meaningful matching results for the operators. © Springer-Verlag Berlin Heidelberg 2010.

A unified classification of manufacturing strategies and design processes

This article categorises manufacturing strategy design processes and presents the characteristics of resulting strategies. This work will therefore assist practitioners to appreciate the implications of planning activities. The article presents a framework for classifying manufacturing strategy processes and the resulting strategies. Each process and respective strategy is then considered in detail. In this consideration the preferred approach is presented for formulating a world class manufacturing strategy. Finally, conclusions and recommendations for further work are given.

Determinants of entry and exit in the foreign owned sector of UK manufacturing

This paper examines the extent to which foreign entry and exit in the UK is related to domestic industry characteristics. The units of analysis are firm numbers, and thus entry and exit at the industry level are treated as being generated by Poisson processes. This therefore uses quasimaximum likelihood estimation, to estimate entry and exit functions simultaneously. The results demonstrate that foreign entry is attracted by industry level profitability and performance, but that firm specific 'ownership' advantages are also important. The results also demonstrate that inward investors that are motivated by the desire to exploit firm-specific assets, are unlikely to be more transient than domestic firms. This however, cannot be said of those foreign entrants who are attracted to the UK by location advantage or investment incentives.