40 research outputs found
Developing a process control strategy for the consistent and scalable manufacture of human mesenchymal stem cells
Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must
therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03· 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process
control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller
rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and
harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality
characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture
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Advanced manufacturing of carbon fiber reinforced PAEK composites : bonding and fracture mechanisms
Fiber reinforced composites are used in applications where lightweight, strength, stiffness, fatigue life, and corrosion resistance are critical. Thermoplastic composites (TPC) offer several advantages over thermosetting ones, including higher toughness, recyclability, weldability, and ease of repair. Over the last decade, the interest in in situ consolidation additive manufacturing (AM) of TPCs has increased exponentially due to their potential for rapid cycle out-of-autoclave processes. To this end, several limitations need to be resolved. Specifically in situ consolidation of TPCs usually result in low interlaminar bonding, weak matrix/fiber adhesion, high void content, and low crystallinity. This dissertation aims to provide a new understanding and solutions to these issues. The material system used in this work is carbon fiber reinforced low-melt polyarlyletherketone (LM-PAEK), processed via two AM methods: fused filament fabrication (FFF) and directed energy deposition (DED) in the form of laser-assisted automated fiber placement (AFP). By combining experiments and modeling, this work aims to develop an understanding of how processing parameters affect interlaminar bonding, void development, and crystallinity for both processes. This dissertation also investigates in situ consolidation of TPCs by examining the underlying physics that control bond strength and fracture toughness at inter-layer interfaces. Overall, this dissertation contributes to the knowledge base in the field, toward the adoption of low-energy and high-rate processes for manufacturing high-performance TPC structures.Mechanical Engineerin
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Design and testing of a device for evaluating rapid gas decompression performance in elastomers
This thesis reports on the design and assessment of a high-pressure test device for the evaluation of rapid gas decompression (RGD) performance of elastomers. RGD damage is a common problem in elastomer seals used in industry applications. RGD results from when gas is diffused in elastomers at high pressures and then is rapidly released from the elastomer when the ambient pressure is lowered rapidly. The resulting damage includes deformation, swelling, blistering, cracking, and, ultimately, failure of the elastomer
To understand the effects of different gas depressurization rates on the elastomer, a commercial off-the-shelf high-pressure test vessel was obtained and modified. Additionally, components for introducing gas, releasing the gas at different rates, heating and temperature control, pressure sensing, data acquisition, and computer control were specified to create a complete test environment. A series of tests was conducted to evaluate the performance of the system based on the NORSOK M710 standard. Damage observed in the test samples is compared to other studies found in the literature. Qualitative correlations between observed damage and test parameters are also proposed. The test system was demonstrated at pressures to 10 ksi and temperatures to 250°F. The use of an actuated micrometering valve allowed depressurization linearly at 300 psi/min. However, the system as designed suffers from a 10% pressure loss during dwell time, does not provide linear depressurization rates below 250 psi/min, and requires a lengthy calibration procedure. Suggestions for addressing these shortcomings include re-machining the system to accept the factory-specified seal and designing a feedback system to control depressurization.Mechanical Engineerin
Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate
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 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 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
Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms
In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation
Multiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells
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/CD105/CD90/HLA-DR/CD34 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. © 2013 Springer Science+Business Media Dordrecht
Development of a process control strategy for the serum-free microcarrier expansion of human mesenchymal stem cells towards cost-effective and commercially viable manufacturing
Human Mesenchymal Stem Cells (hMSCs) are advancing through clinical development with the first allogeneic adult hMSC therapy receiving approval in Europe. To enable successful large-scale manufacture of hMSC therapies, increased product consistency and yield, and a reduced batch-to-batch variation must be achieved. This paper addresses ways to reduce variation by controlling the processing conditions, in particular the dissolved oxygen concentration (dO2), and the culture medium. Bone marrow derived hMSCs were cultured in DASGIP DASbox bioreactors on Plastic P-102 L microcarriers in FBS-containing and serum free (SFM) media at various dO2 values from 100% to 10%, experiencing the same dO2 value throughout the culture process. The superior control of pH and dO2 in the bioreactor led to improved performances compared to poorly controlled spinner flasks, particularly at reduced dO2 concentrations. At 25% dO2, there was a 300 % increase in the BM-hMSC yield in the bioreactor across the two donor BM-hMSCs in SFM compared to FBS-containing medium. Overall, the process yield increased by an average of around 500% for both donors under controlled conditions in SFM at 25% dO2 in the bioreactor compared to the poorly controlled expansion at atmospheric conditions in FBS-containing medium in spinner flasks. Process control significantly reduced the BM-hMSC variation in yield from 79.1% in FBS-containing medium in spinner flasks to < 15% in controlled SFM bioreactor culture
Agitation and aeration of stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development
The impact of agitation rate and sparged aeration on BM-hMSC expansion in conventional stirred tank bioreactors was assessed. It was found that a decrease in impeller speed to below NJS caused sampling difficulties, clumping and an increase to ~2 NJS decreased the growth rate though an intermediate value of ~1.3 NJS did not. Additionally, over this range of agitation intensities, cell quality remained unchanged post-harvest suggesting that poor growth performance at the highest speed was due to a failure of the cells to attach efficiently to microcarriers rather than damage to the cells due to fluid dynamic stress. Further it was shown that direct aeration of the culture medium both with and without Pluronic F68 via a sparger at NJS was detrimental to BM-hMSC growth. Again, this reduction in growth seems to be associated with poor attachment rather than cell damage, which due to the mechanism of PluronicTM F68 reducing the cell hydrophobicity and thus the affinity of the BM-hMSCs to attach to the microcarrier, leads to a poorer performance in the presence of the surfactant. Certain post-harvest quality characteristics are also detrimentally impacted compared to headspace aeration. This problem is discussed in terms of the need to facilitate future large-scale process development where headspace aeration at NJS may not be sufficient to meet culture needs at higher cell densities
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
Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers
The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, εT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high εT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L