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.

Mechanical properties of sheet metal under forming conditions

In the bulge test, a sheet metal specimen is clamped over a circular hole in a die and formed into a bulge by the hydraulic pressure on one side of the specirnen. As the unsupported part of the specimen is deformed in this way, its area is increased, in other words, the material is generally stretched and its thickness generally decreased. The stresses causing this stretching action are the membrane stresses in the shell generated by the hydraulic pressure, in the same way as the rubber in a toy balloon is stretched by the membrane stresses caused by the air inside it. The bulge test is a widely used sheet metal test, to determine the "formability" of sheet materials. Research on this forming process (2)-(15)* has hitherto been almost exclusively confined to predicting the behaviour of the bulged specimen through the constitutive equations (stresses and strains in relation to displacements and shapes) and empirical work hardening characteristics of the material as determined in the tension test. In the present study the approach is reversed; the stresses and strains in the specimen are measured and determined from the geometry of the deformed shell. Thus, the bulge test can be used for determining the stress-strain relationship in the material under actual conditions in sheet metal forming processes. When sheet materials are formed by fluid pressure, the work-piece assumes an approximately spherical shape, The exact nature and magnitude of the deviation from the perfect sphere can be defined and measured by an index called prolateness. The distribution of prolateness throughout the workpiece at any particular stage of the forming process is of fundamental significance, because it determines the variation of the stress ratio on which the mode of deformation depends. It is found. that, before the process becomes unstable in sheet metal, the workpiece is exactly spherical only at the pole and at an annular ring. Between the pole and this annular ring the workpiece is more pointed than a sphere, and outside this ring, it is flatter than a sphere. In the forming of sheet materials, the stresses and hence the incremental strains, are closely related to the curvatures of the workpiece. This relationship between geometry and state of stress can be formulated quantitatively through prolateness. The determination of the magnitudes of prolateness, however, requires special techniques. The success of the experimental work is due to the technique of measuring the profile inclination of the meridional section very accurately. A travelling microscope, workshop protractor and surface plate are used for measurements of circumferential and meridional tangential strains. The curvatures can be calculated from geometry. If, however, the shape of the workpiece is expressed in terms of the current radial (r) and axial ( L) coordinates, it is very difficult to calculate the curvatures within an adequate degree of accuracy, owing to the double differentiation involved. In this project, a first differentiation is, in effect, by-passed by measuring the profile inclination directly and the second differentiation is performed in a round-about way, as explained in later chapters. The variations of the stresses in the workpiece thus observed have not, to the knowledge of the author, been reported experimentally. The static strength of shells to withstand fluid pressure and their buckling strength under concentrated loads, both depend on the distribution of the thickness. Thickness distribution can be controlled to a limited extent by changing the work hardening characteristics of the work material and by imposing constraints. A technique is provided in this thesis for determining accurately the stress distribution, on which the strains associated with thinning depend. Whether a problem of controlled thickness distribution is tackled by theory, or by experiments, or by both combined, the analysis in this thesis supplies the theoretical framework and some useful experimental techniques for the research applied to particular problems. The improvement of formability by allowing draw-in can also be analysed with the same theoretical and experimental techniques. Results on stress-strain relationships are usually represented by single stress-strain curves plotted either between one stress and one strain (as in the tension or compression tests) or between the effective stress and effective strain, as in tests on tubular specimens under combined tension, torsion and internal pressure. In this study, the triaxial stresses and strains are plotted simultaneously in triangular coordinates. Thus, both stress and strain are represented by vectors and the relationship between them by the relationship between two vector functions. From the results so obtained, conclusions are drawn on both the behaviour and the properties of the material in the bulge test. The stress ratios are generally equal to the strain-rate ratios (stress vectors collinear with incremental strain vectors) and the work-hardening characteristics, which apply only to the particular strain paths are deduced. Plastic instability of the material is generally considered to have been reached when the oil pressure has attained its maximum value so that further deformation occurs under a constant or lower pressure. It is found that the instability regime of deformation has already occurred long before the maximum pressure is attained. Thus, a new concept of instability is proposed, and for this criterion, instability can occur for any type of pressure growth curves.