Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency

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 2 = 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

The Warnie volcanic province : Jurassic intraplate volcanism in Central Australia

We wish to thank Santos Ltd. for providing us with the Snowball 3D seismic survey. In particular we wish to thank Jenni Clifford and Lance Holmes who provided helpful feedback and 2D seismic lines covering the Lambda 1, Orientos 2 and Warnie East 1 wells. We also wish to thank Beach Energy, in particular Rob Menpes, for the helpful discussions and feedback on the manuscript in addition to helping us with the analysis of the magnetic data. The work contained in this paper contains work conducted during a PhD study undertaken as part of the Natural Environment Research Council (NERC) Centre for Doctoral Training (CDT) in Oil & Gas [grant number NEM00578X/1] and is fully funded by NERC whose support is gratefully acknowledged. Lastly, the two anonymous reviews of the manuscript are thanked for their insightful and constructive comments that significantly improved the work presented.Peer reviewedPostprin

Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury

Quantification of basin-floor fan pinchouts: examples from the Karoo Basin, South Africa

The topography of the seabed (orientation and gradient) and rheology of the flows greatly influences the character of basin-floor turbidity current deposits. Therefore, submarine fan pinchouts can help to constrain seabed topography and flow behavior at the time of deposition. Although the depositional architecture of submarine lobe pinchouts has been documented in various basin-fills, the quantification of the rates of change at pinchouts in different paleogeographic positions and basin configurations has not been attempted previously. Here, we utilize extensive outcrops and research boreholes from the oblique up-dip pinchout of Fans 3 and 4 and the lateral pinchout of Fan 3 in the Tanqua depocenter, Karoo Basin, South Africa, to compare sedimentary facies and to quantify the rates of change in gross interval thickness. At the oblique up-dip pinchout, Fan 3 thins abruptly at a rate of 12 m/km, while Fan 4 thins at a rate of 4 m/km. Marked differences between Fans 3 and 4 in sedimentary facies and architecture toward the up-dip pinchout, with termination of lobes in Fan 3 and a channel-lobe transition zone and external levee in Fan 4, suggests progradation of the system. The thinning rate of the lateral pinchout of Fan 3 is 2 m/km, with the presence of hybrid beds in the lower part of Fan 3, while the upper part is dominated by structured sandstones and thin-bedded heterolithics. The variations in facies suggest that lobe-scale frontal and lateral pinchouts are stacked at the lobe complex-scale lateral pinchout of Fan 3, highlighting the importance of a hierarchical understanding when studying basin-floor fan pinchouts. The quantified rates of change in fan thickness and sedimentology on the oblique up-dip and lateral fan pinchouts are markedly different. Contrasting pinchout architecture above slopes with subtle differences in gradient and orientation cautions against the simple definition of reservoir input parameters for stratigraphic traps in submarine fan systems

Shelf-margin clinothem progradation, degradation and readjustment: Tanqua Depocentre, Karoo Basin (South Africa)

Degradation of basin-margin clinothems around the shelf-edge rollover zone may lead to the generation of conduits through which gravity flows transport sediment downslope. Many studies from seismic-reflection datasets show these features, but they lack small-scale (centimetre to metre) sedimentary and stratigraphic observations on process interactions. Exhumed basin-margin clinothems in the Tanqua depocentre (Karoo Basin) provide seismic-reflection-scale geometries and internal details of architecture with depositional dip and strike control. At the Geelhoek locality, clinothem parasequences comprise siltstone-rich offshore deposits overlain by heterolithic prodelta facies and sandstone-dominated deformed mouth bars. Three of these parasequences are truncated by a steep (6 to 22°), 100 m deep and 1.5 km wide asymmetrical composite erosion surface that delineates a shelf-edge canyon. The fill, from base to top comprises: (i) thick-bedded sandstone with intrabasinal clasts and multiple erosion surfaces; (ii) scour-based interbedded sandstone and siltstone with tractional structures; and (iii) inverse-graded to normal-graded siltstone beds. An overlying 55 m thick coarsening-upward parasequence fills the upper section of the canyon and extends across its interfluves. Younger parasequences display progressively shallower gradients during progradation and healing of the local accommodation. The incision surface resulted from initial oversteepening and high sediment supply triggering deformation and collapse at the shelf edge, enhanced by a relative sea-level fall that did not result in subaerial exposure of the shelf edge. Previous work identified an underlying highly incised, sandstone-rich shelf-edge rollover zone across-margin strike, suggesting that there was migration in the zone of shelf-edge to upper slope incision over time. This study provides an unusual example of clinothem degradation and re-adjustment with three-dimensional control in an exhumed basin margin succession. The work demonstrates that large-scale erosion surfaces can develop and migrate due to a combination of factors at the shelf-edge rollover zone, and proposes additional criteria to predict clinothem incision and differential sediment bypass in consistently progradational systems

Evolutionary diversification of new caledonian Araucaria

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a ‘museum’ for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.Mai Lan Kranitz, Edward Biffin, Alexandra Clark, Michelle L. Hollingsworth, Markus Ruhsam, Martin F. Gardner, Philip Thomas, Robert R. Mill, Richard A. Ennos, Myriam Gaudeul, Andrew J. Lowe, Peter M. Hollingswort