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

Ecological and evolutionary processes at expanding range margins

Many animals are regarded as relatively sedentary and specialized in marginal parts of their geographical distributions. They are expected to be slow at colonizing new habitats. Despite this, the cool margins of many species' distributions have expanded rapidly in association with recent climate warming. We examined four insect species that have expanded their geographical ranges in Britain over the past 20 years. Here we report that two butterfly species have increased the variety of habitat types that they can colonize, and that two bush cricket species show increased fractions of longer-winged (dispersive) individuals in recently founded populations. Both ecological and evolutionary processes are probably responsible for these changes. Increased habitat breadth and dispersal tendencies have resulted in about 3- to 15-fold increases in expansion rates, allowing these insects to cross habitat disjunctions that would have represented major or complete barriers to dispersal before the expansions started. The emergence of dispersive phenotypes will increase the speed at which species invade new environments, and probably underlies the responses of many species to both past and future climate change

The effect of metapopulation processes on the spatial scale of adaptation across an environmental gradient

We show that the butterfly Aricia agestis (Lycaenidae) is adapted to its thermal environment in via integer changes in the numbers of generations per year (voltinism): it has two generations per year in warm habitats and one generation per year in cool habitats in north Wales (UK). Voltinism is an “adaptive peak” since individuals having an intermediate number of generations per year would fail to survive the winter, and indeed no populations showed both voltinism types in nature. In spite of this general pattern, 11% of populations apparently possess the “wrong” voltinism for their local environment, and population densities were lower in thermally intermediate habitat patches. Population dynamic data and patterns of genetic differentiation suggest that adaptation occurs at the metapopulation level, with local populations possessing the voltinism type appropriate for the commonest habitat type within each population network. When populations and groups of populations go extinct, they tend to be replaced by colonists from the commonest thermal environment nearby, even if this is the locally incorrect adaptation. Our results illustrate how stochastic population turnover can impose a limit on local adaptation over distances many times larger than predicted on the basis of normal dispersal movements

Geochemical-geophysical investigations, Fairbanks district

Trace element distribution in a subarctic valley in the Cleary Hill area of the Fairbanks gold district has been studied. Zinc and arsenic have been found excellent pathfinder elements for auriferous deposits. Methods of analysis for copper, lead, zinc, molybdenum, silver and arsenic as well as heavy metals are discussed. The University of Alaska method #2 has been improved, Terrain, slope, and frozen ground have little effect upon the distribution of trace elements associated with the Cleary H i l l vein. A new method for the determination of zinc using dilute acid is proposed. Analysis of geochemical data by trend surface procedures proved effective for localization of anomalies

Chromospheric oscillations

We show results from SO/Sacramento Peak data to discuss three issues: (i)--the spatial occurrence of chromospheric 3--min oscillations; (ii)--the validity of Ca II H&K line-center Doppler Shift measurements; (iii)--the signi ?cance of oscillation power and phase at frequencies above 10 mHz

Use of experimental design to investigate processing conditions and K value effects in poly(vinyl chloride) window profile extrusion

Statistical experimental design has been used on a laboratory scale, twin screw extruder to investigate how processing conditions and polymer K value affect both the extrusion process and important extrudate properties: viz. Charpy impact strength, colour, gloss, and degree of gelation. It is demonstrated how this approach can be used to define the optimum processing window. Although in production situations it may be impractical or too expensive to vary large numbers of variables over a wide processing range, it is possible to use the experimental design approach without initiating unwieldy experimental programmes by using Evolutionary Operation. This is illustrated with reference to extrusion line trials

Precise numerical results for limit cycles in the quantum three-body problem

The study of the three-body problem with short-range attractive two-body forces has a rich history going back to the 1930's. Recent applications of effective field theory methods to atomic and nuclear physics have produced a much improved understanding of this problem, and we elucidate some of the issues using renormalization group ideas applied to precise nonperturbative calculations. These calculations provide 11-12 digits of precision for the binding energies in the infinite cutoff limit. The method starts with this limit as an approximation to an effective theory and allows cutoff dependence to be systematically computed as an expansion in powers of inverse cutoffs and logarithms of the cutoff. Renormalization of three-body bound states requires a short range three-body interaction, with a coupling that is governed by a precisely mapped limit cycle of the renormalization group. Additional three-body irrelevant interactions must be determined to control subleading dependence on the cutoff and this control is essential for an effective field theory since the continuum limit is not likely to match physical systems ({\it e.g.}, few-nucleon bound and scattering states at low energy). Leading order calculations precise to 11-12 digits allow clear identification of subleading corrections, but these corrections have not been computed.Comment: 37 pages, 8 figures, LaTeX, uses graphic

Massive hiatus hernia: Evaluation and surgical management

AbstractObjective: Paraesophageal hernias represent advanced degrees of sliding hiatus hernia with intrathoracic displacement of the intraesophageal junction. Gastroesophageal reflux disease occurs in most cases, resulting in acquired short esophagus, which should influence the type of repair selected. Methods: Between 1960 and 1996, 94 patients with massive, incarcerated paraesophageal hiatus hernia were operated on at the Toronto General Hospital. The mean age was 64 years (39 to 85 years), with a female to male ratio of 1.8:1. Organoaxial volvulus was present in 50% of cases. Clinical presentation in these patients included postprandial pain in 56%, dysphagia in 48%, chronic iron deficiency anemia in 38%, and aspiration in 29%. Symptomatic reflux, either present or remote, was recorded in 83% of cases. All patients underwent endoscopy by the operating surgeon. In 91 of 94 patients, the esophagogastric junction was found to be above the diaphragmatic hiatus, denoting a sliding type of hiatus hernia. Gross, endoscopic peptic esophagitis was observed in 36% of patients: ulcerative esophagitis in 22% and peptic esophagitis with stricture in 14%. A complete preoperative esophageal motility study was obtained for 41 patients. The lower sphincter was hypotensive in 21 patients (51%), and the amplitude of peristalsis in the distal esophagus was diminished in 24 patients (59%). These abnormalities are both features of significant gastroesophageal reflux disease. In 13 recent, consecutive patients with paraesophageal hernia, the distance between the upper and lower esophageal sphincters was measured during manometry. The average distance was 15.4 ± 2.33 cm (11 to 20 cm), which is consistent with acquired short esophagus. The normal distance is 20.4 cm ± 1.9 (p < 0.0001). Results: All 94 patients were treated surgically: 97% had a transthoracic repair with fundoplication. A gastroplasty was added in 75 cases (80%) because of clearly defined or presumed short esophagus. There were two operative deaths, and two patients were never followed up. Among the 90 available patients, the mean follow-up was 94 months; median follow-up was 72 months. Seventy-two patients (80%) are free of symptoms (excellent result); 13 (13%) have inconsequential symptoms requiring no therapy (good result); and three patients (4%) are improved but have symptoms requiring medical therapy or interval dilatation (fair result). Two patients had poor results because of recurrent hernia and severe reflux. Both were successfully treated by reoperation with the addition of gastroplasty because of acquired shortening, which was not recognized at the first operation. Conclusions: Most of these 94 patients had symptoms or endoscopic, manometric, and operative findings that were consistent with a sliding hiatus hernia. There was a high incidence of endoscopic reflux esophagitis and of acquired short esophagus. True paraesophageal hernia, with the esophagogastric junction in a normal abdominal location, appears rare. Our observations were supported by measurements obtained at preoperative endoscopy and manometry, and by findings at the time of surgical repair. These observations support the choice of a transthoracic approach for repair in most patients. (J Thorac Cardiovasc Surg 1997;115:53-62

Phenomena exposure from the large scale gas injection test (Lasgit) dataset using a bespoke data analysis toolkit

The Large Scale Gas Injection Test (Lasgit) is a field-scale experiment designed to study the impact of gas buildup and subsequent migration through an engineered barrier system. Lasgit has a substantial experimental dataset containing in excess of 21 million datum points. The dataset is anticipated to contain a wealth of information, ranging from long-term trends and system behaviours to small-scale or ‘second-order’ features. In order to interrogate the Lasgit dataset, a bespoke computational toolkit, designed to expose difficult to observe phenomena, has been developed and applied to the dataset. The preliminary application of the toolkit, presented here, has resulted in a large number of phenomena being indicated/quantified, including highlighting of second-order events (small gas flows, perturbations in stress/pore-water sensors, etc.) and quantification of temperature record frequency content. Localized system behaviour has been shown to occur along with systematic aberrant behaviours that remain unexplained