Synthesis With Hypergraphs

Many problems related to synthesis with intelligent tutoring may be phrased as program synthesis problems using AI-style search and formal reasoning techniques. The _x000C_first two results in this dissertation focus on problem synthesis as an aspect of intelligent tutoring systems applied to STEM-based education frameworks, specifically high school geometry. Given a geometric _x000C_figure as input, our technique constructs a hypergraph representing logical deduction of facts, and then traverses the hypergraph to synthesize problems and their corresponding solutions. Using similar techniques, our third result is focused on exhaustive synthesis of molecules. This synthesis process involves bonding sets of basic, molecular `fragments\u27 according to chemical constraints to create molecules of increasing size. For each input set of fragments, synthesis results in a significant set of molecules. Due to big data constraints we give special consideration in how to construct a corresponding molecular hypergraph based on a target, template molecule. Synthesis of the target molecule in a laboratory environment then corresponds to any path in the molecular hypergraph from the set of fragments to the target molecule

Information Flow and Staff Contact: A Quick Evaluation of Financial Aid

The authors examine the relationship between financial aid staff and students and attempt to analyze how the experience at one institution might provide some insight. The problem is viewed from the client perspective based upon day to day contact with various services provided to students by the financial aid office

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

Expansion of human mesenchymal stem cells on microcarriers

The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, NJS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml−1 (ca. 1 g dry weight l−1), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at NJS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence

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

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

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

Qualitative and quantitative demonstration of bead-to-bead transfer with bone marrow-derived human mesenchymal stem cells on microcarriers:utilising the phenomenon to improve culture performance

Human mesenchymal stem cells (hMSCs) are a key candidate for advanced cell therapies with numerous clinical trials investigating their potential to treat acute and chronic indications. However, important translational and manufacturing challenges need to be addressed to improve our capability for scalable production of fully functional cells. In this study, we have demonstrated, both qualitatively and quantitatively, the ability of bone marrow-derived hMSCs to migrate from one microcarrier to another, and, to populate fresh microcarriers when added into suspension culture. Additionally, we have shown that compared to inoculating a culture with cells in free suspension, inoculating 10% of near-confluent microcarriers from an initial seed microcarrier culture resulted in an increase in the cell growth rate and overall cell yield and a significant reduction in the lag phase. These findings were consistent across cells from three different BM-hMSC donors and across different culture medium conditions, foetal bovine serum-supplemented medium, human platelet lysate-supplemented medium and serum-free medium. This new cells-on-beads inoculation method is an effective means of process intensification with the potential to decrease manufacturing times and potentially costs of hMSC-based therapies

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

Conservation Status of the Southern Appalachian Herpetofauna

Seventy one species of amphibians (55 salamanders, 16 anurans) and 46 species of reptiles (15 turtles, 8 lizards, 23 snakes) inhabit a five state area (Kentucky, North Carolina, Tennessee, Virginia, West Virginia) in the southern Appalachian region bordered by the Potomac River, the Blue Ridge Mountains, and the western margin of the Appalachian Plateau. Of these, 47.9 % of the amphibian fauna and 52.2 % of the reptilian fauna are listed as being of conservation concern by federal, state, and Natural Heritage programs in all or a portion of their ranges of this region. The Shenandoah salamander (Plethodon Shenandoah) is listed as Endangered and the Cheat Mountain salamander (Plethodon Shenandoah) is listed as Endangered and the Cheat Mountain salamander (Plethodon nettingi) is listed as Threatened under the U.S. Endangered Species Act. Nine others are classified as federal species at risk. State endangered species number 1-3 (per state), threatened 1-4, and special concern or declining 6-19. Three to 6 species per state are additionally listed as natural heritage S1 and 2-13 as S2. We review the existing and potential threats to species and populations (e.g., timbering, urbanization, collection for the wildlife trade, acid precipitation, introduced species) and provide an assessment of the conservation status of the southern Appalachian herpetofauna based on land ownership