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.

Design and development of a diagonal-airflow batch dryer for spatial drying homogeneity

Das Verfahren der Lebensmitteltrocknung wird häufig angewendet, um ein Produkt für längere Zeit haltbar zu machen. Obst und Gemüse sind aufgrund ihres hohen Wassergehalts leicht verderblich durch biochemische Vorgänge innerhalb des Produktes, nicht sachgemäße Lagerung und unzureichende Transportmöglichkeiten. Um solche Verluste zu vermeiden wird die direkte Trocknung eingesetzt, welche die älteste Methode zum langfristigen haltbarmachen ist. Diese Methode ist jedoch veraltet und kann den heutigen Herausforderungen nicht gerecht werden. In der vorliegenden Arbeit wurde ein neuer Chargentrockner, mit diagonalem Luftstömungskanal entlang der Länge des Trocknungsraumes und ohne Leitbleche entwickelt. Neben dem unbestreitbaren Nutzen der Verwendung von Leitblechen, erhöhen diese jedoch die Konstruktionskosten und führen auch zu einer Erhöhung des Druckverlustes. Dadurch wird im Trocknungsprozess mehr Energie verbraucht. Um eine räumlich gleichmäßige Trocknung ohne Leitbleche zu erreichen, wurden die Lebensmittelbehälter diagonal entlang der Länge des Trockners platziert. Das vorrangige Ziel des diagonalen Kanals war, die einströmende, warme Luft gleichmäßig auf das gesamte Produkt auszurichten. Die Simulation des Luftstroms wurde mit ANSYS-Fluent in der ANSYS Workbench Plattform durchgeführt. Zwei verschiedene Geometrien der Trocknungskammer, diagonal und nicht diagonal, wurden modelliert und die Ergebnisse für eine gleichmäßige Luftverteilung aus dem diagonalen Luftströmungsdesign erhalten. Es wurde eine Reihe von Experimenten durchgeführt, um das Design zu bewerten. Kartoffelscheiben dienten als Trocknungsgut. Die statistischen Ergebnisse zeigen einen guten Korrelationskoeffizienten für die Luftstromverteilung (87,09%) zwischen dem durchschnittlich vorhergesagten und der durchschnittlichen gemessenen Strömungsgeschwindigkeit. Um den Effekt der gleichmäßigen Luftverteilung auf die Veränderung der Qualität zu bewerten, wurde die Farbe des Produktes, entlang der gesamten Länge der Trocknungskammer kontaktfrei im on-line-Verfahren bestimmt. Zu diesem Zweck wurde eine Imaging-Box, bestehend aus Kamera und Beleuchtung entwickelt. Räumliche Unterschiede dieses Qualitätsparameters wurden als Kriterium gewählt, um die gleichmäßige Trocknungsqualität in der Trocknungskammer zu bewerten. Entscheidend beim Lebensmittel-Chargentrockner ist sein Energieverbrauch. Dafür wurden thermodynamische Analysen des Trockners durchgeführt. Die Energieeffizienz des Systems wurde unter den gewählten Trocknungsbedingungen mit 50,16% kalkuliert. Die durchschnittlich genutzten Energie in Form von Elektrizität zur Herstellung von 1kg getrockneter Kartoffeln wurde mit weniger als 16,24 MJ/kg und weniger als 4,78 MJ/kg Wasser zum verdampfen bei einer sehr hohen Temperatur von jeweils 65°C und Scheibendicken von 5mm kalkuliert. Die Energie- und Exergieanalysen für diagonale Chargentrockner wurden zudem mit denen anderer Chargentrockner verglichen. Die Auswahl von Trocknungstemperatur, Massenflussrate der Trocknungsluft, Trocknerkapazität und Heiztyp sind die wichtigen Parameter zur Bewertung der genutzten Energie von Chargentrocknern. Die Entwicklung des diagonalen Chargentrockners ist eine nützliche und effektive Möglichkeit um dei Trocknungshomogenität zu erhöhen. Das Design erlaubt es, das gesamte Produkt in der Trocknungskammer gleichmäßigen Luftverhältnissen auszusetzen, statt die Luft von einer Horde zur nächsten zu leiten.

Diagnostic Tests and their Application in the Management of Soil- and Water-borne Oomycete Pathogen Species

Oomycete diseases cause significant losses across a broad range of crop and aquaculture commodities worldwide. These losses can be greatly reduced by disease management practices steered by accurate and early diagnoses of pathogen presence. Determinations of disease potential can help guide optimal crop rotation regimes, varietal selections, targeted control measures, harvest timings and crop post-harvest handling. Pathogen detection prior to infection can also reduce the incidence of disease epidemics. Classical methods for the isolation of oomycete pathogens are normally deployed only after disease symptom appearance. These processes are often-time consuming, relying on culturing the putative pathogen(s) and the availability of expert taxonomic skills for accurate identification; a situation that frequently results in either delayed application, or routine ‘blanket’ over-application of control measures. Increasing concerns about pesticides in the environment and the food chain, removal or restriction of their usage combined with rising costs have focussed interest in the development and improvement of disease management systems. To be effective, these require timely, accurate and preferably quantitatve diagnoses. A wide range of rapid diagnostic tools, from point of care immunodiagnostic kits to next generation nucleotide sequencing have potential application in oomycete disease management. Here we review currently-available as well as promising new technologies in the context of commercial agricultural production systems, considering the impacts of specific biotic and abiotic and other important factors such as speed and ease of access to information and cost effectiveness

Monitoring Infection Risk for Air and Soil Borne Fungal Plant Pathogens using Antibody and DNA Techniques and Mathematical Models describing Environmental Parameters

Economic losses resulting from disease development can be reduced by accurate and early detection of plant pathogens. Early detection can provide the grower with useful information on optimal crop rotation patterns, varietal selections, appropriate control measures, harvest date and post harvest handling. Classical methods for the isolation of pathogens are commonly used only after disease symptoms. This frequently results in a delay in application of control measures at potentially important periods in crop production. This paper describes the application of both antibody and DNA based systems to monitor infection risk of air and soil borne fungal pathogens and the use of this information with mathematical models describing risk of disease associated with environmental parameters.

Interface of the Environment and Occurrence of Botrytis cinerea in Pre-symptomatic Tomato Crops

Botrytis cinerea (Grey mould) is a necrotrophic fungus infecting over 230 plant species worldwide. It can cause major pre- and post-harvest diseases of many agronomic and horticultural crops. Botrytis cinerea causes annual economic losses of 10–100 billion US dollars worldwide and instability in the food supply (Jin and Wu, 2015). Grey mould losses, either at the farm gate or later in the food chain, could be reduced with improved knowledge of inoculum availability during production. In this paper, we report on the ability to monitor Botrytis spore concentration in glasshouse tomato production ahead of symptom development on plants. Using a light weight and portable air sampler (microtitre immunospore trap) it was possible to quantify inoculum availability within hours. Also, this study investigated the spatial aspect of the pathogen with an increase of B. cinerea concentration in bio-aerosols collected in the lower part of the glasshouse (0.5 m) and adjacent to the trained stems of the tomato plants. No obvious relationship was observed between B. cinerea concentration and the internal glasshouse environmental parameters of temperature and relative humidity. However the occurrence of higher outside wind speeds did increase the prevalence of B. cinerea conidia in the cropping environment of a vented glasshouse. Knowledge of inoculum availability at time periods when the environmental risk of pathogen infection is high should improve the targeted use and effectiveness of control inputs.

Fish handling, processing and storage in Uganda: the role of the extension worker

The handling and processing of fish in Uganda has until recently been carried out exclusively by the artisanal fishermen and fish processors. Their operations have left much to be desired as the product is often of low quality 'and its keeping time is limited. The handling of fresh fish has been without refrigeration but with the recent establishment of commercial fish processing plants a cold chain of fish distribution is being set up for domestic and export markets. Some of the fishermen are beginning to ice their catch immediately after reaching the shore. It is hoped that fishmongers will increasingly find it more profitable to market their products iced. This will make fish available to a large sector of the population and in the process there will be reduced post-harvest losses.

Development and Application of Predictive Models for Survival, Growth, and Death of Enteric Pathogens in Leafy Greens Supply Chain

Leafy greens are essential part of a healthy diet. Because of their health benefits, production and consumption of leafy greens has increased considerably in the U.S. in the last few decades. However, leafy greens are also associated with a large number of foodborne disease outbreaks in the last few years. The overall goal of this dissertation was to use the current knowledge of predictive models and available data to understand the growth, survival, and death of enteric pathogens in leafy greens at pre- and post-harvest levels. Temperature plays a major role in the growth and death of bacteria in foods. A growth-death model was developed for Salmonella and Listeria monocytogenes in leafy greens for varying temperature conditions typically encountered during supply chain. The developed growth-death models were validated using experimental dynamic time-temperature profiles available in the literature. Furthermore, these growth-death models for Salmonella and Listeria monocytogenes and a similar model for E. coli O157:H7 were used to predict the growth of these pathogens in leafy greens during transportation without temperature control. Refrigeration of leafy greens meets the purposes of increasing their shelf-life and mitigating the bacterial growth, but at the same time, storage of foods at lower temperature increases the storage cost. Nonlinear programming was used to optimize the storage temperature of leafy greens during supply chain while minimizing the storage cost and maintaining the desired levels of sensory quality and microbial safety. Most of the outbreaks associated with consumption of leafy greens contaminated with E. coli O157:H7 have occurred during July-November in the U.S. A dynamic system model consisting of subsystems and inputs (soil, irrigation, cattle, wildlife, and rainfall) simulating a farm in a major leafy greens producing area in California was developed. The model was simulated incorporating the events of planting, irrigation, harvesting, ground preparation for the new crop, contamination of soil and plants, and survival of E. coli O157:H7. The predictions of this system model are in agreement with the seasonality of outbreaks. This dissertation utilized the growth, survival, and death models of enteric pathogens in leafy greens during production and supply chain.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

In vitro experimental environments are used to study interactions between microorganisms, and predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to closely match the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation was studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25oC and 30oC). Competition experiments showed interaction between main effects of aflatoxin accumulation and environment at 25oC, but not so at 30oC. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-hour incubation in different experimental environments. Whereas, all fungi incubated within the soil environment survived; in the cotton-wool environment, none of the competitors of A. flavus survived at 30 oC. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post harvest.