Multiplexed microfabricated cell culture device for stem cell process development

In this thesis, a multiplexed micro-fabricated cell culture device is presented to probe the soluble micro-environment of stem cells. A large number of biological, physical and chemical variables determine the micro-environment of stem cells and affect therefore their fate. The soluble micro-environment is believed to play a pivotal role in controlling stem cell fate and its optimisation may therefore allow exploitation of applications such as regenerative medicine or drug discovery. However, novel tools are required to probe and optimise the soluble micro-environment. The ability to move small volumes of liquid and the minimal use of resources are important characteristics of microfluidic systems and thus, they are perfectly suited to study the micro-environment of stem cells. A microfluidic cell culture device must ful fil three important requirements to be of utility in stem cell bioprocess development. The first aspect addresses the need for adaptability and flexibility to implement changes in microfluidic designs to take account of the rapid progress in stem cell research. To this end, a packaging solution specifi cally designed for a microfluidic cell culture device has been developed. The packaging system has thus been complemented with a rapid fabrication method using a micro-milling machine to quickly fabricate disposable and easily reconfi gurable microfluidic chips. The packaging solution has a maximum burst pressure of approximately 7.5 bar and the fabrication method has a dimensional fidelity with less than 10% deviation from the nominal value. The second aspect focuses on the scalability and comparability of results and the feasibility of continuous culture of stem cells - both critical elements for the success of a microfluidic cell culture system for stem cell bioprocess development. A novel cell seeding method has been developed using a pipette to directly and carefully seed cells into a culture chamber within a microfluidic cell culture device. To prevent a wash-out of viable cells during continuous culture, a low hydrodynamic shear stress microfluidic chip has been developed. The cell culture device has been successfully tested using human embryonic stem cells (hESC) on feeder cells. The third aspect concerns the automated monitoring of stem cell bioprocesses in the cell culture device. A multiplexed micro fluidic bioreactor platform with time-lapse imaging has been developed to obtain data-rich experimental sets. The platform consisting of a cooled media reservoir and a pumping mechanism has been characterised and tested using mouse embryonic stem cells (mESC) as a proof of concept. The combination of these three aspects provides a basis towards a multiplexed microfabricated cell culture device, which allows data-rich experimentation and comparability with current benchscale culture vessels for stem cell expansion and di fferentiation

Development of a Multiplexed Microfluidic Platform for the Automated Cultivation of Embryonic Stem Cells.

We present a multiplexed platform for a microfabricated stem cell culture device. The modular platform contains all the components to control stem cell culture conditions in an automated fashion. It does not require an incubator during perfusion culture and can be mounted on the stage of an inverted fluorescence microscope for high-frequency imaging of stem cell cultures. A pressure-driven pump provides control over the medium flow rate and offers switching of the flow rates. Flow rates of the pump are characterized for different pressure settings, and a linear correlation between the applied pressure and the flow rate in the cell culture devices is shown. In addition, the pump operates with two culture medium reservoirs, thus enabling the switching of the culture medium on-the-fly during a cell culture experiment. Also, with our platform, the culture medium reservoirs are cooled to prevent medium degradation during long-term experiments. Media temperature is then adjusted to a higher controlled temperature before entering the microfabricated cell culture device. Furthermore, the temperature is regulated in the microfabricated culture devices themselves. Preliminary culture experiments are demonstrated using mouse embryonic stem cells

O aumento da frequência de salmonella clínica Brasil.

A atividade suinícola é um expoente econômico e cultural que gera renda para famílias de agricultores, emprego direto nas agroindústrias e indireto nas atividades relacionadas, também contribui sobremaneira para o balanço comercial e PIB do país. A manutenção e acesso a mercados exigem a superação de desafios dentre os quais merecem atenção a sustentabilidade das propriedades, o bem estar dos animais, a melhoria da biosseguridade das granjas e o status sanitário do rebanho. O maior obstáculo sanitário deriva do crescente número de animais nos plantéis suinícolas aliado ao reaparecimento de enfermidades de grande impacto na cadeia de produção. Dentre as inúmeras patologias que afetam os suínos, a salmonelose clínica desponta com grande importância nos últimos anos. A contaminação por salmonelas na suinocultura se caracteriza por dois problemas: a presença de sorovares patogênicos, adaptados ao suíno, que provocam gastroenterites e septicemias e a presença de sorovares que não causam doença nos animais, mas são as principais fontes de contaminação das carcaças nos abatedouros e que podem infectar seres humanos. Porém, o foco deste relato é o aumento e caracterização da salmonelose clínica que vem ocorrendo nos últimos anos no Brasil

Comparing Galaxy Morphology at Ultraviolet and Optical Wavelengths

We have undertaken an imaging survey of 34 nearby galaxies in far-ultraviolet (FUV, ~1500A) and optical (UBVRI) passbands to characterize galaxy morphology as a function of wavelength. This sample, which includes a range of classical Hubble types from elliptical to irregular with emphasis on spirals at low inclination angle, provides a valuable database for comparison with images of high-z galaxies whose FUV light is redshifted into the optical and near- infrared bands. Ultraviolet data are from the UIT Astro-2 mission. We present images and surface brightness profiles for each galaxy, and we discuss the wavelength-dependence of morphology for different Hubble types in the context of understanding high-z objects. In general, the dominance of young stars in the FUV produces the patchy appearance of a morphological type later than that inferred from optical images. Prominent rings and circumnuclear star formation regions are clearly evident in FUV images of spirals, while bulges, bars, and old, red stellar disks are faint to invisible at these short wavelengths. However, the magnitude of the change in apparent morphology ranges from dramatic in early--type spirals with prominent optical bulges to slight in late-type spirals and irregulars, in which young stars dominate both the UV and optical emission. Starburst galaxies with centrally concentrated, symmetric bursts display an apparent ``E/S0'' structure in the FUV, while starbursts associated with rings or mergers produce a peculiar morphology. We briefly discuss the inadequacy of the optically-defined Hubble sequence to describe FUV galaxy images and estimate morphological k-corrections, and we suggest some directions for future research with this dataset.Comment: Accepted for publication in the ApJS. 15 pages, 17 JPEG figures, 10 GIF figures. Paper and full resolution figures available at http://nedwww.ipac.caltech.edu/level5/Kuchinski/frames.htm

An Investigation of Diffuse Interstellar Gas toward a Large, Low Extinction Window into the Inner Galaxy

Halpha and Hbeta spectroscopy with the Wisconsin H-Alpha Mapper (WHAM) reveals a strong concentration of high velocity emission in a ~ 5 by 5 deg area centered near (l.b) = (27,-3), known as the Scutum Cloud. The high velocities imply that we are detecting optical emission from near the plane of the Galaxy out to the tangent point at heliocentric distances of D \gtrsim 6 kpc, assuming the gas participates in circular Galactic rotation. The ratio of the Halpha to Hbeta emission as a function of velocity suggests that dust along these lines of sight produces a total visual extinction of A_v ~ 3 at D ~ 6 kpc. This makes it possible to use optical emission lines to explore the physical conditions of ionized gas in the inner Galaxy. At a Galactocentric distance R_G ~ 4 kpc, for example, we find that the H^+ has an rms midplane density of ~ 1 cm^-3 with a vertical scale height of ~ 300 pc. We also find evidence for an increase in the flux of Lyman continuum photons and an increase in the ratio of ionized to neutral hydrogen toward the inner Galaxy. We have extended the measurements of E(B-V) in this direction to distances far beyond what has been accessible through stellar photometry and find E(B-V)/N_H to be near the local mean of 1.7 x 10^-22 cm^2 mag, with evidence for an increase in this ratio at R_G ~ 4 kpc. Finally, our observations of [NII] 6583, [SII] 6716, and [OIII] 5007 toward the window reveal that in the inner Galaxy the temperature of the gas and the ionization state of oxygen increase with increasing height from the midplane.Comment: ApJ, accepted. 28 pages, 13 figures, 1 tabl

Salmonella clinical isolates from Brazilian pig herds: genetic relationship and antibiotic resistance profiling.

Abstract In Brazil, since 2011 clinical cases of salmonellosis has been increasing substantially. Nevertheless, few information is available about the antimicrobial profile, distribution, serotypes and genetic relationship among the strains. The objectives of this study were: to identify the Salmonella serotypes, to characterize the in vitro antimicrobial resistance profiles and to determine the genetic relationship of clinical isolates in Brazil. During 2016, clinical isolates of Salmonella (111) from nine States were sent to Embrapa Swine and Poultry for complementary analysis. First, isolates were serotyped by Kauffmann White Scheme. In parallel, the strains were tested against fifteen antimicrobials by disk diffusion method and genotyping was performed by Pulsed Field Gel Electrophoresis (PFGE) using the XbaI restriction enzyme. As expected, the main serovars found were Typhimurium and Choleraesuis. Four strains showed resistance to only one antimicrobial and 76.5% (85/111) were considered multiresistant. The highest level of resistance was found against to tetracycline. More than 80% of the strains were susceptible to fosfomycin, lincomycin/spectinomycin and norfloxacin. It was possible to identify one major Choleraesuis clonal group present in different Brazilian States. Further, several small clonal groups were obtained for Typhimurium. In conclusion, clinical salmonellosis caused by Typhimurium and Choleraesuis is endemic in pig production areas and the majority of the strains are multi-resistantSafePork 2017

The diagnostic value of liver biopsy

BACKGROUND: Since the introduction of molecular diagnostic tools such as markers for hepatitis C and different autoimmune diseases, liver biopsy is thought to be useful mainly for staging but not for diagnostic purposes. The aim was to review the liver biopsies for 5 years after introduction of testing for hepatitis C, in order to evaluate what diagnostic insights – if any – remain after serologic testing. METHODS: Retrospective review of all liver biopsies performed between 1.1.1995 and 31.12.1999 at an academic outpatient hepatology department. The diagnoses suspected in the biopsy note were compared with the final diagnosis arrived at during a joint meeting with the responsible clinicians and a hepatopathologist. RESULTS: In 365 patients, 411 diagnoses were carried out before biopsy. 84.4 % were confirmed by biopsy but in 8.8 %, 6.8 % and 10.5 % the diagnosis was specified, changed or a diagnosis added, respectively. Additional diagnoses of clinical relevance were unrecognized biliary obstruction and additional alcoholic liver disease in patients with chronic hepatitis C. Liver biopsy led to change in management for 12.1 % of patients. CONCLUSION: Even in the era of advanced virological, immunological and molecular genetic testing, liver biopsy remains a useful diagnostic tool. The yield is particularly high in marker negative patients but also in patients with a clear-cut prebiopsy diagnosis, liver biopsy can lead to changes in patient management

Microfabricated modular scale-down device for regenerative medicine process development.

The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development