37 research outputs found
Fundamental differentiation and growth characterization of murine embryonic stem cells in varied culture conditions
No full textThesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references (leaves 81-83).Although embryonic stem (ES) cells and their pluripotent capability have been elucidated for decades, little study has been done on obtaining the pluripotency profile of ES cells in the incipient stages of differentiation. In this research, an ES cell line with transfected green fluorescent protein (GFP) co-expressed by an Oct-4 promoter was analyzed by fluorescence-activated cell sorter (FACS) to obtain such profile. As Oct-4 is an ES cell differentiation marker whose expression varies with pluripotency, GFP expression could simply be measured in these cells to determine how pluripotent they are as a population. The differentiation characterization of ES cells was also conducted with different culture conditions of reduced serum and glucose concentrations both in the presence and absence of leukemia inhibitory factor (LIF) which prevents spontaneous differentiation, as well as at varied LIF concentrations and seeding densities. In addition, fundamental growth kinetic and metabolic profiles were obtained to get a more complete picture of how ES cells behave under these varied culturing conditions. The doubling time (t[sub]d) of R1 Oct4-GFP cell line was found to be 13 hours in LIF⁺ culture and 8 hours in culture with LIF addition after 7 days of LIF withdrawal, implying that cell proliferation rate is higher for cells receiving a sudden upregulation of genes controlling cell division through LIF addition. Although the upregulation of the genes is rapid, the downregulation of these genes through LIF withdrawal was found to take 6-7 days, while 3-4 days were required to downregulate the pou5f gene (which controls Oct4 expression). Higher concentration of LIF resulted in higher ES cell proliferation rate, but GFP⁺ expression was unaffected by(cont.) concentration. Higher seeding density resulted in greater improvement in GFP⁺ expression for LIF⁺ culture but lower non significant reduction in GFP⁺ expression in LIF⁻ culture. Low level of glucose in medium led to reduction in the rate of ES cellular mechanisms and lower Y[sub]lac/gluc (8-49 % versus 40-60 % in high glucose), but metabolic rates were consistent with cells grown in high glucose medium, implying more efficient glucose metabolism through oxidative phosphorylation. The level of serum in medium had no effect on GFP⁺ expression or cell proliferation rate in LIF⁺ cultures, but reduction in GFP⁺ expression level was higher and t[sub]d was longer in low-serum culture (71 [plus-minus] 33 hours versus 35 [plus-minus] 9 hours) in the absence of LIF.by Yasunori Hashimura.M.Eng
Human pluripotent stem cell expansion in vertical-wheel bioreactors
No full textHuman induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates.
In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system.
In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 µm to 397 ± 119 µm after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line.
This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine.
[1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), “Scalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under Xeno‐Free Conditions Using Single‐Use Vertical‐Wheel™ Bioreactors”, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573
Scale up of pluripotent stem cell cultivation and differentiation in a novel single-use bioreactor system
No full textFlexion-extension motion assistance using an upper limb motion-assist robot based on trajectory estimation of reaching movement
No full textScale up of pluripotent stem cell cultivation and directed differentiation in a novel single-use bioreactor system
No full textEstudo da formação de rebarbas no processo de furação
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro TecnologicoInvestigação da formação de rebarbas na usinagem, mais precisamente na furação, com o objetivo de adequar o processo, visando a minimização das rebarbas. Estudo dos efeitos da velocidade de corte, do avanço e da geometria da broca sobre o tamanho e a forma das rebarbas produzidas. Experimentos realizados em chapas de alumínio 6063-O com 3 mm de espessura. Análise das rebarbas da saída do furo; por serem maiores, de mais difícil acesso, e conseqüentemente mais problemáticas em termos de facilidade de remoção. As brocas utilizadas nos experimentos são de aço rápido, com ângulos de ponta de 100º, 118º e 135º, e ângulos de hélice de 25º, 35º e 45º, todas com 10 mm de diâmetro. A avaliação das rebarbas é feita pela suas dimensões, pelo peso e pela forma que esta apresenta. Verifica-se a ocorrência de uma diminuição substancial no tamanho da rebarba quando são empregados avanços pequenos e quando se aumenta o ângulo de hélice da broca
Técnica de Wilson no tratamento do Hálux Valgus.
No full textTrabalho de Conclusão de Curso - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Departamento de Clínica Cirúrgica, Curso de Medicina, Florianópolis, 199
Distribution of LH-RH nerve endings in the median eminence of proestrus female rats: fluorescence and peroxidase anti-peroxidase(PAP) immunohistochemistry.
No full textDiseño de una Estrategia Comercial Para la Compañía Deviteck SAS
No full textSe plantea el diseño de una estrategia comercial para tener como objetivo incrementar las
ventas en cualquier época del año, se realizara la comparación con el año inmediatamente
anterior en la empresa Deviteck S.A.S para poder observar el comportamiento de las ventas a
lo largo del año, esta empresa tiene como actividad principal la importación de equipos y
tecnología para vías, túneles y peajes, por consiguiente sus ingresos provienen de las
importaciones que realizan de países como Estados Unidos, Italia, China y Alemania, la
empresa se especializa en brindar soluciones tecnológicas en las diferentes áreas de los
sistemas de transporte.
Sus productos están enfocados a fortalecer e innovar eléctricamente sistemas como:
estaciones de peajes, sistemas de control de accesos, clasificación vehicular, transporte masivo,
sistemas de visión y seguridad, vías y túneles, equipos de modernización de los sistemas de
transporte; estas tecnologías cuentan con: radares de ocupación y velocidad, sensores de
tráfico, sensores para túneles con tecnología de punta para modernizar, monitorear y controlar
el tráfico vehicular.
Se toma como referencia Deviteck S.A.S porque actualmente presenta una variedad de
falencias para llevar acabo su actividad comercial eficientemente, como primera medida se observa que al pertenecer al sector importador, suele presentar demoras en la entrega de
pedidos ya sea por retrasos en fábrica o en los procesos de nacionalización de la mercancía, por
lo general un pedido tarda en entrar al territorio nacional, entre 30 y 40 días lo que es un
inconveniente para el cliente, porque en ocasiones requieren los productos para entrega
inmediata, esto ha causado la pérdida de clientes, el quebranto de las relaciones comerciales, y
demuestra que la empresa no cuenta con políticas adecuadas de atención al cliente, por otra
parte el cambio de infraestructura vial, por el que está atravesando el país en el momento, ha
generado que los peajes estén en reestructuración, lo que significa que se encuentran en
cambios transitorios de administración, impidiendo que estas realicen compras frecuentes; por
estas razones se ha reflejado un decrecimiento en la ventas de la empresa.
Por medio del diseño de una estrategia comercial se pretende lograr un incremento en ventas
de la compañía, que se pueda utilizar en cualquier época del año, para el planteamiento del
diseño de la estrategia comercial, se toman inicialmente los datos proporcionado bs por los
representantes de la empresa, como los son: la información pertinente de los procesos de sus
productos, asesores comerciales, datos de sus clientes, datos históricos de ventas y estados
financieros, para la recolección de información con el fin llevar a cabo el diseño de la
estrategia, se realizará el impacto directamente en el área comercial, ya que desde allí se
integra la solución, para obtener una relación estable y duradera con los consumidores de los
diferentes productos, que oferta la empresa, teniendo en cuenta que se pretende captar,
convencer y conservar tanto los clientes actuales como los posibles prospectos, además se hará
la valoración de los productos que cuentan con una mayor rotación en inventario, lo que permitirá conocer las necesidades de los clientes, y los productos que se requieren tener a
disposición inmediata.PregradoProfesional en Finanzas y Negocios InternacionalesGestión Empresaria
