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Hematopoietic differentiation of mouse embryonic stem cells in rotary and stirred tank bioreactorsFridley, Krista Marie 14 February 2012 (has links)
Embryonic stem (ES) cells provide a potentially unlimited cell source for cellular therapies; however, reliable methods must be developed to provide clinically-relevant numbers of homogeneous therapeutic cell populations. Dynamic cultures may encourage ES cell differentiation and amenable to large-scale cell production. Our goal was to optimize dynamic culture parameters (bioreactor type, speed, cell seeding density, conditioned medium, and hypoxia) to maximize the generation of hematopoietic stem and progenitor cells (HSPCs) from ES cells and also to investigate the ability of dynamic culture-derived HSPCs to generate terminally differentiated hematopoietic cells. Our results indicate that varying cell seeding density and speed in two different bioreactors significantly affects embryoid body formation and ES cell differentiation efficiency into progenitor cells. In general, increased cell seeding density generated higher percentages of HSPCs in both bioreactors. In addition, rotary (Synthecon) bioreactors produced more sca-1⁺ progenitors, and spinner flasks generated more c-kit⁺ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that unique gene expression profiles were observed in the two bioreactors with the expression of specific hematopoietic genes more up regulated in the Synthecon cultures compared to spinner flasks. Combining bioreactor cultures with directed differentiation strategies via conditioned medium and hypoxic culture may further encourage hematopoietic differentiation. Dynamically cultured ES cell-derived hematopoietic stem and progenitor cells were further differentiated into a phenotype typical of dendritic cells which had the ability to process antigen. Additionally, microarray analysis of isolated ES cell-derived HSPCs demonstrated differences in the gene expression from native HSCs isolated from the fetal liver or bone marrow of mice. Insight gained from this work should be continued by comparing the differentiation efficiency of HSPCs derived in dynamic and traditional static culture methods into functional, terminally differentiated hematopoietic cells to generate clinically-relevant numbers of transplantable, therapeutic cells. / text
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Flow Characterization and Modeling of Cartilage Development in a Spinner-Flask BioreactorSucosky, Philippe 30 March 2005 (has links)
Bioreactors are devices used for the growth of tissues in a laboratory environment. They exist in many different forms, each designed to enable the production of high-quality tissues. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. Chondrocytes, the building blocks of articular cartilage, for example, are stimulated by mechanical stresses such as shear, as compared with those in tissues grown under static incubation conditions. On the other hand, high shear can damage cells. Consequently the shear-stress level has to be controlled in order to optimize the design and the operating conditions of bioreactors.
Spinner flasks have been used for the production of articular cartilage in vitro. Assuming the existence of a relation between the cellular glycosaminoglycan (GAG) synthesis and the local shear stresses on the construct surfaces, this research focuses on the development of a model for cartilage growth in such devices. The flow produced in a model spinner flask is characterized experimentally using particle-image velocimetry (PIV). A computational fluid dynamic (CFD) model validated with respect to the laboratory measurements is constructed in order to predict the local shear stresses on the construct surfaces. Tissue growth experiments conducted in the prototype bioreactor permit construct histologies and GAG contents to be analyzed and then correlated with the shear-stress predictions. The integration of this relation into the CFD model enables the prediction of GAG synthesis through convective effects. Coupling this convective model to an existing diffusive model produces a complete cartilage-growth model for use in aiding the optimization of existing bioreactors, and in the design of new ones.
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Cultivo de célula BHK-21 C13 em meio de cultura livre de soro fetal bovino adaptada para crescimento em suspensão / Cell bhk-21 c13 culture in the means of free culture of fetal bovine serum adapted for suspension growthLeme, Jaci 14 December 2016 (has links)
Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.
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Cultivo de célula BHK-21 C13 em meio de cultura livre de soro fetal bovino adaptada para crescimento em suspensão / Cell bhk-21 c13 culture in the means of free culture of fetal bovine serum adapted for suspension growthJaci Leme 14 December 2016 (has links)
Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.
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Development of a culture system for modeling of pH effects in CHO cells / Utveckling av ett odlingssystem för modellering av pH-effekter i CHO-cellerHagrot, Erika January 2011 (has links)
pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37 °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (> 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells. / pH är en viktig parameter i optimeringen av animalcellsprocesser och har sammankopplats med specifika konsumtions- och produktionsmönster rörande extracellulära metaboliter. Det extracellulära pH-värdets effekt på den intracellulära metabolismen är dock inte fullt klarlagd. Metabolisk flux analys är en matematisk metod som kan användas för att generera intracellulära fluxfördelningar i celler, exempelvis som en funktion av någon yttre parameter. Det övergripande målet i detta arbete var att utveckla ett odlingssystem och experimentellt protokoll för odling av CHO-celler som kan användas för att generera data för metabolisk flux analys där målet är att studera effekten av pH på den intracellulära cellmetabolismen. En IgG-producerande CHO-cellslinje odlades först i skakkolv för att välja ut ett akademiskt kemiskt definierat medium med känd sammansättning. Därefter följde försök att anpassa cellerna till det valda mediet. Det visade sig att ett kommersiellt medium behövde tillsättas för att ge godtagbar tillväxt och viabilitet. Effekten av den biologiska bufferten HEPES på cellernas tillväxt och viabilitet, samt pH-stabiliteten under odling, undersöktes också genom odling i skakkolv. HEPES-koncentrationer i det undersökta intervallet (7.5 – 45 mM) hade ingen större effekt på tillväxt och viabilitet. För de högre koncentrationerna var buffertkapaciteten något bättre precis vid inokulering. Dessa koncentrationer var dock ej tillräckliga för att ge stabilt pH under odlingen. Baserat på dessa resultat övergavs tanken på att använda skakkolvsodling för att odla celler vid olika pH-värden. Ett odlingssystem och ett protokoll baserat på en 100 mL Spinnerflaska med pH-reglering specialdesignades istället för projektet. I det färdiga systemet fanns lösningar för kontinuerlig övervakning av pH och DO, stabil temperatur vid 37 °C, justerbar omrörningshastighet, samt valmöjligheten att flöda in luft, O2 och CO2. Dessutom infördes möjligheten att koppla loss flaskenheten från reglersystemet för byte av medium och provtagning. För att demonstrera systemet genomfördes en odling med den anpassade IgG-producerande cellinjen enligt principen för pseudo-perfusion vid pH 7.0. Odlingen pågick under 24 dagar och optimerade reglerinställningar identifierades. Det visades att systemet kunde understödja cellkoncentrationer upp till 11 miljoner celler per milliliter, samt hög viabilitet (> 90 %). Under den senare delen av odlingen uppnåddes stabil tillväxt, vid specifika tillväxthastigheter omkring 0.7 per dygn. Den specifika konsumtions- och produktionshastigheten för metaboliterna glukos, glutamin, laktat och NH4+, samt 20 aminosyror analyserades. Majoriteten av hastigheterna stämde överens med typisk CHO-cellsmetabolism. Den förväntade konsumtionen av majoriteten av de essentiella aminosyrorna och huvudsakliga kolkällorna glukos och glutamin konfirmerades, såväl som den associerade produktionen av bi-produkterna laktat och NH4+. Odlingssystemet och det experimentella protokollet som utvecklades i detta arbete kan användas i framtida experiment för att generera data som beskriver metaboliska profiler som funktion av extracellulärt pH. Dessa data kan sedan användas i metabolisk flux analys för att dra slutsatser om pH-effekter i CHO-celler.
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