Spelling suggestions: "subject:"serum free medium"" "subject:"rerum free medium""
1 |
Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free culturesEriksson, Ulrika January 2005 (has links)
<p>The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.</p><p>Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.</p><p>Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.</p><p>In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.</p><p>Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.</p>
|
2 |
Produção e purificação da glicocerebrosidase humana recombinante expressa por célula CHO em meio livre de soro fetal bovino / Production and purification of recombinant human glucocerebrosidase expressed by CHO cells in serum free mediumCassundé, Bruna Cristine Fernandes 02 February 2017 (has links)
A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.
|
3 |
Produção e purificação da glicocerebrosidase humana recombinante expressa por célula CHO em meio livre de soro fetal bovino / Production and purification of recombinant human glucocerebrosidase expressed by CHO cells in serum free mediumBruna Cristine Fernandes Cassundé 02 February 2017 (has links)
A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.
|
4 |
Produção de anticorpos monoclonais anti-GITR e anti-CD25 através de cultivo de hibridomas e comparação do seu potencial como agentes antitumoraisPrampero, Anna Carolina 24 February 2017 (has links)
Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-10-06T18:08:45Z
No. of bitstreams: 1
DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:39Z (GMT) No. of bitstreams: 1
DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:55Z (GMT) No. of bitstreams: 1
DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Made available in DSpace on 2017-11-28T12:26:25Z (GMT). No. of bitstreams: 1
DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5)
Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nowadays, cancer is one of the most feared diseases, affecting each day more and more people
worldwide. The importance of new cancer treatment researches is very clear since the ones that
has been used are not very effective and may lead to drug resistance, implying in a constant
dose increasing which can lead to toxicity issues. Collateral effects and the instability generated
in the patient’s organism are also reasons why the necessity of discovering new cancer
treatments is imminent. A treatment alternative that has aroused interest is the use of
monoclonal antibodies as immunotherapics, since they act by stimulating the patient’s immune
system neutralizing the tumor cells in a very efficient and specific way. This kind of antibody
can be produced by culturing hybrid animal cells, better known as hybridoma, under strictly
controlled conditions so they can be studied and used in human beings. For this reason, the
major goal of this project was the production of murine monoclonal antibodies using hybridoma
cell culture in order to stablish an efficient culture methodology for hybridomas PC-61 or DTA1
producers of monoclonal antibodies anti-CD25 and anti-GITR, respectively, with high quality
and enough amounts using Fetal Bovine Serum (FBS) free medium to, in the future, carry out
animal model studies of their potential as therapeutic agents for cancer treatment. Both
hybridomas were cultivated on a small scale with RPMI medium and addition of SFB, for
comparative purposes and only one was selcted for the second step. The sequential adaptation
methodology test, consisted in a gradual percent’s reduction of medium with serum at the same
time that increase the percentage of commercial medium without serum, and was selected the
medium without SFB in which the hybridoma was better adapted. After was carried out on a
laboratory scale in a system type spinner flask (500 ml) with the commercial medium selected
in the previous step, in controlled conditions of temperature (37 ° C) and pH (7.2). Based on
analyzes of cell culture results, amino acid consumption and monoclonal antibodies
quantification , SFM commercial medium SFB-free provided better results for culturing the
PC-61 hybridoma, allowing the pilot scale culture reached even higher cell densities than in the
standard medium with addition of FBS. / O câncer é uma das doenças mais temidas da atualidade, e atinge cada vez mais pessoas em
todo o mundo. A importância de pesquisas sobre novos tratamentos na luta contra o câncer é
clara e consensual, uma vez que os que vem sendo utilizados, não são muito eficientes, causam
resistência à medicação utilizada o que implica na utilização de doses crescentes que por sua
vez podem gerar problemas de toxicidade. Os efeitos colaterais e a instabilidade gerada no
organismo do paciente, também são fatores da necessidade de pesquisar novos caminhos para
o tratamento do câncer. Uma das alternativas de tratamento que tem despertado interesse é a
utilização de anticorpos monoclonais (mAbs) como imunoterápicos, os quais agem estimulando
o próprio sistema imune do paciente neutralizando a ação das células tumorais de forma
eficiente e específica. A produção de tais anticorpos pode ser feita mediante o cultivo de células
animais híbridas, mais conhecidas como hibridomas, sobre condições estritamente controladas
para que possam ser estudados e utilizados em humanos. Por essa razão definiu-se como
objetivo desse trabalho a produção anticorpos monoclonais murinos por meio de cultivo de
hibridomas com a finalidade de estabelecer uma metodologia eficiente de cultivo dos
hibridomas PC-61 ou DTA1 secretores dos mAbs anti-CD25 e antiGITR respectivamente, com
qualidade e em quantidades suficientes utilizando meios livres de soro fetal bovino (SFB), para
a seguir efetuar estudos em modelo animal de seu potencial como agentes terapêuticos no
tratamento de câncer. Os dois hibridomas foram cultivados em pequena escala utilizando meio
RPMI-1640 e adição de SFB, para fins comparativos e somente um foi selecionado para a
próxima etapa. O teste da metodologia de adaptação sequencial, onde houve a redução gradativa
da porcentagem de meio RPMI-1640 com 10% de SFB e o aumento da porcentagem de meio
comercial sem SFB, e foi selecionado o meio livre de SFB em que o hibridoma melhor se
adaptou. Posteriormente foi realizado o cultivo do hibridoma em escala laboratorial em sistema
de frasco agitado biorreator do tipo Spinner (500 mL) com o meio livre de SFB selecionado na
etapa anterior, sob condições bem controladas de temperatura (37ºC), pH (7,2). Com base nas
análises dos resultados dos cultivos celulares, metabolismo de aminoácidos e quantificação de
mAbs, o meio comercial SFM livre de SFB proporcionou melhor resultados para o cultivo do
hibridoma PC-61, permitindo que o cultivo em escala laboratorial atingisse densidades
celulares ainda maiores que no meio padrão com adição de SFB. Como consequência desse
vasto crescimento celular em quantidades abundantes de mAbs foram conseguidas para iniciar
num futuro próximo ensaios em modelos animais.
|
5 |
Étude des procédés d’amplification de cellules souches mésenchymateuses humaines / Study on expansion processes for human mesenchymal stem cellMartin, Céline 08 December 2016 (has links)
L'essor des thérapies régénératives au cours des 10 dernières années a entraîné un effort de recherche important, mais l'obtention des cellules souches humaines en quantité suffisante reste cependant encore problématique, notamment concernant les cellules souches mésenchymateuses (CSM). Ces travaux ont donc mis en œuvre une approche à la croisée de la biologie et du génie des procédés afin d'identifier les verrous limitant la croissance des CSM. L'étude des méthodes d'intensification de culture a été entreprise grâce à l'utilisation de microporteurs et d'une plateforme de minibioréacteurs de 200~mL. Puis le développement d'un milieu de culture sans sérum a été testé dans le but de maximiser la croissance cellulaire dans des conditions biochimiques contrôlées. Les CSM humaines en tant que modèle type en thérapie cellulaire ont été démontrées comme extrêmement sensibles aux phases de congélation/décongélation, aux variations de température, à un vieillissement prématuré et nécessitant un milieu de culture complexe riche en facteurs de croissance et d'adhérence. Suite à cette étude, plusieurs écueils pourront être évités lors de la montée en échelle d'un procédé de culture de CSM afin d'intégrer leurs paramètres biologiques intrinsèques aux paramètres d'ingénierie des bioréacteurs (transfert de chaleur, contraintes hydrodynamiques, surface d'adhérence) / Progress in regenerative medicines over the past ten years have led to an important research mobilisation, but obtaining a sufficient amount of human stem cells remains nonetheless problematic, especially for mesenchymal stem cells (MSC). Hence, this work developed an approach coupling biology and process engineering to identify barriers limiting MSC growth. The study of scaled-up amplification methods was performed using microcarriers and a 200~mL minibioreactors platform. In order to maximise MSC growth in a biochemically controlled environment, a serum free medium development was tested as well. Human MSC as model cell type for cellular therapies have thus been demonstrated as extremely sensitive to freeze/thaw cycles, temperature variations, subject to premature aging and needing a complex medium enriched in multiple growth and adherence factors. Following this study, several pitfalls might be avoided during MSC process scale-up by integrating the cells biology into the bioreactors' process engineering parameters (heat transfer, hydrodamic stress, adhesion surface)
|
6 |
Procédés de cultures de cellules VERO en milieu sans sérum : contributions au développement d'une stratégie PAT / Vero cell culture processes in serum-free medium : contributions to the development of the PAT strategyPetiot, Emma 06 November 2009 (has links)
Ce travail apporte une contribution au développement de la stratégie PAT pour les procédés de culture de cellules animales. Il propose l'amélioration de la compréhension et de la maîtrise de la culture de cellules Vero, dédiées à la production de vaccins viraux, et cultivées sur microporteurs dans un milieu sans sérum. Une première partie a permis de cribler les effets de certains groupes de composés du milieu de culture par le suivi de la croissance en microplaques. Puis, des études cinétiques et métaboliques plus approfondies, réalisées en spinners, ont montré que le métabolisme carboné des cellules Vero est saturé par l'accumulation intracellulaire de pyruvate et qu’il est peu orienté vers la croissance. Alors que le renouvellement du milieu ou l'ajout ponctuel de glutamine amélioraient la croissance cellulaire sans ré-équilibrer le métabolisme, la substitution du glucose et de la glutamine a permis de réduire l’apoptose et d'améliorer les performances métaboliques et de croissance. Par ailleurs, les spectroscopies diélectrique et proche-infrarouge ont été évaluées pour le contrôle en-ligne du procédé, en prenant en compte les particularités des cellules adhérentes. Nous avons montré leur capacité à évaluer les concentrations de cellules, de composés du milieu, et à détecter l'apoptose. Enfin, les principales améliorations par substitution de la glutamine ont été appliquées en bioréacteurs, à la production d'un vaccin prototype contre la dengue, dans des conditions proches de celles d'un procédé industriel. Ceci a permis de limiter les renouvellements de milieu pendant l’expansion cellulaire, sans compromettre la production de particules virales infectieuses / This work contributes to the development of the PAT strategy for animal cell culture processes. The aim of this study was to improve the understanding and the control of Vero cell culture, dedicated to the production of viral vaccines, and grown on microcarriers in serum-free medium. An initial study was performed to screen the effects of certain groups of compounds of the culture medium, by the cell growth monitoring in microplates. Then, kinetic and metabolic studies conducted in spinners flasks allowed to go further and to show that the Vero cell metabolism is saturated through the pyruvate intracellular accumulation and that it is not oriented toward growth. While media renewal or punctual addition of glutamine improve the cell growth without improving the metabolism balance, the substitution of glucose and glutamine allowed to reduce apoptosis and to improve growth and metabolic performances.Furthermore, dielectric and near-infrared spectroscopies have been evaluated for the in-line process monitoring, taking into account the particularities of adherent cells. We have demonstrated their ability to quantify cell concentrations, medium component concentrations, and to detect apoptosis. Finally, major improvements by substitution of glutamine have been applied to bioreactor culture to produce a dengue vaccine prototype, with culture conditions close to industrial process. In these cases, medium renewal during the cell expansion was removed without compromising the production of infectious viral particles
|
7 |
Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free culturesEriksson, Ulrika January 2005 (has links)
The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>
|
8 |
Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino. / Production of recombinant proteins in BHK-21 cells cultured in serum free media.Patiño, Sandra Fernanda Suárez 06 May 2016 (has links)
Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira. / Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.
|
9 |
Adaptção de linhagens celulares humanas para crescimento em suspensão e meios de cultura livres de soro fetal bovino / Serum-free suspension adaptation of human cell linesBiaggio, Rafael Tagé 28 March 2014 (has links)
Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.
|
10 |
Adaptção de linhagens celulares humanas para crescimento em suspensão e meios de cultura livres de soro fetal bovino / Serum-free suspension adaptation of human cell linesRafael Tagé Biaggio 28 March 2014 (has links)
Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.
|
Page generated in 0.0437 seconds