When grown under hypothermic conditions, Chinese Hamster Ovary (CHO) cells become growth arrested in the G₀/G₁ phase of the cell cycle and also often exhibit increased recombinant protein production. In this study, we have validated this hypothesis by stimulating hypothermic growth using basic fibroblast growth factor and fetal bovine serum supplementation. This method led to 7.7- and 4.9-fold increase in total production compared to the 37°C and 32°C control cultures, respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes. / Singapore-MIT Alliance (SMA)

This thesis presents a study on the enhancement of CHO cell growth by Yeast extract, Yeastolate, and Primatone fractions obtained by dead-end ultrafiltration. The total solid, peptide contents, antioxidant capacity and hydrophobicity of the fractions were evaluated. The objective of this project was to evaluate the potential of sequential ultrafiltration as an effective, simple and economical method for the identification of CHO cell growth enhancement components in yeast extract and yeastolate (primatone). The fractionation by sequential ultrafiltration (50 kDa membrane, 3 kDa membrane and 1 kDa membrane) of yeast extract (YE), yeastolate (YET), and primatone (PRI) showed different fouling and fractionation behaviour. Significant fouling was observed with the 50 kDa and 3 kDa membrane while negligible fouling was observed with the 1 kDa membrane. Similar and more significant fouling was observed with the 50 kDa membrane and for YE and PRI in comparison to YET. In contrast, more fouling was observed during the ultrafiltration with the 3kDa MWCO and for YE and YET in comparison to PRI. Finally a relatively constant permeate flux was obtained with the 1 kDa membrane, with PRI the highest and YET the lowest permeate flux. Different total peptide contents were present in the three feeds, 410, 327 and 300 mmol Phe-Gly equivalent/ g total solids for YE, PRI and YET respectively. In spite of different feed equivalent Phe-Gly, all three feeds contained a similar amount of equivalent Phe-Gly with molecular weight larger than 50 kDa, 15-19% of the initial feed stream. This was similar amount to the total solids content. The total peptide content of the retentate obtained for the 3kDa filtration indicated that YE and YET contained ~ 20% of equivalent Phe-Gly larger than 3 kDa but smaller than 50kDa. In contrast, PRI contained only 6% of equivalent Phe-Gly with such molecular weight. The retentate of the 1kDa filtration contained 55% of the feed equivalent Phe-Gly compared to 47% for YE and 38% for YET (p< 0.05). All three feeds have similar total peptide content smaller than 1 kDa. For any given feed, the equivalent Phe-Gly was larger than 1 kDa but smaller than 3 kDa predominated. The total peptide content profile according to size coincides with the total solids distribution for all three feed types. This is the first study that reports on the total peptide content for YE, YET, and PRI subjected to ultrafiltration fractionation. All three feeds and their fractions when freeze-dried had similar antioxidant capacity estimated by the FCR (Folin-Ciocalteu reagent) assay, ~ 40-50 mg Trolox/g sample. The bioactivity of feed and fractions was measured as cell density for CHO (beta-IFN producers) in basal medium supplemented with a combination of the crude non-fractionated feed material and a specific fraction and grown in T25 flasks. PRI showed a similar growth enhancement effect for all fractions when compared to a culture supplemented with the crude non-fractionated. YE showed no growth enhancement for any of the fractions when compared to a culture supplemented with the crude non-fractionated YE. This observation need to be confirmed as a culture supplemented with the crude non-fractionated YE showed a very high growth stimulating effect which was much higher than PRI and YET at the same concentration. Finally, YET 3kDa retentate fraction displayed a 50 % growth enhancement effect. In conclusion, the fractions obtained from the two non-animal protein hydrolysates considered in this study, YE and YET showed limited CHO cell growth enhancement effect when compared to the non-fractionated material. Only the YET 3kDa retentate fraction displayed a good CHO cell growth enhancement effect. YET 3kDa represent an attractive serum substitute for its use in culturing CHO cells. PRI, an animal derived protein hydrolysate showed the best growth enhancement effect for all fractions produced in this study. These results suggest that YET has high potential as a media additive for the development of serum-free media which can promote cell growth and, in the future this work can contribute in production of therapeutic proteins markets.

This thesis presents a study on the enhancement of CHO cell growth by Yeast extract, Yeastolate, and Primatone fractions obtained by dead-end ultrafiltration. The total solid, peptide contents, antioxidant capacity and hydrophobicity of the fractions were evaluated. The objective of this project was to evaluate the potential of sequential ultrafiltration as an effective, simple and economical method for the identification of CHO cell growth enhancement components in yeast extract and yeastolate (primatone). The fractionation by sequential ultrafiltration (50 kDa membrane, 3 kDa membrane and 1 kDa membrane) of yeast extract (YE), yeastolate (YET), and primatone (PRI) showed different fouling and fractionation behaviour. Significant fouling was observed with the 50 kDa and 3 kDa membrane while negligible fouling was observed with the 1 kDa membrane. Similar and more significant fouling was observed with the 50 kDa membrane and for YE and PRI in comparison to YET. In contrast, more fouling was observed during the ultrafiltration with the 3kDa MWCO and for YE and YET in comparison to PRI. Finally a relatively constant permeate flux was obtained with the 1 kDa membrane, with PRI the highest and YET the lowest permeate flux. Different total peptide contents were present in the three feeds, 410, 327 and 300 mmol Phe-Gly equivalent/ g total solids for YE, PRI and YET respectively. In spite of different feed equivalent Phe-Gly, all three feeds contained a similar amount of equivalent Phe-Gly with molecular weight larger than 50 kDa, 15-19% of the initial feed stream. This was similar amount to the total solids content. The total peptide content of the retentate obtained for the 3kDa filtration indicated that YE and YET contained ~ 20% of equivalent Phe-Gly larger than 3 kDa but smaller than 50kDa. In contrast, PRI contained only 6% of equivalent Phe-Gly with such molecular weight. The retentate of the 1kDa filtration contained 55% of the feed equivalent Phe-Gly compared to 47% for YE and 38% for YET (p< 0.05). All three feeds have similar total peptide content smaller than 1 kDa. For any given feed, the equivalent Phe-Gly was larger than 1 kDa but smaller than 3 kDa predominated. The total peptide content profile according to size coincides with the total solids distribution for all three feed types. This is the first study that reports on the total peptide content for YE, YET, and PRI subjected to ultrafiltration fractionation. All three feeds and their fractions when freeze-dried had similar antioxidant capacity estimated by the FCR (Folin-Ciocalteu reagent) assay, ~ 40-50 mg Trolox/g sample. The bioactivity of feed and fractions was measured as cell density for CHO (beta-IFN producers) in basal medium supplemented with a combination of the crude non-fractionated feed material and a specific fraction and grown in T25 flasks. PRI showed a similar growth enhancement effect for all fractions when compared to a culture supplemented with the crude non-fractionated. YE showed no growth enhancement for any of the fractions when compared to a culture supplemented with the crude non-fractionated YE. This observation need to be confirmed as a culture supplemented with the crude non-fractionated YE showed a very high growth stimulating effect which was much higher than PRI and YET at the same concentration. Finally, YET 3kDa retentate fraction displayed a 50 % growth enhancement effect. In conclusion, the fractions obtained from the two non-animal protein hydrolysates considered in this study, YE and YET showed limited CHO cell growth enhancement effect when compared to the non-fractionated material. Only the YET 3kDa retentate fraction displayed a good CHO cell growth enhancement effect. YET 3kDa represent an attractive serum substitute for its use in culturing CHO cells. PRI, an animal derived protein hydrolysate showed the best growth enhancement effect for all fractions produced in this study. These results suggest that YET has high potential as a media additive for the development of serum-free media which can promote cell growth and, in the future this work can contribute in production of therapeutic proteins markets.

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 (&gt; 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 (&gt; 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.

O projeto de novos materiais para aplicações tecnológicas em biomateriais e bioengenharia é altamente dependente de como as células aderem à superfície de um material. A adesão e crescimento em biomateriais depende de propriedades do substrato, tais como molhabilidade da superfície, a topografia e a composição química de superfície. O objetivo deste estudo foi investigar as interações de diversos materiais com culturas celulares de células epitelial CHO (Ovário de Hamster Chinês). Os materiais utilizados foram SU-8 2005 (elétron-resiste, Microchem), PDMS (Poli (dimetil siloxano), Down Corning), DLC (Diamond-like Carbon) e vidro foi utilizado como referência. Superfícies de vidro, SU-8, PDMS e DLC lisas (planas) e isentas de modificação ou tratamento específico foram avaliadas quanto ao cultivo de células CHO. Valores médios dos fatores de forma (Ff) de 450 células foram calculados para cada uma das culturas realizadas sobre os 4 substratos. Foram obtidos Ff próximos a 0,52 para o vidro, o SU-8 liso e o DLC, demonstrando um bom espraiamento das células nessas superfícies. A superfície de PDMS apresentou valor unitário para o fator de forma (Ff), que está relacionado a um baixo espraiamento das células. A energia de superfície (ES) obtida para o PDMS é compatível com o resultado de fator de forma (Ff), uma vez que o menor valor para ES é coerente com a baixa adesão celular, o que gerou células com elevado fator de forma (Ff). O SU-8 foi modificado por implantação iônica com uma dose de 1,2x1016 átomos/cm2 e a energia de implantação foi de 8 keV, como referência foi utilizada uma superfície lisa de SU-8 sem implantação. Os resultados mostraram que o número de células vivas por unidade de área foi superior na superfície de SU-8 com prata implantada, mostrando o bom desempenho da cultura nesse substrato. As superfícies de DLC modificadas por tratamento com plasma de oxigênio (DLC-O) e com plasma de hexafluoreto de enxofre (DLC-F) foram utilizadas para cultura celular, os resultados de três experimentos independentes de contagem de número de núcleos (marcados com DAPI) por unidade de área confirmaram os resultados obtidos através do teste de viabilidade (marcados com trypan blue). A superfície de DLC-O, apresentou um maior número de núcleos por unidade de área, quando comparado à superfície DLC-F, da mesma forma que nos resultados obtidos pelo teste de viabilidade. As energias de superfície para as amostras de DLC-F e DLC-O indicaram que a superfície DLC-O é mais hidrofílica do que a superfície DLC-F, que está coerente com o que é conhecido da literatura e com os resultados obtidos em nosso trabalho. Cultura de células CHO foram realizadas em superfícies litografadas com estruturas hexagonais periódicas com o parâmetro 2R (diâmetro do círculo inscrito) sendo 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. Estas superfícies foram caracterizadas por microscopia óptica de fluorescência com relação ao número de núcleos (marcados com o fluoróforo DAPI) por unidade de área, isto é, núcleos/mm2. Obteve-se histogramas com o número médio de núcleos por mm2 em três experimentos independentes, onde o número núcleos/mm2 foi consideravelmente maior para 80 µm. As superfícies contendo cavidades periódicas de 12 µm e 30 µm apresentaram dificuldade para as células CHO aderirem à superfície. Em uma outra etapa realizou-se culturas celulares em triplicata dos substratos com as superfícies 12 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As células em cada uma das superfícies foram analisadas por microscopia óptica (MO) para avaliação da viabilidade celular, utilizando marcador trypan blue. Obteve-se histogramas com os valores médios para o número de células vivas/mm2 para as culturas celulares que corrobora os resultados obtidos no histograma da cultura celular que tiveram os núcleos marcados pelo fluoróforo DAPI. Assim, fica confirmado o melhor desempenho da cultura celular no substrato 80 µm que apresentou o maior número de células vivas/mm2 As micrografias obtidas através de marcação por DAPI foram analisadas através da função de correlação com intuito de se entender como as células estavam organizadas. Isso foi feito para cada uma das superfícies litografadas, 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As superfícies dos substratos 80 µm apresentaram os menores valores de distâncias para primeiros e segundos vizinhos, ou seja, as células estão mais próximas umas das outras. As demais superfícies tendem a separar mais as células. Obteve-se também os valores de raio de aglomerado (rc), distância entre os aglomerados (dc) e o número de primeiros vizinhos (Np) através do ajuste da função de correlação. A análise de correlação mostrou com clareza o que não era evidenciado apenas visualizando-se as imagens. Ela mostra que as células, mesmo em SU-8 liso tem a forte tendência de formar aglomerados de células com raio de aproximadamente 45 µm. No caso de substratos lisos, células CHO apresentaram a melhor adesão na superfície do SU-8, seguido do DLC, enquanto que o PDMS foi a pior situação, devido à baixa molhabilidade do material. No caso de superfícies com microestrutura, SU-8 contendo microcavidades hexagonais de 12 e 30 µm mostraram ser as situações mais adversas para o crescimento de células CHO, provavelmente por causa da topografia das cavidades serem de menor tamanho quando comparadas ao tamanho das células CHO. Em vez disso, SU-8, contendo microcavidades hexagonais de 80 µm foi a superfície mais favorável para o crescimento de células CHO. / The design of new materials for technological applications in biomaterials and bioengineering is highly dependent on how the cells adhere to the material surface. The cells adhesion and growth on biomaterials depends on substrate properties such as surface wettability, topography and the chemical composition. The aim of this study was to investigate the interactions of various materials with cell cultures of epithelial cells CHO (Chinese Hamster Ovary). The materials used were SU-8 2005 (electron resists, Microchem), PDMS (poly (dimethyl siloxane), Dow Corning), DLC (Diamond-like Carbon) and glass was used as reference. Unmodified and flat surfaces of glass, SU-8, PDMS and DLC were evaluated for the culture of CHO cells. Form factor (Ff) values were calculated as average of 450 cells for each of the cultures performed on the four substrates. Ff close to 0.52 was obtained for flat surfaces of glass, SU-8 and DLC, showing a good cell spreading on these surfaces. The surface of PDMS presented a form factor (Ff) near unity, which is related to low spreading cell. The surface energy (ES) obtained for the PDMS is coherent with the Ff result, since the smallest value of ES is consistent with the low cell adhesion, which resulted in cells with a high Ff. The SU-8 was modified by ion implantation using a dose of 1.2x1016 atoms/cm2 and an implantation energy of 8 keV, unmodified flat SU-8 was used as a reference. The cell culture results showed that the number of live cells per unit area was greater in the SU-8 surface implanted with silver, showing a good performance in the culture substrate. The DLC surfaces modified by plasma treatment with oxygen (DLC-O) and sulfur hexafluoride (DLC-F) were used for cell culture. The results of three independent experiments, counting the number of nuclei (marked with DAPI) per unit area, confirmed the results obtained by the viability test (marked with trypan-blue). The surface of the DLC-O had higher number of nuclei per unit area when compared to the surface of the DLC-F, similarly to the results obtained for the viability test. The surface energies of the DLC-F and DLC-O samples indicated that the DLC-O surface is more hydrophilic than the DLC-F surface, which is consistent with results obtained with our work and with the literature. CHO cell culture were performed on surfaces with periodic hexagonal structures with the diameter of inscribed circle (2R) given by 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. These surfaces were characterized by fluorescence optical microscopy with respect to the number of nuclei (marked with fluorophore DAPI) per unit area, i.e. nuclei/mm2. Histograms were obtained for the average number of nuclei per mm2 in three independent experiments, where the substrate with periodic hexagonal structures with 2R = 80 µm presented considerably higher nuclei/mm2. Surfaces containing periodic cavities of 2R =12 µm and 30 µm were adverse for CHO cells adhesion. In another approach, cell culture were analyzed by light microscopy (LM) for evaluation of cell viability using trypan-blue marker. This was carried out in triplicate cell culture on substrates with surfaces 12 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. Histograms were generated for average number of living cells/mm2 for each substrate, which corroborates with the results obtained for the cell culture marked with fluorophore DAPI. Thus, it is confirmed the better performance of the cell culture on substrates with 2R = 80 µm, presenting the highest number of living cells/mm2. The micrographs obtained with cells marked with DAPI were analyzed through the correlation function with the aim of understanding how the cells were organized. This was performed for each of the lithographed surfaces 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also flat SU-8. The surfaces of the substrates with 2R = 80 µm had the lowest values for length between its neighbors, that is, the cells are closer to each other. The remaining surfaces tend to separate the cells. Also were obtained the cluster radius values (rc), the distance between the clusters (dc) and the number of nearest neighbors (Np) through the correlation function fitting. The correlation analysis clearly showed what was not possible to observe by viewing the images. It shows that the cells, even in flat SU-8, have a strong tendency to form clusters of cells within about 45 micrometers. In the case of flat substrates, CHO cells exhibited better adhesion to the surface of SU-8, followed by the DLC, while the PDMS was worse due to low wettability of the material. In the case of surfaces with microstructures, SU-8 containing hexagonal microstructures of 12 and 30 µm showed to be the most adverse conditions for the CHO cell growth, probably because of the topography of the cavities being smaller in size compared to the size of CHO cells. SU-8 with 80 µm hexagonal microstructures was more favorable surface for the growth of CHO cells.

O projeto de novos materiais para aplicações tecnológicas em biomateriais e bioengenharia é altamente dependente de como as células aderem à superfície de um material. A adesão e crescimento em biomateriais depende de propriedades do substrato, tais como molhabilidade da superfície, a topografia e a composição química de superfície. O objetivo deste estudo foi investigar as interações de diversos materiais com culturas celulares de células epitelial CHO (Ovário de Hamster Chinês). Os materiais utilizados foram SU-8 2005 (elétron-resiste, Microchem), PDMS (Poli (dimetil siloxano), Down Corning), DLC (Diamond-like Carbon) e vidro foi utilizado como referência. Superfícies de vidro, SU-8, PDMS e DLC lisas (planas) e isentas de modificação ou tratamento específico foram avaliadas quanto ao cultivo de células CHO. Valores médios dos fatores de forma (Ff) de 450 células foram calculados para cada uma das culturas realizadas sobre os 4 substratos. Foram obtidos Ff próximos a 0,52 para o vidro, o SU-8 liso e o DLC, demonstrando um bom espraiamento das células nessas superfícies. A superfície de PDMS apresentou valor unitário para o fator de forma (Ff), que está relacionado a um baixo espraiamento das células. A energia de superfície (ES) obtida para o PDMS é compatível com o resultado de fator de forma (Ff), uma vez que o menor valor para ES é coerente com a baixa adesão celular, o que gerou células com elevado fator de forma (Ff). O SU-8 foi modificado por implantação iônica com uma dose de 1,2x1016 átomos/cm2 e a energia de implantação foi de 8 keV, como referência foi utilizada uma superfície lisa de SU-8 sem implantação. Os resultados mostraram que o número de células vivas por unidade de área foi superior na superfície de SU-8 com prata implantada, mostrando o bom desempenho da cultura nesse substrato. As superfícies de DLC modificadas por tratamento com plasma de oxigênio (DLC-O) e com plasma de hexafluoreto de enxofre (DLC-F) foram utilizadas para cultura celular, os resultados de três experimentos independentes de contagem de número de núcleos (marcados com DAPI) por unidade de área confirmaram os resultados obtidos através do teste de viabilidade (marcados com trypan blue). A superfície de DLC-O, apresentou um maior número de núcleos por unidade de área, quando comparado à superfície DLC-F, da mesma forma que nos resultados obtidos pelo teste de viabilidade. As energias de superfície para as amostras de DLC-F e DLC-O indicaram que a superfície DLC-O é mais hidrofílica do que a superfície DLC-F, que está coerente com o que é conhecido da literatura e com os resultados obtidos em nosso trabalho. Cultura de células CHO foram realizadas em superfícies litografadas com estruturas hexagonais periódicas com o parâmetro 2R (diâmetro do círculo inscrito) sendo 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. Estas superfícies foram caracterizadas por microscopia óptica de fluorescência com relação ao número de núcleos (marcados com o fluoróforo DAPI) por unidade de área, isto é, núcleos/mm2. Obteve-se histogramas com o número médio de núcleos por mm2 em três experimentos independentes, onde o número núcleos/mm2 foi consideravelmente maior para 80 µm. As superfícies contendo cavidades periódicas de 12 µm e 30 µm apresentaram dificuldade para as células CHO aderirem à superfície. Em uma outra etapa realizou-se culturas celulares em triplicata dos substratos com as superfícies 12 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As células em cada uma das superfícies foram analisadas por microscopia óptica (MO) para avaliação da viabilidade celular, utilizando marcador trypan blue. Obteve-se histogramas com os valores médios para o número de células vivas/mm2 para as culturas celulares que corrobora os resultados obtidos no histograma da cultura celular que tiveram os núcleos marcados pelo fluoróforo DAPI. Assim, fica confirmado o melhor desempenho da cultura celular no substrato 80 µm que apresentou o maior número de células vivas/mm2 As micrografias obtidas através de marcação por DAPI foram analisadas através da função de correlação com intuito de se entender como as células estavam organizadas. Isso foi feito para cada uma das superfícies litografadas, 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As superfícies dos substratos 80 µm apresentaram os menores valores de distâncias para primeiros e segundos vizinhos, ou seja, as células estão mais próximas umas das outras. As demais superfícies tendem a separar mais as células. Obteve-se também os valores de raio de aglomerado (rc), distância entre os aglomerados (dc) e o número de primeiros vizinhos (Np) através do ajuste da função de correlação. A análise de correlação mostrou com clareza o que não era evidenciado apenas visualizando-se as imagens. Ela mostra que as células, mesmo em SU-8 liso tem a forte tendência de formar aglomerados de células com raio de aproximadamente 45 µm. No caso de substratos lisos, células CHO apresentaram a melhor adesão na superfície do SU-8, seguido do DLC, enquanto que o PDMS foi a pior situação, devido à baixa molhabilidade do material. No caso de superfícies com microestrutura, SU-8 contendo microcavidades hexagonais de 12 e 30 µm mostraram ser as situações mais adversas para o crescimento de células CHO, provavelmente por causa da topografia das cavidades serem de menor tamanho quando comparadas ao tamanho das células CHO. Em vez disso, SU-8, contendo microcavidades hexagonais de 80 µm foi a superfície mais favorável para o crescimento de células CHO. / The design of new materials for technological applications in biomaterials and bioengineering is highly dependent on how the cells adhere to the material surface. The cells adhesion and growth on biomaterials depends on substrate properties such as surface wettability, topography and the chemical composition. The aim of this study was to investigate the interactions of various materials with cell cultures of epithelial cells CHO (Chinese Hamster Ovary). The materials used were SU-8 2005 (electron resists, Microchem), PDMS (poly (dimethyl siloxane), Dow Corning), DLC (Diamond-like Carbon) and glass was used as reference. Unmodified and flat surfaces of glass, SU-8, PDMS and DLC were evaluated for the culture of CHO cells. Form factor (Ff) values were calculated as average of 450 cells for each of the cultures performed on the four substrates. Ff close to 0.52 was obtained for flat surfaces of glass, SU-8 and DLC, showing a good cell spreading on these surfaces. The surface of PDMS presented a form factor (Ff) near unity, which is related to low spreading cell. The surface energy (ES) obtained for the PDMS is coherent with the Ff result, since the smallest value of ES is consistent with the low cell adhesion, which resulted in cells with a high Ff. The SU-8 was modified by ion implantation using a dose of 1.2x1016 atoms/cm2 and an implantation energy of 8 keV, unmodified flat SU-8 was used as a reference. The cell culture results showed that the number of live cells per unit area was greater in the SU-8 surface implanted with silver, showing a good performance in the culture substrate. The DLC surfaces modified by plasma treatment with oxygen (DLC-O) and sulfur hexafluoride (DLC-F) were used for cell culture. The results of three independent experiments, counting the number of nuclei (marked with DAPI) per unit area, confirmed the results obtained by the viability test (marked with trypan-blue). The surface of the DLC-O had higher number of nuclei per unit area when compared to the surface of the DLC-F, similarly to the results obtained for the viability test. The surface energies of the DLC-F and DLC-O samples indicated that the DLC-O surface is more hydrophilic than the DLC-F surface, which is consistent with results obtained with our work and with the literature. CHO cell culture were performed on surfaces with periodic hexagonal structures with the diameter of inscribed circle (2R) given by 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. These surfaces were characterized by fluorescence optical microscopy with respect to the number of nuclei (marked with fluorophore DAPI) per unit area, i.e. nuclei/mm2. Histograms were obtained for the average number of nuclei per mm2 in three independent experiments, where the substrate with periodic hexagonal structures with 2R = 80 µm presented considerably higher nuclei/mm2. Surfaces containing periodic cavities of 2R =12 µm and 30 µm were adverse for CHO cells adhesion. In another approach, cell culture were analyzed by light microscopy (LM) for evaluation of cell viability using trypan-blue marker. This was carried out in triplicate cell culture on substrates with surfaces 12 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. Histograms were generated for average number of living cells/mm2 for each substrate, which corroborates with the results obtained for the cell culture marked with fluorophore DAPI. Thus, it is confirmed the better performance of the cell culture on substrates with 2R = 80 µm, presenting the highest number of living cells/mm2. The micrographs obtained with cells marked with DAPI were analyzed through the correlation function with the aim of understanding how the cells were organized. This was performed for each of the lithographed surfaces 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also flat SU-8. The surfaces of the substrates with 2R = 80 µm had the lowest values for length between its neighbors, that is, the cells are closer to each other. The remaining surfaces tend to separate the cells. Also were obtained the cluster radius values (rc), the distance between the clusters (dc) and the number of nearest neighbors (Np) through the correlation function fitting. The correlation analysis clearly showed what was not possible to observe by viewing the images. It shows that the cells, even in flat SU-8, have a strong tendency to form clusters of cells within about 45 micrometers. In the case of flat substrates, CHO cells exhibited better adhesion to the surface of SU-8, followed by the DLC, while the PDMS was worse due to low wettability of the material. In the case of surfaces with microstructures, SU-8 containing hexagonal microstructures of 12 and 30 µm showed to be the most adverse conditions for the CHO cell growth, probably because of the topography of the cavities being smaller in size compared to the size of CHO cells. SU-8 with 80 µm hexagonal microstructures was more favorable surface for the growth of CHO cells.

Des hormones gonadotropiques sont utilisées pour la maîtrise de la reproduction dans le domaine vétérinaire. Ces hormones sont actuellement extraites de tissus ou de fluides animaux. L’entreprise CEVA Santé Animale a récemment fait le choix stratégique de produire ces hormones par voie recombinante. L’objectif de cette étude était d’obtenir une gonadotropine chorionique équine recombinante, reCG, pure et biologiquement active, à partir d’une lignée de cellules mammifères CHO. Les étapes de production, de purification et de caractérisation de l’hormone recombinante ont été développées. Les cellules CHO ont été cultivées en fiole d’Erlenmeyer dans différents milieux de culture. Le suivi de la croissance cellulaire et de la quantité d’hormone produite a permis de sélectionner deux milieux. Le procédé de production, avec ces deux milieux, a été optimisé en bioréacteur en contrôlant les paramètres de culture (température, pH). Les protéines produites dans le surnageant, de ces deux cultures, ont été nommées reCG 1 et reCG 2. Un procédé de purification en 3 étapes a été mis au point pour la reCG 1. Plusieurs résines et conditions chromatographiques ont été criblées en microplaques. Les résines multimodales utilisées ont permis d’éliminer des contaminants majeurs grâce à leur sélectivité. Les agrégats de la reCG ont été éliminés grâce à une résine anionique. Le procédé de purification global a été validé pour la reCG 1 et la reCG 2. Il a permis d’obtenir une pureté de 98 % avec un rendement de 80 %. L’activité biologique de la reCG 1 et la reCG 2, in vitro et in vivo, est comparable à celle de la protéine naturelle. L’activité biologique in vivo des reCG est cohérente avec l’étude réalisée sur les glycosylations des hormones et notamment avec leur degré de sialylation. / The gonadotrophic hormones are used for reproduction control in farming animals. Up to now, these hormones were extracted from animal fluids or tissues. The company CEVA Santé Animal has recently decided to produce recombinant versions of these hormones. The objective of this study was to obtain a pure and biologically active recombinant equine chorionic gonadotropin (reCG) after expression in CHO mammalian cells. The production, purification and characterization steps have been developed. CHO cells were grown in Erlenmeyer flasks with different culture media. Two media were selected based on their cell growth potency and of the amount of reCG produced. By using a bioreactor to control key parameters (temperature, pH), the production process was then optimized. The recombinant proteins that accumulated in the supernatant of the two conditions were called reCG 1 and reCG 2. A 3-steps purification process was then developed using reCG 1. Several resins and chromatographic conditions were screened in microplates. Multimodal resins were used to eliminate the main contaminants thanks to their selectivity. reCG aggregates were efficiently eliminated by a chromatographic step with an anionic resin. The overall purification process was finally validated for reCG 1 and reCG 2. Purity and yield were respectively, 98 % and 80 % for the two reCG. We verified that the in vitro and in vivo activities of reCG 1 and reCG 2 were comparable to those of the CG extracted from natural sources. The in vivo assays also confirmed previous studies showing that the degree of glycosylation of an hormone, and most notably their level of sialytation, is important for their biological activity.

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

No description available.