The purpose of this study was to rationally design animal component free, chemically defined serum free media (ACF-CD-SFM) for MRC-5 and Vero cells while adhering to the Quality by Design guidelines. This was achieved by using the Modified Vero Serum Free Medium (MVSFM) as the basal formulation and supplementing it with various combinations of growth factors (LONG® EGF, LONG® R3 IGF-I, rTransferrin, bFGF, TGF-3 and PDGF-AA), lipids (linoleic acid, cholesterol, and dexamethasone), lipid precursors (ethanolamine and phosphoethanolamine) and vitamins (all-trans retinoic acid, -tocopherol and ascorbic acid). Media development was achieved by conducting a series of steps using different experimental methodologies with the end goal of satisfying the requirements of each cell line. MRC-5 and Vero cells were each cultured in specific media containing unique concentrations of supplements that were prepared according to the different statistical design methodologies. The original objective was to create a SFM, however due to the stringent nutritious requirements of anchorage dependent cell lines, only a reduction to 0.5% FBS was achieved. For MRC-5 cells, the one-factor-at-a-time (OFAT) generated the Prototype + 0.5% FBS medium. The Definitive Screening Design (DSD) gave rise to the Delta 1 + 0.5% FBS, which was the optimum medium formulation for MRC-5 cells as it had comparable cell yields to DMEM + 10 % FBS. This result was confirmed by the Genetic Algorithms-Hill Climbing (GA-HC) method. In the case of Vero cells, the OFAT and the DSD confirmed that MVSFM + 0.5 % FBS was the most optimal formulation. The morphology in both media for both cell lines was comparable to that in DMEM-10% FBS. It was concluded that the DSD method successfully achieved a reduction of the serum concentration from 10% to 0.5% FBS. / October 2017

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

Cell culture media used in the manufacture of biopharmaceuticals conventionally contain many animal derived components. These components can harbor adventitious agents which can be transmitted through biotherapeutics, employed in the medical treatment of immunocompromised patients. An ADCF (animal derived component free) medium formulation obviates this concern. A rational method for the rapid and efficient screening and optimization of ADCF media while preserving, if not enhancing, cellular growth and protein productivity is needed. CHO (Chinese Hamster Ovary) cells, widely used as a production platform in industry, expressing a recombinant protein, were employed as a model system. Design of Experiment (DOE) and statistical analysis were employed to assess the impact of media formulation on cellular physiology. Metabolic flux, cellular growth, and protein productivity were evaluated as the measures of ADCF media formulation success. Measurements of extracellular metabolites were determined by HPLC and enzymatic methods. Recombinant protein production was measured by HPLC. This research demonstrates the successful screening and optimization of four plant hydrolysate mixtures (2 soy and 2 wheat) as a replacement for animal derived components. / NSERC, ABIN, MABNET

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.

The global demand for animal proteins is projected to rise by 14% by 2030, amplifying the environmental toll of conventional animal-based protein production. Cultivated meat technology can alleviate the growing demand for protein and address the environmental and ethical concerns associated with conventional livestock farming. However, it faces a critical challenge: the high cost of cell culture media, primarily due to the use of Fetal Bovine Serum (FBS). Substituting serum with protein hydrolysates reduces the production expense of cultivated meat products and promotes establishing a sustainable food system. This study explores black soldier fly larvae (Hermetia illucens) as an emerging ethical and cost-effective alternative protein source to replace serum in media, particularly for cultivated meat production. The development of BSFL protein isolate involved defatting the larva, followed by protein extraction. The protein isolate was then hydrolyzed using an enzyme to produce BSFL hydrolysates. The goal was to supplement the protein isolate and hydrolysates with a serum-free media (B8) and determine their efficacy in replacing the 20% serum requirement for the cell culture of Bovine Satellite Cells. The BSFL protein isolate developed had a crude protein content of 80.42% and an amino acid composition conducive to cell proliferation. Experimental concentrations, ranging from 0.006 mg/ml for hydrolysate to 0.06 mg/ml for protein isolate, exhibited enhanced cell growth. Data from dsDNA quantification revealed no significant difference in growth between cells fed serum-containing growth media (BSC-GM) and BSFL protein hydrolysate (BSFLH_1h) over a short-term study. Results from the multi-passage growth study revealed that BSFLH_1h significantly improved cell growth compared to B8 over 4 passages. However, its doubling time was slower than BSC-GM. Additionally, it was observed that the protein isolate and hydrolysate were cytotoxic at higher concentrations. In the future, identifying and removing the cytotoxic compounds can further optimize the media composition. Immunostaining using Pax7 and DAPI identified supplemented media-maintained satellite cell identity of Bovine satellite cells, offering crucial insights into cellular proliferation. Furthermore, since each cell type requires varying serum and nutrients, testing these isolates and hydrolysates on different cell lines can provide better insight into creating a universal serum-free media. / Master of Science in Life Sciences / The global demand for animal proteins is projected to rise by 14% by 2030, amplifying the environmental toll of conventional animal-based protein production. Meat, dairy, aquaculture, and eggs significantly contribute to food-related emissions and occupy a vast portion of global farmland. Cultivated meat production can alleviate the growing demand for protein and address the environmental and ethical concerns associated with conventional livestock farming. Currently, the production of cultivated meat faces a significant hurdle: the high cost of culture media, primarily attributed to the use of Fetal Bovine Serum (FBS). Substituting serum with protein isolates or hydrolysates reduce the production expense of cultivated meat products and promotes a sustainable food system. Protein isolate and hydrolysates derived from black soldier fly larvae (Hermetia illucens) are rich in protein and essential amino acids and can be used as a cost-effective alternative to serum in cell culture media. The protein isolate and hydrolysates derived from BSFL were tested as supplements to a serum-free media (B8) to evaluate their effectiveness in supporting the growth of Bovine Satellite Cells. The protein hydrolysate demonstrated enhanced cell growth at experimental concentrations. However, it could not completely replace serum requirements without slowing cell growth. Despite challenges such as cytotoxicity at higher concentrations, our study suggests that further refinements and application on various cell types can assist in creating a sustainable and affordable serum-free media for cultivated meat production.

Células HEK293T e Huh7 foram adaptadas em meios livres de soro fetal bovino (SFM). Parâmetros metabólicos e de crescimento foram avaliados, além da expressão gênica heteróloga, utilizando um sistema de expressão que produz pseudo-partículas (ppHCV), derivadas do vírus da Leucemia Murina (MLV) e da Hepatite C (HCV). A adaptação foi realizada através de diluição sequencial para SFM. A linhagem HEK293T foi adaptada em dois SFM: Hybridoma-SFM e CHO-S-SFMII, a linhagem Huh7 foi adaptada nos quatro SFM escolhidos. O consumo de substratos para cada linhagem foi diferente entre os SFM, apesar de o crescimento celular ter sido semelhante. Para a análise da expressão gênica, três vetores foram co-transfectados em células HEK293T. Foi observado que para a produção de ppHCV, o tempo de coleta foi de 48 horas. O método de co-transfecção por lipofectamina produziu mais cópias de vírus, sendo que quantificações de 5,30x103 cópias RNA/&#956;L foram encontradas para vírus produzidos em células adaptadas no meio Hybridoma-SFM através de qRT-PCR. Estas ppHCV foram usadas para infectar células Huh7, células infectadas produziram cerca de 10 ng de proteína recombinante/106 células. / HEK 293-T and Huh7 cells were adapted in serum free mediu (SFM). Metabolic and growth parameters were assessed, as well as heterologous gene expression, using an expression system that produces pseudo-particles (ppHCV), derived from the murine leukemia virus (MLV), and Hepatitis C (HCV). The adaptation was performed by sequential dilution in SFM. The HEK- 293T line was adapted in two SFM: Hybridoma-SFM and CHO-S-SFMII, the Huh7 line was adapted in four chosen SFM. The consumption of substrates were different for each line in SFM, while cell growth was similar. For the analysis of gene expression, three vectors were co-transfected into HEK-293T cells. It was observed that for the production of ppHCV, the collection time was 48 hours. The method of co-transfection with lipofectamine produced more copies of the virus into the cells, 5,30 x103 RNA copies/&#956;L were found to virus produced in the cells adapted in Hybridoma- SFM, by qRT-PCR. These ppHCV were used to infect Huh 7, infected cells produced around 10 ng recombinant protein /106 cells.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-11T15:21:43Z No. of bitstreams: 1 Arquivototal.pdf: 4776508 bytes, checksum: 39f64f970100a5b5b15e3f1db0c3812c (MD5) / Made available in DSpace on 2018-04-11T15:21:43Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 4776508 bytes, checksum: 39f64f970100a5b5b15e3f1db0c3812c (MD5) Previous issue date: 2017-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cyberculture has caused changes in communication processes in the contemporary world, especially in relation to the poles of information production and diffusion, giving significant visibility to free media and post-mass movements, aided by the communication established through portable devices, (Wi-Fi, 3G, 4G) and other digital tools such as social networking sites as well as streaming and collaborative platforms. This new media communication phenomenon, with P2P communication, carries hybrid, convergent and multimedia elements, through which the subjects carry out new media practices and, in this way, put in tension the relations of power in the mediatic ecosystem by acting in Postmodernity post-mass movements, such as midialivrism. In order to understand how this phenomenon occurs, we analyze the configuration of midialivrism (MALINI; ANTOUN, 2013) in a convergent way to social manifestations that have occurred in Brazil since 2013, with the free collectives Mídia NINJA and Jornalistas Livres (Free Journalists), also converging to cyberculture, ruled by the cyberculture laws (LEMOS, 2003). These collectives emerge from this scenario, which mixes cyber-presence activism, providing the basis for reflecting on the points of tension and connection between free-media practices and corporate media, pointing to post-journalism. From the participant observer method, and with the aid of the grounded theory (DIC, 2005), we collect and analyze the material produced by midialivists in cyberculture. Through analysis and interpretation, it resulted in a mapping of a set of midialivist practices. This path made it easier to identify the midialivist's activities in the internet and street networks, and how the three factors (consumption / sharing / production) conditioned each other, ended up connecting, in a demonstration of what this new relationship of free media with Journalism as an alternative media in the mediatized society and how it interacts, in a collaborative way, on the social protest and movements. / A cibercultura ocasionou mudanças nos processos de comunicação na contemporaneidade, principalmente no tocante aos polos de produção e difusão de informação, dando significativa visibilidade à mídia livre e aos movimentos pós-massivos, auxiliados pela comunicação estabelecida através dos dispositivos portáteis, conexões móveis (Wi-Fi, 3G, 4G) e outras ferramentas digitais, como sites de redes sociais, além das transmissões via streaming e plataformas colaborativas. Este novo fenômeno comunicacional midiático, com comunicação P2P, carrega elementos híbridos, convergentes e multimídia, através dos quais os sujeitos protagonizam novas práticas midiáticas e, desta maneira, põem em tensão as relações de poder no ecossistema midiático pela atuação em movimentos pós-massivos da pós-modernidade, como o Midialivrismo. Com o objetivo de entender como acontece esse fenômeno, analisamos a configuração do midialivrismo – a partir do conceito de Fábio Malini e Henrique Antoun (2013) – de maneira convergente às manifestações sociais ocorridas no Brasil a partir de 2013, com os coletivos livres Mídia NINJA e Jornalistas Livres, convergente, também, à cibercultura, tendo como norte às leis da cibercultura de André Lemos (2003). Esses coletivos emergem deste cenário, que mescla ativismo ciber-presencial, possibilitando bases para refletirmos sobre os pontos de tensão e conexão entre práticas de mídia livre e a mídia corporativa, apontando na direção de um pós-jornalismo. A partir do método do observador participante, e com o auxílio da teoria fundamentada (DIC, 2005), coletamos e analisamos o material produzido pelos midialivristas na cibercultura. Mediante a análise e interpretação, resultou num mapeamento de um conjunto de práticas midialivristas. Esse percurso facilitou identificarmos as atividades do midialivrista nas redes da internet e na rua, e como os três fatores (consumo/compartilhamento/produção) condicionados entre si, acabaram se conectando, numa demonstração do que vem a ser essa nova relação da mídia livre com o jornalismo, como mídia alternativa, e de como ela interage na sociedade midiatizada, de maneira colaborativa, ao universo dos protestos e movimentos sociais.

Células HEK293T e Huh7 foram adaptadas em meios livres de soro fetal bovino (SFM). Parâmetros metabólicos e de crescimento foram avaliados, além da expressão gênica heteróloga, utilizando um sistema de expressão que produz pseudo-partículas (ppHCV), derivadas do vírus da Leucemia Murina (MLV) e da Hepatite C (HCV). A adaptação foi realizada através de diluição sequencial para SFM. A linhagem HEK293T foi adaptada em dois SFM: Hybridoma-SFM e CHO-S-SFMII, a linhagem Huh7 foi adaptada nos quatro SFM escolhidos. O consumo de substratos para cada linhagem foi diferente entre os SFM, apesar de o crescimento celular ter sido semelhante. Para a análise da expressão gênica, três vetores foram co-transfectados em células HEK293T. Foi observado que para a produção de ppHCV, o tempo de coleta foi de 48 horas. O método de co-transfecção por lipofectamina produziu mais cópias de vírus, sendo que quantificações de 5,30x103 cópias RNA/&#956;L foram encontradas para vírus produzidos em células adaptadas no meio Hybridoma-SFM através de qRT-PCR. Estas ppHCV foram usadas para infectar células Huh7, células infectadas produziram cerca de 10 ng de proteína recombinante/106 células. / HEK 293-T and Huh7 cells were adapted in serum free mediu (SFM). Metabolic and growth parameters were assessed, as well as heterologous gene expression, using an expression system that produces pseudo-particles (ppHCV), derived from the murine leukemia virus (MLV), and Hepatitis C (HCV). The adaptation was performed by sequential dilution in SFM. The HEK- 293T line was adapted in two SFM: Hybridoma-SFM and CHO-S-SFMII, the Huh7 line was adapted in four chosen SFM. The consumption of substrates were different for each line in SFM, while cell growth was similar. For the analysis of gene expression, three vectors were co-transfected into HEK-293T cells. It was observed that for the production of ppHCV, the collection time was 48 hours. The method of co-transfection with lipofectamine produced more copies of the virus into the cells, 5,30 x103 RNA copies/&#956;L were found to virus produced in the cells adapted in Hybridoma- SFM, by qRT-PCR. These ppHCV were used to infect Huh 7, infected cells produced around 10 ng recombinant protein /106 cells.