Les anticorps monoclonaux (AcM) sont des protéines ayant une reconnaissance antigénique spécifique utilisée pour le développement de réactifs thérapeutiques et diagnostiques. La production commerciale est réalisée en cultivant des cellules hôtes dans des bioréacteurs spécifiques. La densité cellulaire et le métabolisme cellulaire sont des paramètres clés pour le rendement élevé des AcM. Bioréacteur à fibres creuses (HFB), une cartouche contenant des fibres poreuses emballées, est l'un des systèmes de production disponibles dans le commerce. Si la densité de cellules obtenue peut conduire à un rendement élevé, le coût de l'ensemble du dispositif, y compris les pompes et les cartouches très coûteuses, empêche son utilisation de petites unités. Comme une alternative économique, nous avons proposé ici d'étudier le potentiel des modules de dialyse de polysulfone du commerce, classiquement employés dans le traitement de l'insuffisance rénale en phase terminale. Cependant, la membrane de polysulfone native a démontré une adsorption de protéines non spécifique significative préjudiciable à la production d'AcMs. De plus des enjeux normatifs viennent se greffer à ces problématiques scientifiques et technico-économiques, avec le cas des normes (ISO 13485/ AC S99-104/ GMP FDA /BPF…) qui imposent des méthodes de travail normalisées. Ce travail de thèse consiste en la conception d’un bioréacteur jetable, sur la base d’une cartouche de dialyse médicale à fibres creuses. Ce système doit offrir toutes les garanties en termes de production, de facilité d’utilisation, de stérilité, et permettre de concentrer les produits de cytoculture. La méthodologie scientifique a été couplée à une démarche qualité. La gestion de ce projet a été couplé à l’analyse de risques. En effet ce projet a été divisé en ses 5 composantes élémentaires décrite par Ishikawa par la méthode des 5 M. L’analyse de risque a consisté au calcul d’indice de criticité par la méthode AMDEC de chacune de ses familles de risques. Cette approche nous a permis de formaliser deux axes de recherche : i) la mise en œuvre d’une oxygénation efficace du milieu de culture (chapitre 4) et ii) les moyens de limiter le colmatage dans le module à fibres creuses pour obtenir une culture cellulaire conforme aux objectifs (chapitre 5). Le taux d’oxygénation est un facteur à prendre en compte dans un processus de culture cellulaire. L’oxygène peut être supplémenté selon deux modes, le mode passif ou le mode actif [Ozturk et Palsson 1990; Zhang S. et al 1992]. Il existe 2 types de système d’oxygénation : les système dit passif ou les échanges se font a travers une paroi de silicone et un système actif par aération directe dans le milieu de culture. Ce système est de loin l'opération la plus simple pour fournir de l'oxygène. Cependant, lorsque celui-ci est utilisé pour apporter de l’oxygène à des cultures de cellules mammifères, cela peut engendrer des altérations cellulaires. Des agents protecteurs chimiques peuvent être utilisés pour réduire les dommages cellulaires et la formation de mousse [Kamase et Moo-yung 1990; van Der pol L.A et al 1993]. Nos études ont démontré que l'addition d'agents anti-mousse peut entraîner une diminution du coefficient de transfert de masse d’O2 en phase liquide (Kl) [Kamase et Moo-yung 1990]. Nous avons établi l’efficacité de l’utilisation d’un polymère silice/silicone pour éliminer la mousse sur des cultures bactériennes et de cellules mammifères. Afin de limiter ces phénomènes de colmatage, les fibres de polysulfone ont été traitées avec plusieurs tensioactifs (acide pluronique F127, D-limonen et différentes huiles de silicone) qui ont conduit à une diminution significative de l'adsorption protéique. L'effet de ces surfactants sur les performances de filtration et sur la cytotoxicité a été étudié. Certains d'entre eux n'ont pas influencé ces paramètres alors que d'autres ont présenté des effets négatifs. / Monoclonal Antibodies (mAbs) are proteins with specific antigen recognition used for development of both therapeutic and diagnostic reagents. Commercial production is achieved by growing host cells in specific bioreactors. Cell density and cell metabolism are key parameters for high yield of mAbs. Hollow fiber bioreactor (I-IFB), a cartridge containing packed porous fibres, is one of the system for production commercially available. If the cell density achieved can lead to high yield, the cost of the whole device, including pumps and very expensive cartridges prevents its use of small units. As an economical alternative, we proposed here to investigate the potential of commercial polysulfone dialysis modules, classically employed in the treatment of end stage renal failure. However, the native polysulfone membrane demonstrated a significant non-specific protein adsorption detrimental to mAbs production. Moreover normative issues are added to these scientific and techno-economic issues, with the case of standards (ISO 13485 / AC S99-104 / GMP FDA / BPF ...) which impose standard working methods. This thesis consists of the design of a disposable bioreactor, based on a hollow-fiber medical dialysis cartridge. This system must offer all the guarantees in terms of production, ease of use, sterility, and allow to concentrate the cytoculture products. Scientific methodology has been coupled with a quality approach. The management of this project was coupled with the risk analysis. Indeed this project was divided into its 5 elementary components describe by Ishikawa by the 5M method. The risk analysis consisted in the calculation of the criticality index by the AMDEC method of each of its families of risks. This approach allowed us to formalize two research axes: i) the implementation of an effective oxygenation of the culture medium (chapter 4) and ii) the means of limiting the clogging in the hollow fiber module to obtain a culture consistent with the objectives (Chapter 5). The rate of oxygenation is a factor to be taken into account in a cell culture process. Oxygen can be supplemented in two modes, passive mode or active mode [Ozturk and Palsson 1990; Zhang S. et al 19921. There are two types of oxygenation system: the so-called passive system or the exchanges are made through a silicone wall and an active system by direct aeration in the culture medium. This system is by far the simplest operation for providing oxygen. However, when it is used to supply oxygen to mammalian cell cultures, this can cause cellular damage. Chemical protective agents can be used to reduce cell damage and DKamase and Moo-yung 1990 foam formation; van Der pol L.A. et al 1993 Cl. Our studies have shown that the addition of antifoaming agents can lead to a decrease in the liquid phase (K2) mass transfer coefficient of D Kamase and Moo-yung 1990C]. We have established the effectiveness of using a silica / silicone polymer to remove foam on bacterial and mammalian cell cultures. In order to limit these adsorption phenomena, polysulfone fibers were treated with several surfactants (pluronic acid F 127, D-limonen, and different silicone oils) which led to a significant decrease in protein adsorption. The effect of such surfactants on the filtration performances and on cytotoxicity were investigated. Some of the them did not influence these parameters while some presented negative effects. Finally, different cell culture parameters (cells densities, production yield, flow properties, fouling) were studied, as well as the performance of the bioreactor in perfusion continuous mode. The bioreactor was maintained in continuous mode for fifteen days and the production yield per batch was 250 mg of AcMs. The results obtained in this work allowed us to define the next steps to be taken, and are the subject of the Perspectives section.

La thèse est consacrée à l’étude de l’influence de l’aération sur le colmatage demembranes fibres creuses, en application aux bioréacteurs à membranes (BAMs)traitant les eaux usées. Un pilote expérimental permet des expériences de filtrationpar plusieurs faisceaux de fibres creuses immergées, en filtration externe-interne, lacompacité des fibres est proche de la configuration de modules membranairesindustriels. Les fibres ont une surface totale de 1,2 m. Les filtrations sont menéessur une suspension synthétique complexe dont les caractéristiques rhéologiquessont comparables à celles des boues activées de BAM. Les différents paramètresde l’aération testés sont le débit d’air, le lieu d’injection des bulles, et le type debulles injectées (grosses ou fines), pour un flux de filtration constant égalementparamètre d’étude. Pour des conditions d’aération montrant des performances defiltration contrastées, il est effectué la caractérisation locale de la phase gaz parbisonde optique, à l’intérieur des faisceaux de fibres. La rétention gazeuse et lesprofils de vitesse (tant la vitesse moyenne des bulles, que les fluctuations de vitesse)sont mesurés et analysés en fonction des paramètres d’aération. La synthèse des résultats met en évidence deux mécanismes hydrodynamiqueslocaux liés à l’aération, et permettant d’expliquer les performances de filtrationconsidérées. Les perspectives de l’étude proposent des emplacements pour lesaérateurs ce qui pourrait mener à une meilleure utilisation de l’air pour la réductiondu colmatage dans les bioréacteurs à membranes. / This thesis is dedicated to better understand the role of aeration to limit fouling onsubmerged hollow fibers membrane fouling, for wastewater treatment industry. Thisstudy uses an innovative experimental setup to perform outside-in filtrationexperiments thanks to several submerged loose hollow fibers organized in confinedbundles. The total membrane area is about 1.2 m2; the configuration of the bundlesis close to the one found in Membrane Bioreactors (MBRs). A complex synthetic fluidwith rheological characteristics close to MBRs sludge is used. Different aerationconditions such as the air flowrate, the location of the air injection, the type ofbubbles injected (whether they are coarse or fine bubbles injected in the reactor)were tested and membrane performances were analysed under a low or a ratherhigh flux of filtration. In specific aeration conditions showing a different behaviour interm of fouling, local characterization of the dispersed phase in an air and watersystem was undertaken by optical probes. Gas hold up and both mean and rootmean square bubble velocity profiles were determined and analysed regarding thelocation of air injection, the air flowrate and the type of bubbles. This study helps understanding the link between local hydrodynamics and foulingmechanisms and could lead to input for aeration device design and location in orderto decrease operating costs due to aeration in Membrane Bioreactors technology.

Orientadora: Prof.ª Dra Luisa Helena dos Santos Oliveira / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental, 2016. / Biorreatores com Membranas Submersas (BRMS) são, atualmente, reconhecidos como opção viável para o tratamento de esgotos sanitários e o reúso de águas. Apesar disso, a tecnologia é geralmente vista como de alto investimento quando comparado a sistemas convencionais de tratamento de esgoto, sobretudo pelo maior gasto energético com aeração do sistema. Porém, BRMS quando operados sob condições específicas para nitrificação e desnitrificação simultâneas (NDS), é possível obter um efluente de elevada qualidade com menor custo, devido a menor necessidade de aeração. Nesse sentido, a operação do sistema de BRMS com o processo de NDS pode aumentar a utilização desta tecnologia no tratamento de esgoto sanitário. Neste contexto, este trabalho teve por objetivo principal avaliar a remoção de nitrogênio via NDS em diferentes concentrações de oxigênio dissolvido (OD) em uma unidade piloto de um Biorreator de Membranas Submersas tratando esgoto sanitário. Para isso, o experimento foi dividido em três Fases de acordo com a concentração de oxigênio dissolvido no licor misto: Fase I (2,3 mgO2/L), Fase II (0,8 mgO2/L e Fase III (0,3 mgO2/L). Foram alcançadas elevadas eficiências de remoção de material orgânico, independente das variações na concentração de OD, DBO5 com valores médios de 98%, 97% e 98% e DQO de 95%, 96% e 95% para as Fases I, II e III, respectivamente. A remoção média de nitrogênio total obtida foi 33% na Fase I, 60% na Fase II e 50% na Fase III. Onde a remoção via NDS foi responsável por 60%, 78% e 74% do total removido para as Fases I, II e III, respectivamente. A remoção de nitrogênio amoniacal não foi limitada devido as baixas concentrações de OD e houve acúmulo de NO2-. O sistema operou sob baixos valores de pressão transmembrana durante todo o período, porém foi encontrada diferença significativa nos valores da Fase III em relação às Fases I e II. Houve uma redução significativa na permeabilidade da membrana quando a concentração de OD no licor misto foi reduzida para 0,3 mg/L, porém sem efeito significativo quando a concentração de OD foi de 0,8 mg/L. Foram observados maiores valores de SPE ligado na Fase III em relação às demais, principalmente na fração de carboidratos que pode estar diretamente ligada a taxa de fouling. Foi possível alcançar remoção de NT em OD de 0,8 mg/L (Fase II) sem causar um impacto negativo no desempenho das membranas, sendo esta fase a mais adequada para a ocorrência do processo de NDS em BRMS. / Submerged Membrane Bioreactors (SMBR) are actually, recognized as viable option for wastewater treatment and reuse. Nevertheless, this technology is generally viewed as a high investment compared to conventional sewage treatment systems, especially for greater energy requirement for aeration system. However, SMBR when operated under specific conditions for simultaneous nitrification and denitrification (SND) it is possible to obtain a high quality effluent with lower compared to the conventional configuration because of less need for aeration. Therefore, the operation of the system SMBR with the NDS process can increase the utilization of this technology in the wastewater treatment. In this context, this study aimed to evaluate the removal of nitrogen via SND under different dissolved oxygen concentrations (DO) in a pilot of a SMBR treating wastewater. For this, the experiment was divided into three phases according to the concentration of dissolved oxygen in the mixed liquor: Phase I (2,3 mgO2/L), Phase II (0,8 mgO2/L and Phase III (0,3 mgO2/L). Were achieved high organic matter removal efficiencies regardless of fluctuations in DO concentration in terms of BOD5 average values of 98%, 97% and 98% and COD average values of 95%, 96% and 95% for Phases I, II and III respectively. The average total nitrogen removal was 33% in Phase I, 60% in Phase II and Phase III 50%. Where the removal means SND is responsible for 60%, 78% and 74% of the total removed for Phases I, II and III respectively. The removal of ammoniacal nitrogen is not limited by the low DO concentrations and there was accumulation of NO2- in Phase III. The system operated under low transmembrane pressure values throughout the period, but significant difference was found in the values of Phase III in relation to phases I and II. There was a significant reduction in membrane permeability when the DO concentration in the mixed liquor was reduced to 0.3 mg/L, but without significant effect when the DO concentration was 0.8 mg/L. It was observed highest bound EPS values in Phase III in relation to the others, mainly in the fraction of carbohydrates that can be directly linked to fouling rate. Satisfactory NT removal was achieved when concentration DO was 0.8 mg/L (Phase II) without causing a negative impact on the performance of membranes, being this phase the most suitable for the occurrence of the SND process in SMBR.

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)

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2016-10-10T12:43:24Z No. of bitstreams: 1 TeseGYR.pdf: 8156092 bytes, checksum: 727d9cd338ae7ed90fe24238b42228bd (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-11T20:34:55Z (GMT) No. of bitstreams: 1 TeseGYR.pdf: 8156092 bytes, checksum: 727d9cd338ae7ed90fe24238b42228bd (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-11T20:35:04Z (GMT) No. of bitstreams: 1 TeseGYR.pdf: 8156092 bytes, checksum: 727d9cd338ae7ed90fe24238b42228bd (MD5) / Made available in DSpace on 2016-10-11T20:42:33Z (GMT). No. of bitstreams: 1 TeseGYR.pdf: 8156092 bytes, checksum: 727d9cd338ae7ed90fe24238b42228bd (MD5) Previous issue date: 2015-03-25 / Outra / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Pneumatic bioreactors are devices free of moving parts which have the purpose of converting raw materials in bio-products of commercial interest by the action of enzymes or cells. They are promising in the biochemical industry, ensuring good oxygen transfer and consuming less energy. Global performance parameters such as global gas hold up and the volumetric oxygen transfer coefficient are important criteria in the design and selection among different geometries of the mentioned devices. In the present work it was carried out modeling and simulation of pneumatic bioreactors based on Computational Fluid Dynamics (CFD) in order to estimate the global gas hold up and the volumetric oxygen transfer coefficient in three different geometries of pneumatic bioreactors: bubble column, concentric tube airlift and split tube airlift. The simulated results of each performance parameter were verified by comparison with the experimental values reported by Thomasi et al. (2010) and Mendes and Badino (2015) for the fluids distilled water, glycerol solution 10 cP and xanthan gum solution 0.2% w/v (weight/volume) in a wide range of specific air flow rate (0 to 5 min- 1). Application suite ANSYS® 14.5 was used for numerical simulations in CFD. Important parameters such as the bubble diameter played a great influence on results of the volumetric oxygen transfer coefficient. It can be observed by the experimental and simulated results that the concentric tube airlift bioreactor was the best alternative to the global gas hold up and the volumetric oxygen transfer coefficient (reaching 14% and 0.06 s-1 for distilled water, respectively). It was found that the results obtained via the CFD agreed with the majority trend of experimental data, capturing the most important hydrodynamic phenomena and mass transfer characteristics, showing that the modeling of different systems with different fluids fulfilled the main objective of obtaining reliable models design and performance of other geometries of pneumatic bioreactors. / Os biorreatores pneumáticos são equipamentos industriais isentos de partes móveis que têm a finalidade de converter matérias-primas em bioprodutos de interesse comercial pela ação de enzimas ou células. São promissores na indústria bioquímica, pois garantem boa transferência de oxigênio consumindo menos energia. Parâmetros globais de desempenho, como a retenção gasosa global e o coeficiente volumétrico de transferência de oxigênio são critérios importantes no projeto e seleção entre geometrias diferentes dos equipamentos mencionados. No presente trabalho foi realizada a modelagem e a simulação de biorreatores pneumáticos baseada na Fluidodinâmica Computacional (CFD - Computational Fluid Dynamics) de forma a estimar a retenção gasosa global e o coeficiente volumétrico de transferência de oxigênio em três geometrias distintas: coluna de bolhas, airlift de cilindros concêntricos e airlift split. Os resultados simulados de cada parâmetro de desempenho foram verificados comparando-se com os valores experimentais reportados nos trabalhos de Thomasi et al. (2010) e Mendes e Badino (2015) para os fluidos água destilada, solução de glicerol 10 cP e solução de goma xantana 0,2% m/v (massa/volume) e vazão de alimentação específica de ar numa ampla faixa (0 a 5 min-1). Foi empregada a suíte de aplicativos ANSYS® 14.5 para as simulações numéricas em CFD. Parâmetros importantes, como o diâmetro de bolha, exerceram grande influência nos resultados referentes ao coeficiente volumétrico de transferência de oxigênio. Destaca-se, pelos resultados experimentais e simulados, que o biorreator pneumático do tipo airlift de cilindros concêntricos apresentou-se como a melhor alternativa para a retenção gasosa global e para o coeficiente volumétrico de transferência de oxigênio (atingindo 14% e 0,06 s-1 para a água destilada, respectivamente). Verificou-se que os resultados obtidos via CFD concordaram com a tendência majoritária dos dados experimentais, capturando os fenômenos mais relevantes das características hidrodinâmicas e da transferência de massa, mostrando que a modelagem dos diferentes sistemas com diferentes fluidos atendeu ao principal objetivo de obter modelos confiáveis para o projeto e comparação de desempenho de outras geometrias de biorreatores pneumáticos. / CNPq: 478472/2011-0 / CNPq: 140466/2011-8

Orientador: Prof. Dr. Eduardo Lucas Subtil / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental, Santo André, 2018. / O lançamento de águas urbanas servidas (AUS) sem tratamento causa problemas associados à saúde pública e à deterioração dos recursos hídricos. Neste contexto, os Biorreatores com Membranas Submersas (BRMS) e Biofilme (BRMS-BF), operados sob condições específicas para nitrificação e desnitrificação simultâneas (NDS), são uma opção viável para o tratamento de água AUS e seu potencial reúso. Este projeto de pesquisa teve como objetivos avaliar a remoção de nitrogênio via NDS, caracterizar o processo de fouling das membranas e identificar os principais microrganismos associados ao ciclo do nitrogênio em um sistema piloto BRMS-BF operado em diferentes concentrações de oxigênio dissolvido (OD) para o tratamento de AUS. O volume útil do reator foi de 156 L com módulo de 17 membranas (sintetizadas em fluoreto de vinilideno, PVDF) tipo placa plana de ultrafiltração de 0,1 ?m. O material suporte foi de poliuretano (gel termoplástico). O efluente utilizado foi da residência estudantil e o restaurante universitário da Universidade de São Paulo. Foram propostas duas fases de operação com concentrações de OD acima de 2, e 0,8 mgO2/L. Os valores de temperatura (T), Potencial de Redox (ORP), e pressão transmebranar (PTM) foram monitorados e os de pH, vazão (Q) e OD controlados, por meio de sensores. A aeração foi realizada controlando o fluxo através de rotação por meio de dois difusores de ar de 10 e 15 L/min. Os resultados para a fase I e II apresentaram os valores médios a seguir, respetivamente: T (24 e 23 ?), ORP (186 e -11 mV), PTM (0,02 e 0,04 bar), pH (6,9 e 6,7), Q (22 e 16 L/h), OD (2,2 e 0,9 mgO2/L) e SST (5,7 e 5,6 g/L). A remoção de matéria orgânica (DQO e DBO5 mg/L) e de turbidez foi maior de 96% e de 99%, respetivamente nas duas fases. A remoção de nitrogênio total foi de 33% para a Fase I e 74% na Fase II, apresentando melhoras na remoção a baixa concentração de OD. No entanto, a Fase II apresentou acúmulo de nitirto, que pode estar relacionado com a desnitrificação parcial, devido a ausência de carbono suficiente em presença de baixas concentrações de OD. As comindades de microrganismos observadas perteneceram principalmente ao filo Petrobacteria (abundância relativa > 70% na biomassa suspensa e o biofilme para a Fase I e II). O gênero Nitrospira foi o principal gênero identificado que influenciou o processo de oxidação de nitirito. Não foi encontrada diferença significativa entre os microrganismos presentes segundo o tipo de biomassa e a concentração de OD. Não houve uma redução significativa na permeabilidade das membranas, associada ao fouling, quando a concentração de OD foi reduzida para 0,9 mg/L. / Urban waters served (UWS) without any kind of treatment causes problems which are related to public health and hydric resources deterioration. In this context, the Submerged Membranes Bioreactor (MBRS) and Biofilm (MBRS-BF), operated under specific conditions for simultaneous nitrification-denitrification (SND), are a viable option for UWS treatment and their potential reuse. The aim of this project is to evaluate nitrogen removal via SND, characterize the process of fouling of the membranes and identify the main microorganisms related to nitrogen cycle in a MBRS-BF pilot-system which was operated on different concentrations of dissolved oxygen (DO) for UWS treatment. Reactor useful volume was 156 L with 17-membranes module - (synthesized in a Polyvinylidene Fluoride - PDVF) - 0,1 ?m ultrafiltration flat-plate type. Support material was composed of polyurethane (thermoplastic gel). The used effluent has come as from student residence as USP's food court. Two operation-phases were proposed in which concentration was over 2, and 0,8 mgO2/L. Temperatures values (T), Redox potential (ORP) and trasmembrane pressure(TMP) was monitored as well as pH, leakage (L) and DO was controlled by sensors. Aeration was carried out by controlling flow in rotation through two air diffusers with 10 e 15 L/min capacity. The results for phases I and II performed the following average values, respectively: T (24 and 23), ORP (186 and -11 mV), PTM (0,02 and 0,04 bar), pH (6,9 and 6,7), Q (22 and 16 L/h), OD (2,2 and 0,9 mgO2/L) and SST (5,7 and 5,6 g/L). Organic matters removal (DQO and DBO5 mg/L) and turbidity was bigger, by ranging from 96% to 99%, respectively, in both phases of this operation. Total nitrogen removal was 33% for Phase I and 74% of Phase II, by perfoming best efficiency in concentration of low OD (0,9mgO2/L).However, phase II showed nitrite storage, which can be attributed to partial denitrification, due to absence of carbon enough in OD low concentrations. Observed microorganisms communities belonged to mainly Petrobacteria phylum (relative abundance > 70% in suspended biomass and biofilm for phases I and II). Nitrospira was the main genus found which influenced nitrite oxidation process. It was not found significant difference among these present microorganisms according to their biomass type and DO concentration. There were not any significant reduction on membranes permeability, related to fouling, when DO concentration was reduced to 0,9 mg/L.

O presente trabalho apresenta o biorreator anaeróbio contínuo de leito fixo ordenado como alternativa ao tratamento de efluentes contendo os íons \'CU POT.2+\' e \'CD POT.2+\', sendo que cada metal foi estudado isoladamente. O biorreator operou sempre com um tempo de detenção hidráulica de 12 à 15 horas, com uma água residuária sintética simulando o esgoto sanitário, na concentração de 500 mg \'0 IND.2\'/L (em termos de DQO), a uma temperatura de 30°C. Durante a fase na qual o biorreator operou com água residuária em condições metanogênicas o biorreator foi alimentado, em condições distintas, com concentrações crescentes de \'CU POT.2+\' e \'CD POT.2+\'. O acúmulo dos metais na matriz de imobilização levou ao bioreator apresentar instabilidade na operação, sendo determinadas as concentrações críticas para o \'CU POT.2+\' (50,8 mg \'CU POT.2+\'/L) e para o \'CD POT.2+\' (29,8 mg \'CD POT.2+\'/L). Durante a operação nas condições metanogênicas foi observado o efeito tóxico pela diminuição na eficiência de remoção de matéria orgânica, mas sem perder a estabilidade operacional até que as concentrações críticas foram atingidas. A partir dos valores das concentrações críticas, o biorreator foi operado em condições sulfetogênicas em duas relações DQO/[\'SO IND.4\'POT.2-\'] distintas (0,68 e 1,99 para o uma concentração média de 36 mg \'CU POT.2+\'/L; e 0,69 e 8,02 para uma concentração média de 27 mg \'CD POT.2+\'/L). Nas operações em condições sulfetogênicas, o biorreator operou de maneira estável, no entanto, não foi capaz de remover todo o metal alimentado. / This work presents the anaerobic continuous bioreactor with fixed-structured bed as a viable configuration of treatment of wastewater containing \'CU POT.2+\' and \'CD POT.2+\' ions. Each metallic ion were studied separately. The bioreactor was operated with 12 to 15 hours of hydraulic retention time, with a synthetic wastewater simulating domestic sewage (500 mg \'0 IND.2\'/L, in COD terms), at 30°C. In methanogenic conditions, the bioreactor was fed with crescent amount of \'CU POT.2+\' and \'CD POT.2+\'. The metal accumulation in the irnrnobilization matrix caused instability on bioreactor when achieving the critical concentrations for \'CU POT.2+\' (50,8 mg \'CU POT.2+\'/L) and for \'CD POT.2+\' (29,8 mg \'CD POT.2+\'/L). Also it was observed that this crescent metal concentration affected the efficiency of bioreactor in removing organic matter, although without operational instability until the system reach these critical metal concentrations. The bioreactor was operated in sulfidogenic conditions fed with a wastewater containing \'CU POT.2+\' and \'CD POT.2+\' at critical concentrations considering two different COD/[\'SO IND.4\'POT.2-\'] (0,68 and 1,99 for a mean influent \'CU POT.2+\' concentration of 36 mg \'CU POT.2+\'/L; and 0,69 and 8,02 para for a mean influent \'CD POT.2+\' concentration of 27 mg \'CD POT.2+\'/L). During the sulfidogenic conditions operations the bioreactor operated showing stability, however the system was not able to achieve the maximum efficiency in metal removal.

Aujourd’hui, la transplantation est le seul traitement efficace proposé aux patients souffrant d’une insuffisance hépatique fulminante. La nécessité de disposer d’un système de suppléance hépatique transitoire apparaît donc indispensable. C’est dans cet axe que se sont développés les systèmes qualifiés de foies bio artificiels (BAL). Leur principale caractéristique est d’incorporer un bioréacteur hébergeant des cellules pouvant restaurer l’activité hépatiques dans son ensemble. A l’heure actuelle, les hépatocytes primaire humains (HEP) issus de foies de donneurs non transplantables sont considérées comme le meilleur choix. Cependant, leur utilisation reste limitée par leur faible disponibilité et la difficulté à les maintenir différenciés en culture in vitro. Pour remédier à ce dernier point, l’approche la plus prometteuse semble être une co-culture des hépatocytes avec les cellules non parenchymateuses afin de recréer un environnement proche des sinusoïdes hépatiques. Ce travail de thèse repose sur la mise en place d’une nouvelle approche de co-culture tridimensionnelle sous la forme de sphéroïdes, d’HEP primaires avec les principaux types de cellules non-parenchymateuses (les cellules de Kupffer, les cellules endothéliales et les cellules étoilées) selon des proportions spécifiques. Puis de leurs encapsulations dans des billes d’alginate et leurs cultures au sein d’un bioréacteur à lit fluidisé. Ce modèle s’est révélé pertinent et approprié à maintenir les fonctions hépatiques dans le temps. Bien que beaucoup d’optimisation reste à définir, ce travail exploratoire témoigne de l’intérêt de cette approche intéressante pour le progrès des systèmes BAL. / Liver shortage makes transplantation inapplicable to all acute liver failure patients. Bioartificial Iiver (BAL) devices represent a temporary solution for these patients which are thereby bridged tilt Iiver transplantation or regeneration BAL treatment offers blood purification and substitution of metabolic functions through the activity of hepatocytes (HEPs), which are integrated in the device within acclimating containers, so-called bioreactors. Primary human hepatocytes are the ideal cell type to use in BAL, but they are scarcely available and difficult to maintain in vitro. Co-culture of HEPs with supporting cells has been proposed as the most promising strategy for preserving HEP behaviors in in vitro conditions. In fact, assisting cells types hold their ability to influence functional responses of the HEPs by providing them with cues of the native organ.This PhD work proposed a novel approach of co-culture for the functional sustain and preservation of the HEPs in the environment of the fluidized bed bioreactor (designed in our Iaboratory). Definition of this model took inspiration from the cellular organization in the organ; therefore, it employed three major sinusoidal non-parenchymal cell populations (liver sinusoidal, Kupffer, and hepatic stellate cells) which, together with HEPs, were cultured with three-dimensional arrangement (spheroids) and according to specific proportions. The resulting model was characterized in terms of functional benefits for the HEPs, and then applied in the microenvironment of alginate beads, which provide cells with immunological and mechanical protection in the fluidized bed bioreactor. This spheroidal multi-cultured model revealed its potentiality in sustaining in vitro HEP behaviors over time. Although much remains to be refined, this model may represent an interesting approach for the progress of BAL

La nécessité de nouveaux modes de suppléance hépatique se fait clairement ressentir pour maintenir en vie les patients en attente d’une greffe. Des traitements, basés sur l’utilisation de cellules cultivées dans un environnement adéquat, pourraient même permettre le rétablissement de certains patients en insuffisance hépatique sévère et ainsi éviter la greffe et les traitements immunosuppresseurs associés. Une suppléance hépatique efficace pourrait aussi servir au rétablissement des patients récemment greffés ou ayant subi une hépatectomie. L’objectif de cette thèse a été de développer, créer et valider un nouveau foie bioartificiel avec une prise en compte des contraintes réglementaires des médicaments combinés de thérapie innovante. Ce BAL doit pouvoir inclure des bioréacteurs à lit fluidisé perfusés par du plasma et contenant des sphéroïdes d’hépatocytes encapsulés. Des microparticules de verre, ajoutés aux billes d’alginate pour les alourdir, permettent d’obtenir une fluidisation optimale dans du plasma pathologique sans que des effets délétères ne soient observables ni pour les cellules ni pour les propriétés mécaniques des billes. Une méthode de culture cellulaire utilisant un revêtement anti-adhérent sur des boites de Petri en verre permet de produire un nombre important de sphéroïdes viables in vitro. Ces sphéroïdes encapsulés peuvent être maintenus vivants et métaboliquement actifs dans un bioréacteur à lit fluidisé pendant au minimum 4 jours.Pour assurer l’efficacité du BAL et la sécurité des patients, une circulation extracorporelle complexe a été mise au point pour être compatible avec une machine d’épuration extracorporelle commerciale, la Prismaflex® de la société Gambro, déjà utilisée en soins intensifs. Une étude préclinique sur un modèle ovin a montré que le traitement était bien toléré en ce qui concerne les aspects hémodynamiques. La prochaine étape concerne la mise en place d’une étude dans un modèle porcin d’insuffisance hépatique, avant de pouvoir procéder aux premiers essais cliniques. / The need for new liver support devices is clearly felt to allow keeping alive patients waiting for a transplant. Treatments, based on the use of cells cultured in an adequate environment, may even allow the recovery of some patients suffering from acute liver failure and avoid graft and associated immunosuppressive therapies. A hepatic substitution could also be used to reestablish patients recently transplanted or who had underwent an hepatectomy.. The objective of the thesis was to design, create and validate of a new bioartificial liver with consideration for the regulatory requirements of the Advanced Therapies Medical Product (ATMP). This device has to include fluidized bed bioreactors perfused with plasma and hosting alginate-encapsulated hepatocytes spheroids. Microparticles of glass have been added to weight down alginate beads in order to have an optimal fluidization in pathological plasma without negative effects neither on cells metabolism nor on mechanical properties of the beads. A cellular culture method using non adhesive coating in Petri dish led to the production of a large amount of viable spheroids in vitro. These encapsulated spheroids can be kept alive and metabolically active in a fluidized bed bioreactor during a minimum of four days. To ensure the efficacy of the BAL and the safety of patients, a complex extracorporeal circulation was designed to be compatible with a commercial medical device, the Prismaflex® monitor of the company Gambro, already used in intensive care units. A preclinical study on sheeps has shown that the treatment was well tolerated in terms of hemodynamics considerations. The next step is the establishment of a study in a porcine model of liver failure, before we can proceed to the first clinical trial.

As células estromais mesenquimais (CMMs) se tornaram de grande interesse para a terapia celular devido ao seu potencial de se diferenciar e reconstituir tecidos especializados. Mais recentemente, este interesse tem aumentado significativamente devido à descoberta de que as CMMs são capazes de secretar uma infinidade de mediadores para estimular a regeneração in situ de tecidos lesados. Dessa forma, CMMs podem ser consideradas tanto como um produto terapêutico em si, quanto uma biofábrica de diversas proteínas relevantes do ponto de vista terapêutico. Para atender a estas crescentes demandas, ambas as aplicações requerem o desenvolvimento de processos de expansão celular com alto rendimento, sob condições de cultivo definidas, reprodutíveis, escalonáveis, permitindo a obtenção de produtos com adequada identidade, potência, pureza, segurança e viáveis economicamente. Frente ao exposto, este trabalho teve como objetivos principais o estabelecimento de um processo de expansão de CMMs baseado em biorreatores e a caracterização do secretoma destas células visando aplicações terapêuticas. Para isto, a expansão de CMMs do cordão umbilical (MCUs) foi realizada em frascos multicamadas (MC) e nos biorreatores de leito fixo (LF), tanque agitado com microcarregadores (TA) e fibrasocas (FO). Os resultados mostraram que a taxa de proliferação específica das células foi maior (< tempo de duplicação) no biorreator de FO (36,8 ± 1,7 horas), bem como o fator de expansão (9,8 ± 1,0) e a eficiência na recuperação celular (100%). Um nível similar de produção celular foi observado para o TA, MC e LF com elevado fator de expansão celular (8,8 ± 0,39, 8,7 ± 0,90, 6,9 ± 1,3, respectivamente). No entanto, em termos de eficiência na recuperação celular (%), LF apresentou a menor taxa de recuperação dentre todos os sistemas (18% (± 0,77)), acompanhado pelo TA (61% (± 15,7)). As células mantiveram suas características imunofenotípicas e o potencial de diferenciação em adipócitos, osteócitos e condrócitos em todos os sistemas de cultivo avaliados. Foi também realizada a análise de custos (COG) e avaliação da viabilidade econômica para produção de CMMs visando tratamento da doença do enxerto contra o hospedeiro (DECH) em escala comercial, utilizando os sistemas de cultivo avaliados experimentalmente sob diferentes estratégias de reembolso. Apesar dos resultados experimentais satisfatórios para o biorreator FO, o COG revelou que este sistema tem o maior custo devido aos elevados custos dos consumíveis requeridos e do custo do equipamento. O frasco MC foi considerado como a tecnologia mais rentável e robusta no cenário avaliado e o biorreator TA obteve a segunda posição. O biorreator TA foi escolhido como o mais adequado analisando de maneira conjunta os dados experimentais obtidos, a análise dos custos dos diferentes sistemas de cultivo e a escalonabilidade de cada sistema. Assim, esse biorreator foi eficientemente utilizado para o cultivo de MCUs em condições isentas de SFB e xenoantígenos, sendo possível a produção de uma grande quantidade de células, representando um passo importante no desenvolvimento de um bioprocesso em conformidade com as normas das agências regulatórias. Por fim, com a análise do secretoma das CMMs por espectrometria de massas foi possível a identificação de uma gama enorme de proteínas interessantes (aprox. 2400) envolvidas em importantes processos biológicos. O futuro monitoramento dessas proteínas em biorreatores poderá representar um método inovador e original de produção de produtos livres de células para uso na terapia celular. / Mesenchymal stem/stromal cells (MSC) have become of great interest for cell therapy because of its potential to differentiate and reconstitute specialized tissues. More recently, such interest has significantly increased due to the discovery that MSC are capable of secreting a plethora of mediators to stimulate the in situ regeneration of injured tissues. Thus, MSC can be considered as a therapeutic product itself and as a biofactory of various relevant therapeutic proteins. To meet these increasing demands, both applications require the development of high-yield, reproducible, scalable and cost-effective bioprocesses under defined culture conditions, obtaining products with proper identity, purity and safety. Based on these, the main goal of this work was the establishment of a MSC expansion process based on bioreactors and secretome characterization of these cells targeting therapeutic applications. The MSC expansion was performed using multi-layered flasks (ML) and fixed bed (PB), stirred tank (STR) and hollow fiber (HF) bioreactors. The results showed that the proliferation rate of the cells was higher (< doubling time) in the HF bioreactor (36.8 ± 1.7 hours), as well as the expansion fold-increase (9.8±1.0) and harvesting efficiency (100%). A similar level of cell production was observed for STR, ML and PB with high fold-increase (8.8±0.39, 8.7±0.90, 6.9±1.3, respectively). However, in terms of harvesting efficiency (%), PB bioreactor presented the lowest retrieval rate across all the technologies (18% (±0.77)), followed by STR (61% (±15.7)). The cells retained their functional properties after culture in all the culture systems evaluated. This study was then extended through the use of a bioprocess economics tool for the evaluation of the economic feasibility of producing MSC-based treatment for acute graft vs. host disease (aGvHD) at commercial scale, using the culture systems experimentally evaluated under different reimbursement strategies. Despite the advantageous experimental results of HF bioreactors, the COG analysis has revealed that this is the least cost effective cell culture system to be used, due to its high consumable and equipment costs. ML flasks ranked first as the most cost effective and robust technology in this scenario and microcarrier-based technologies (STR) ranked in second position. The STR bioreactor was chosen as the most suitable for MSC expansion analyzing the experimental data, COG analysis and scalability of each culture system. Thus, STR bioreactor was efficiently tested for MSC expansion under serum and xeno-free conditions and it was possible to produce a large amount of cells. The development of a scalable microcarrier-based stirred culture system using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates. Finally, with the MSC secretome analysis by mass spectrometry it was possible to identify a wide range of interesting proteins (approx. 2400) involved in important biological processes. The future monitoring of these proteins in bioreactors may represent a novel and unique method of producing cell-free products for use in cellular therapy.