Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.

O ribeirão Limeira nasce em Piquete - SP e deságua no rio Paraíba do Sul, em Lorena - SP. Em sua bacia hidrográfica existem indústrias de materiais bélicos e abrasivos, plantações de eucalipto e áreas de pastagem. Para avaliar possíveis impactos do uso e ocupação da área, este estudo avaliou as condições limnológicas e ecotoxicológicas do ribeirão, entre set/07 e jul/08. Ao longo do ribeirão, foram estabelecidos sete pontos amostrais, da nascente até a foz. A água e sedimento foram avaliados quanto à toxicidade aguda e crônica através de bioensaios com os cladóceros Daphnia similis, Ceriodaphnia silvestrii e a alga Pseudokirchneriella subcapitata para a água; e C. silvestrii, D. similis e o quironomídeo Chironomus xanthus para o sedimento. Os parâmetros limnológicos analisados na água foram: turbidez, condutividade, OD, pH, DQO, DBO, nitrato, nitrito, fósforo total, sólidos totais dissolvidos, dureza, metais (Ag, Cd, Cr, Cu, Fe, Mn, Mg, Ni, Pb e Zn) e coliformes termotolerantes. Os parâmetros físico-químicos do sedimento analisados foram: fósforo total, matéria orgânica, granulometria, metais totais e frações biodisponíveis. Bioensaios in situ foram realizados utilizando câmaras de PVC, em pontos previamente selecionados, baseados nos resultados de ecotoxicidade do período de set/07 a jul/08. A sazonalidade influenciou na dinâmica deste ambiente e nas variáveis analisadas, também ocorreu variação espacial indicando influência de ações antrópicas no ribeirão. Conforme os parâmetros analisados na água, alguns pontos amostrais estiveram fora dos padrões estabelecidos pela Resolução CONAMA 357/05 (Classe I e II) em alguns meses. Fontes de poluição difusa dificultaram a identificação das possíveis substâncias causadoras de toxicidade. Ao longo do ribeirão, certos pontos apresentaram valores de metais no sedimento fora dos limites preconizados pela Canadian Council of Ministers of the Environment. O ribeirão Limeira apresentou a qualidade da água regular, segundo o índice para a manutenção da vida aquática (IVA). Os ensaios in situ indicaram que o sedimento do ponto P4 apresentou toxicidade. Esses bioensaios no ambiente se mostraram mais eficientes que os ensaios laboratoriais, refletindo uma melhor realidade em relação à possível presença de substâncias tóxicas no ecossistema aquático. / The Limeira stream is born in Piquete - SP and flows into the Paraiba do Sul river, in Lorena - SP. In this basin there are military and abrasives industries, eucalyptus plantations and pasture areas. To evaluate potential impacts of the use and occupation of the area, this study assessed the ecotoxicological and limnological conditions of the stream between September 2007 and July 2008. Seven sampling points were estabilished along the river from the source to the mouth. The acute and chronic toxicity were evaluated both in the water and sediment, using the cladocerans Daphnia similis and Ceriodaphnia silvestrii, algae Pseudokirchneriella subcapitata for the water and C. silvestrii, D. similis and chironomids Chironomus xanthus for the sediment. The limnological parameters analyzed in water were: turbidity, conductivity, dissolved oxygen, pH, COD, BOD, nitrate, nitrite, total phosphorus, total dissolved solids, hardness, metals (Ag, Cd, Cr, Cu, Fe, Mn, Mg, Ni , Pb and Zn) and fecal coliform; and the physical-chemical parameters of sediment were: total phosphorus, organic matter, granulometry, metals and bioavailable fractions. The in situ bioassays were conducted using PVC chambers at previously selected points, based on the results of ecotoxicity. The results showed that seasonality influenced both the dynamics of the environment and the analyzed variables. The spatial variation indicated influence of human activities along the stream. In some months, the analyzed parameters of water were outside the parameters established by CONAMA Resolution 357/05 for Class I and II. Diffuse pollution sources made it difficult to identify the potential toxicity-causing substances. In some points along the river, the values of metals in sediment were outside the limits recommended by the Canadian Council of Ministers of the Environment. According to the index for the maintenance of aquatic life (VAT), the river showed regular water quality due to the results of acute and chronic toxicity observed in some points.

O ribeirão Limeira nasce em Piquete - SP e deságua no rio Paraíba do Sul, em Lorena - SP. Em sua bacia hidrográfica existem indústrias de materiais bélicos e abrasivos, plantações de eucalipto e áreas de pastagem. Para avaliar possíveis impactos do uso e ocupação da área, este estudo avaliou as condições limnológicas e ecotoxicológicas do ribeirão, entre set/07 e jul/08. Ao longo do ribeirão, foram estabelecidos sete pontos amostrais, da nascente até a foz. A água e sedimento foram avaliados quanto à toxicidade aguda e crônica através de bioensaios com os cladóceros Daphnia similis, Ceriodaphnia silvestrii e a alga Pseudokirchneriella subcapitata para a água; e C. silvestrii, D. similis e o quironomídeo Chironomus xanthus para o sedimento. Os parâmetros limnológicos analisados na água foram: turbidez, condutividade, OD, pH, DQO, DBO, nitrato, nitrito, fósforo total, sólidos totais dissolvidos, dureza, metais (Ag, Cd, Cr, Cu, Fe, Mn, Mg, Ni, Pb e Zn) e coliformes termotolerantes. Os parâmetros físico-químicos do sedimento analisados foram: fósforo total, matéria orgânica, granulometria, metais totais e frações biodisponíveis. Bioensaios in situ foram realizados utilizando câmaras de PVC, em pontos previamente selecionados, baseados nos resultados de ecotoxicidade do período de set/07 a jul/08. A sazonalidade influenciou na dinâmica deste ambiente e nas variáveis analisadas, também ocorreu variação espacial indicando influência de ações antrópicas no ribeirão. Conforme os parâmetros analisados na água, alguns pontos amostrais estiveram fora dos padrões estabelecidos pela Resolução CONAMA 357/05 (Classe I e II) em alguns meses. Fontes de poluição difusa dificultaram a identificação das possíveis substâncias causadoras de toxicidade. Ao longo do ribeirão, certos pontos apresentaram valores de metais no sedimento fora dos limites preconizados pela Canadian Council of Ministers of the Environment. O ribeirão Limeira apresentou a qualidade da água regular, segundo o índice para a manutenção da vida aquática (IVA). Os ensaios in situ indicaram que o sedimento do ponto P4 apresentou toxicidade. Esses bioensaios no ambiente se mostraram mais eficientes que os ensaios laboratoriais, refletindo uma melhor realidade em relação à possível presença de substâncias tóxicas no ecossistema aquático. / The Limeira stream is born in Piquete - SP and flows into the Paraiba do Sul river, in Lorena - SP. In this basin there are military and abrasives industries, eucalyptus plantations and pasture areas. To evaluate potential impacts of the use and occupation of the area, this study assessed the ecotoxicological and limnological conditions of the stream between September 2007 and July 2008. Seven sampling points were estabilished along the river from the source to the mouth. The acute and chronic toxicity were evaluated both in the water and sediment, using the cladocerans Daphnia similis and Ceriodaphnia silvestrii, algae Pseudokirchneriella subcapitata for the water and C. silvestrii, D. similis and chironomids Chironomus xanthus for the sediment. The limnological parameters analyzed in water were: turbidity, conductivity, dissolved oxygen, pH, COD, BOD, nitrate, nitrite, total phosphorus, total dissolved solids, hardness, metals (Ag, Cd, Cr, Cu, Fe, Mn, Mg, Ni , Pb and Zn) and fecal coliform; and the physical-chemical parameters of sediment were: total phosphorus, organic matter, granulometry, metals and bioavailable fractions. The in situ bioassays were conducted using PVC chambers at previously selected points, based on the results of ecotoxicity. The results showed that seasonality influenced both the dynamics of the environment and the analyzed variables. The spatial variation indicated influence of human activities along the stream. In some months, the analyzed parameters of water were outside the parameters established by CONAMA Resolution 357/05 for Class I and II. Diffuse pollution sources made it difficult to identify the potential toxicity-causing substances. In some points along the river, the values of metals in sediment were outside the limits recommended by the Canadian Council of Ministers of the Environment. According to the index for the maintenance of aquatic life (VAT), the river showed regular water quality due to the results of acute and chronic toxicity observed in some points.