Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Modulation of inflammatory mediators by Opuntia ficus-indica and Prunus avium bioproducts using an in vitro cell-based model of intestinal inflammation

Dissertation to obtain a Master Degree in Biotechnology

Bioactive beads for local sensing of proteases in 3D engineered tissues

Dissertação para obtenção do Grau de Doutor em Bioengenharia (MIT)

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

In vitro studies of gum arabic-coated magnetic nanoparticles with mammalian cell cultures

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Evaluation of Opuntia spp. bioactive products as promising natural chemotherapeutical agents- an in vitro approach

Dissertation to obtain a Master Degree in Biotechnology

Insights into human carboxylesterase 2 stability and activity in vivo and in vitro

Dissertation to obtain a Master Degree in Biotechnology

Caracterização in vitro de siRNAs ionicamente modificados

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Avaliação do efeito da simulação in vitro da digestão gastrointestinal nas propriedades antioxidantes das infusões de cidreira e tília

Dissertação para obtenção do Grau de Mestre em Tecnologia e Segurança Alimentar

Avaliação do efeito da digestão in vitro na capacidade antioxidante de infusões medicinais: flor de camomila e flor de laranjeira

Dissertação para obtenção do Grau de Mestre em Tecnologia e Segurança Alimentar

Dispositivos médicos para diagnóstico in vitro : impacto operacional, clínico e económico da sua utilização em instituições de saúde

RESUMO - Introdução: Os dispositivos médicos para diagnóstico in vitro (DMDIV) são testes de diagnóstico rápido que podem ser realizados em diversos contextos, seja na enfermaria, no departamento de urgência, no bloco operatório, no consultório médico, centros de enfermagem, farmácias, lares de terceira idade ou até na própria residência, uma vez que a sua utilização não requer formação especializada em técnicas de laboratório. Os DMDIV têm inúmeras finalidades: triagem de utentes, diagnóstico de situações agudas, monitorização de fármacos ou acompanhamento de doenças crónicas. Objectivos: conhecer as potenciais implicações operacionais, clínicas e económicas da implementação generalizada de DMDIV em instituições de saúde da região de Lisboa e Vale do Tejo, bem como conhecer o nível de utilização e opinião sobre DMDIV de médicos e enfermeiros da região de saúde de Lisboa e Vale do Tejo. Metodologia: foi realizado um estudo observacional, analítico e transversal. Como instrumento de recolha de dados, foi aplicado um questionário a uma amostra de conveniência constituída por médicos e enfermeiros a exercer funções em instituições hospitalares públicas e privadas e em instituições de cuidados de saúde primários da região de saúde de Lisboa e Vale do Tejo. Conclusão: a utilização de DMDIV permite diminuir o tempo de resposta do resultado analítico, o que se traduz numa maior rapidez do diagnóstico e numa intervenção clínica mais célere, com impacto ao nível da redução do número de admissões desnecessárias, do tempo de internamento e do número de consultas médicas, com a consequente redução do consumo de recursos hospitalares. Na região de saúde de Lisboa e Vale do Tejo, um número significativo de instituições de saúde disponibiliza este tipo de equipamentos portáteis que, de uma forma geral, têm uma boa aceitação por parte dos profissionais de saúde.

Evaluation of pathogenic potential of Aeromonas spp. strains using in vitro methodologies

Dissertation to obtain a Master Degree in Molecular Genetics and Biomedicine

Avaliação in vitro e in vivo da atividade antimalárica dos trioxolanos NAC89, LDC67 e LC50 em estirpes sensíveis e resistentes à artemisinina e ao artesunato.

A malária mantem-se como uma das doenças mais importantes do mundo, causando a morte de mais de 1 milhão de pessoas anualmente e elevada morbilidade. Face à propagação da resistência do Plasmodium falciparum à maioria dos medicamentos antimaláricos disponíveis, a Organização Mundial de Saúde (OMS), desde 2006, recomenda a utilização de terapias combinadas com artemisinina (ACTs) como tratamento de primeira linha para a malária não complicada. Em 2008, relatórios clínicos revelaram a falha terapêutica dos ACTs na fronteira Tailândia-Camboja e uma vez que não existem alternativas para o tratamento da malária é fundamental manter linhas de investigação sobre novos e eficazes fármacos. A partir da artemisinina (ART) surgiram novos peróxidos designados trioxolanos que apresentam como farmacóforo a função 1,2,4-trioxano. A acessibilidade, a preparação relativamente económica e a estabilidade da função 1,2,4-trioxano permite a síntese de derivados com estruturas diversas, alargando a possibilidade de desenvolvimento de novos fármacos. Foram realizados testes in vitro de triagem com o MARK III (OMS micro-ensaio), com controlos positivos (artemisinina e dihidroartemisinina) e controlo negativo (sem fármaco). Foram efetuados ensaios diversos com 3 compostos, aqui denominados NAC89, LCD67 e LC50 em culturas da estirpe de P. falciparum (Dd2) para avaliação da atividade antimalárica dos compostos, bem como ensaios utilizando o modelo de malária de murino, Plasmodium chabaudi, com 4 estirpes, denominadas AS-3CQ, AS-ATN, respectivamente sensível e resistente ao artesunato (ATN) e AS-30CQ e AS-ART respectivamente sensível e resistente à (ART). Também foi avaliada a citotoxicidade dos compostos, utilizando células HepG2 de hepatoma humano pelo ensaio com método colorimétrico metil-tiazol-tetrazólico (MTT). No modelo murino compararam-se também duas vias de administração dos novos compostos, sendo uma por via subcutânea nos 3 compostos e outra por via tópica apenas para LC50. A verificação de cura foi efetuada por observação microscópica de esfregaços sanguíneos corados pelo método de Giemsa e determinação da parasitemia. Os resultados observados foram: a) baixa citotoxicidade dos três compostos; b) o composto LC50 eliminou a parasitémia nos ensaios in vitro em cultivos de P. falciparum bem como eliminou P. chabaudi nos tratamentos por via subcutânea e tópica na dose de 50 mg/kg e na dose de 10 mg/Kg na via subcutânea; c) o NAC89 mostrou boa atividade no mesmo ensaio in vivo, na dose de 10 mg/Kg e 50 mg/Kg por via subcutânea; d) fraca atividade para LCD67 na dose de 50 mg/Kg. O LC50 e o NAC89 foram muito eficazes contra parasitas resistentes ao ATN e à ART sugerindo novos mecanismos de ação. Assim, este trabalho de investigação trouxe resultados promissores na àrea de potenciais novos antimaláricos.

Analysis of the interaction of polycyclic aromatic compounds in a model organism: integration of genotoxic and histopathological effects

Due to their toxicity, especially their carcinogenic potential, polycyclic aromatic hydrocarbons (PAHs) became priority pollutants in biomonitoring programmes and environmental policy, such as the European Water Framework Directive. The model substances tested in this study, namely benzo[b]fluoranthene (B[b]F), considered potentially carcinogenic to humans and an effector carcinogenic PAH to wildlife, and phenanthrene (Phe), deemed a non-carcinogenic PAH, are common PAHs in coastal waters, owning distinct properties reflected in different, albeit overlapping, mechanisms of toxicity. Still, as for similar PAHs, their interaction effects remain largely unknown. In order to study the genotoxic effects of caused by the interaction of carcinogenic and non-carcinogenic PAHs, and their relation to histopathological alterations, juvenile sea basses, Dicentrarchus labrax, a highly ecologically- and economically-relevant marine fish, were injected with different doses (5 and 10 μg.g-1 fish ww) of the two PAHs, isolated or in mixture, and incubated for 48 h. Individuals injected with B[b]F and the PAH mixture exhibited higher clastogenic/aneugenic effects and DNA strand breakage in blood cells, determined through the erythrocytic nuclear abnormalities (ENA) and Comet assays, respectively. Also, hepatic histopathological alterations were found in all animals, especially those injected with B[b]F and the PAH mixture, relating especially to inflammation. Still, Phe also exhibited genotoxic effects in sea bass, especially in higher doses, revealing a very significant acute effect that was accordant with the Microtox test performed undergone in parallel. Overall, sea bass was sensitive to B[b]F (a higher molecular weight PAH), likely due to efficient bioactivation of the pollutant (yielding genotoxic metabolites and reactive oxygen species), when compared to Phe, the latter revealing a more significant acute effect. The results indicate no significant additive effect between the substances, under the current experimental conditions. The present study highlights the importance of understanding PAH interactions in aquatic organisms, since they are usually present in the aquatic environment in complex mixtures.