Process engineering of liver cells for drug testing applications

• Autor(es): Tostões, Rui Manuel Lucas Gameiro Domingues
• Data: 2012
• Identificador Persistente: http://hdl.handle.net/10362/7612
• Origem: Repositório Institucional da UNL
• Assunto(s): CYP450; Hepatocytes; 3D; Long term
• Tipo de Documento: Tese de doutoramento
• Idioma: Inglês
• Orientador(es): Alves, Paula
• Contribuidor(es): RUN

Descrição

Dissertação para obtenção do Grau de Doutor em Bioengenharia

The primary culture of human hepatocytes is a requirement in drug development tests. This application is currently hampered by two problems: the limited proliferation of the hepatocytes and the rapid loss of liver-specific phenotype of these cells, when cultured in vitro. This thesis aimed at minimizing this latter issue by cultivating hepatocytes, as spheroids, in fully controlled bioreactors. The state of the art of the primary cultures of hepatocytes is reviewed in Chapter 1, after a brief introduction to the liver physiology the drug development process. The improvement of the bioreactor cultures of hepatocyte spheroids was initially done using freshly isolated rat hepatocytes; the effects of alginate microencapsulation, perfusion culture and their synergy on the maintenance of the hepatocyte spheroids liver-specific phenotype were assessed in Chapters 2 and 3; it was concluded that the perfusion culture and alginateencapsulation had a positive synergic effect on such hepatic phenotype. The perfusion bioreactor developed in Chapter 3 was used in Chapter 4 for the extended culture of freshly isolated human hepatocytes, as spheroids, from three different donors. These cultures responded to repeated dose drug treatments as expected from mature and differentiated hepatocytes, in up to 4 weeks culture time. In Chapter 5, human embryonic stem cell-derived hepatic progenitors were cultured as spheroids and further differentiated into hepatocyte-like cells; the differential expression of hepatic genes between this spheroid population and a monolayer differentiated hepatocyte-like cell population showed a more efficient differentiation under spheroid culture. The bioengineering improvements of this thesis, as well as the future work, were discussed in Chapter 6. This thesis has led to the establishment and validation of primary cultures of hepatocyte spheroids, in perfusion bioreactors, which can be used for long-term, repeated dose tests in drug development.