Establishment of 3D cell cultures under dynamic conditions using a multi-compartment holder for spinner flask

Abstract

In the last years, cell cultures in three-dimensional (3D) scaffolds have gained expression in the field of tissue engineering and regenerative medicine as a more physiological and reliable in vitro approach of the in vivo situation. Cell morphology, proliferation, differentiation and regulation were proven to change dramatically from standard two-dimensional (2D) conditions to 3D environment. However, the standard culture in static conditions is not the most suitable for 3D cell culture, due to lack of nutrients and oxygen diffusion towards the scaffolds center. Therefore, dynamic cell culture should be a more physiological approach. This thesis addresses the potential of a multi-compartment container, designed and patented at INEB, to support cell culture in 3D scaffolds under dynamic conditions. The container was conceived to culture cells in 3D fragile hydrogel-like matrices, overcoming the lack of solutions for this type of matrices in the market. As a model system, human mesenchymal stem cells (hMSCs) cultured in chitosan (Ch) 3D scaffolds was compared in static and dynamic conditions. The effect of the following parameters was addressed: agitation rate, media feeding regime and the period of cell attachment before dynamic culture. Cell cultures were analyzed for: cell viability, cell proliferation, cell metabolic activity and cell distribution in 3D architecture. In addition, cell capacity to undergo osteogenic differentiation was also analyzed. The data collected clearly suggested higher cell proliferation, more homogenous cell distribution and cell migration towards scaffolds center, under dynamic conditions, when compared to static. Cell metabolic analysis did not reveal major differences between culture conditions. Furthermore, hMSCs differentiation into osteogenic lineage appeared to occur earlier when dynamic conditions are used, as suggested by alkaline phosphatase (ALP) activity. Overall, this study highlights some benefits of dynamic culture conditions, particularly using the proposed multi-compartment holder, for 3D culture of hMSCs

Date of Award	2012
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Raquel Madeira Gonçalves (Supervisor) & Cristina Carvalho Barrias (Co-Supervisor)