Breakthroughs in health care over the coming decades may well consist of cell based therapeutics, potentially allowing the repair and replacement of damaged tissues and organs. Recently developed induced pluripotency methods as well as advances in stem cell biology show hope of such a reality. But such prospects come with the need to have a robust understanding and control over cell development and differentiation processes. Cells in the body develop in tightly controlled and specialized micro-environments that provide many complex cues and interactions that guide cell fate by directing gene expression. But current cell culture techniques that are most widely used to study the cell outside the body have not significantly changed from when first started over a century ago, using simple containers and flasks with plastic surfaces. Many studies have shown that three-dimensions (3D) over conventional 2D cell culture alone, showed to have increased levels of cell organization, morphology and function. By combining novel biomaterials and microfabrication methods, this project aims to create a tool that allows the study of cells within a simplified 3D environment. With the potential to build up the physical and soluble complexity of this environment in a tailor made fashion, a "dissection" of the functions and influences of external factors may be understood. Such a tool can also be used for the development of cell based models, highly relevant for drug discovery application as well as potentially for the development of cell based therapeutics. We have developed a novel microfluidic 3D cell culture platform that allows controlled soluble factor perfusion in an enzymatically formed and cell responsive PEG-based hydrogel. Soft lithographic processes were used to mold microchannels on a layer of hydrogel. In a second step, a flat hydrogel layer is placed on top to enclose and form the microchannels . The system allows the control over soluble factor delivery and it is able to perform long-term cell culture and generate user controlled gradients of soluble factors. With continuous perfusion, metabolic waste is constantly removed and cells are supplied with fresh medium. The platform also can allow creation of tailor made environments building up complexity in a controlled fashion. Higher throughput is also achieved by arraying individual culture chambers. The device was applied towards the culture of mouse embryonic stem cells (mESC) in order to demonstrate their self-renewal capacity on the microfluidic platform. The effects of some cytokine factors were also studied. Meaningful readouts such as fluorescence are easily used for analysis and cells can possibly be extracted from the device for further downstream analysis and processing. Through this device we show that microfluidics technologies combined with biomaterials enables the creation of platforms that are biologically relevant for the study and understanding of cell processes from cancer to stem cell development, processes which are fundamentally regulated by interactions with the microenvironment. The platform developed may also be used to develop human tissue models perhaps more relevant than current animal based testing, providing new tools for drug development and also potentially new approaches for developing cell based therapeutics