Abstract/Summary

Standard 2D cell culture does not recreate the complex features of the in vivo environment, such as soluble factor gradients, cell migration into multiple planes, or cell-cell and cell-matrix interactions.1–3 3D cell culture addresses these limitations by using 3D biomaterial scaffolds, such as alginate hydrogels, to recreate the in vivo cell microenvironment in vitro. 4, 5 3D platforms can be used to create gradients of soluble factors, vary the biomaterial substrate stiffness, permit cell-matrix interactions or promote cell migration.6, 7 Currently available 3D platforms are prone to the burst release of soluble factors from the biomaterials, making it difficult to tightly control the soluble factor concentration.7 This limits the use of 3D platforms for investigating processes such as patterned neuronal differentiation, or cell fate specification in response to small changes in soluble factor concentration. This project proposes a novel 3D alginate platform for patterned differentiation. The first part of this thesis describes experiments to optimise alginate hydrogels for the encapsulation, aggregation and differentiation of embryonic stem cells (ESCs), and demonstrates that encapsulated ESCs form embryoid bodies containing cells from the three germ layers. Exogenous retinoic acid (RA) is used for in vitro neuronal differentiation protocols, but exogenous RA is not stable in cell culture and is easily degraded by light. The second part of the thesis outlines experiments to validate a cell-derived source of RA, which produces a stable concentration of RA in vitro and addresses the limitations of exogenous RA. The final section describes the novel 3D platform that combines the results from the previous sections using an adapted gradient maker protocol, to create 3D co-culture alginate tubes. The tubes support patterned differentiation of ESCs in response to the concentration gradient of cell-derived RA incorporated into the platform. The novel 3D platform produced in this project contributes a novel tool to the field of 3D cell culture. The 3D platform is a tool for investigating ESC differentiation in response to a 3D concentration gradient of a cell-derived source of retinoic acid. For experiments that require a gradient of RA, the ability to maintain a stable source of RA over several days is an advantage of using this 3D platform over the currently available alternatives. In addition, alginate hydrogels are highly tunable. Thus, the ability to tune the scaffold properties, change the cell types encapsulated, or introduce gradients of alternative soluble factors makes this a versatile tool for 3D culture.