The viability, proliferation and insulin production of RIN-m5F cells when loaded into alginate beads to form a 3D culture system has been investigated. The mechanism of alginate cross-linking ( calcium ions vs barium ions), the addition of poly(L-lysine) (PLL) and poly(L-ornithine (PLO) and presence of different extra-cellular matrix proteins (ECM) influence the RIN-m5F cell behaviour. Cells in calcium alginate beads ( CAB) proliferated and produced more insulin per cell than monolayer culture, but the physical properties of the beads were poor and they ruptured within a few days of culture. Barium alginate beads (BABs) provided a stable encapsulation method. Addition of PLL and PLO at concentrations above 0.1% w/v with the culture medium increased cell proliferation. With the addition of ECMs after bead formation there was a further increase in cell proliferation for certain combinations of ECM and PLO. It was concluded that RIN-m5F-loaded Ba-alginate beads ( BABs), when incorporated with varying concentrations of poly (L) lysine ( PLL), poly ( L) ornithine ( PLO) in the presence of extra-cellular matrix proteins ( ECMs) were superior to both tissue culture and RIN-m5F-loaded Ca-alginate beads (CABs) in terms of physical stability, cell proliferation and insulin production.