Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand β-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in β-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants.