The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.