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Objective: Long-term in vitro maintenance of human insulin-secreting insulinoma cells. Methods: (1) Cell culture of ex vivo-derived insulinoma cell suspensions from 8 individual human donors, using various cell culture medium supplementations; (2) determination of insulin synthesis and secretion using immunocytochemistry and insulin and pro-insulin radioimmunoassays; (3) nestin-immunostaining of long-term in vitro grown insulinoma cell suspensions, and (4) phase-contrast light microscopy for analyzing the in vitro growth characteristics of the insulinoma cells. Results: (1) Parallel persistence of in vitro insulinoma cell proliferation as well as insulin-synthesizing and -secreting capacity depended on both the co-culture of insulinoma cells with human fibroblasts and the supplementation of the cell culture medium with tissue culture supernatant derived from the rodent pituitary adenoma cell line GH-3; (2) immunostaining for insulin and secretagogin confirmed the neuroendocrine origin of the insulinoma cells grown in vitro; (3) insulin secretion capability persisted up to an observation period of 25 weeks; (4) insulin secretion rates after 6 weeks of in vitro growth ranged from 3.5 to 83.3 µU/ml/h/60,000 cells plated, and (5) after long-term in vitro growth of insulinoma-derived cell suspensions with persistent insulin-secreting capacity, nestin staining was observed predominantly in co-cultured fibroblasts. Conclusion: Our data describe for the first time the long-term in vitro culture of insulin-secreting human insulinomas and highlight the importance of β-cell trophic factors for insulinoma cell growth.