β-cell dysfunction in pancreatic islets, characterized as either the loss of β-cell mass or the resistance of β-cell to glucose, is the leading cause of progression to diabetes. Islet transplantation became a promising approach to replenish functional β-cell mass. However, not much known about changes in islets used for transplantation after isolation. We have subjected human islets into long-term in vitro culture (LTC) and characterized those survived islets. While most of the dysregulated genes were downregulated during LTC, specific groups of mRNA or miRNA were upregulated, and they are involved in specific pathways. In general, α-cells and β-cells of LTC-islets have elevated expressions of MAFB and MAFA genes, respectively. We also found that exocrine cells were eliminated faster than endocrine cells, and β-cells were lost at a higher rate than α-cells. Interestingly, one specific group of cells that have characteristics of immature α-cells or β-cells, were enriched in LTC-islets, revealing the possibility of transdifferentiation of α-cells to β-cells, or dedifferentiation of β-cells to α -cells, under in vitro culture. Our results suggest that there are intrinsic cellular and molecular mechanisms in pancreatic cells that are associated with their maturity and correlated with their survival ability under unfavorable living conditions.