Few studies report whole pancreatic tissue culture, as it is a difficult task using traditional culture methods. Here, a factorial design was used to investigate the singular and combinational effects of flow, dissolved oxygen concentration (D.O.) and pulsation on whole mechanically disrupted rat pancreata in a perfusion bioreactor. Whole rat pancreata were cultured for 72 h under defined bioreactor process conditions. Secreted insulin was measured and histological (haematoxylin and eosin (H&E)) as well as immunofluorescent insulin staining were performed and quantified. The combination of flow and D.O. had the most significant effect on secreted insulin at 5 h and 24 h. The D.O. had the biggest effect on tissue histological quality, and pulsation had the biggest effect on the number of insulin-positive structures. Based on the factorial design analysis, bioreactor conditions using high flow, low D.O., and pulsation were selected to further study glucose-stimulated insulin secretion. Here, mechanically disrupted rat pancreata were cultured for 24 h under these bioreactor conditions and were then challenged with high glucose concentration for 6 h and high glucose + IBMX (an insulin secretagogue) for a further 6 h. These cultures secreted insulin in response to high glucose concentration in the first 6 h, however stimulated-insulin secretion was markedly weaker in response to high glucose concentration + IBMX thereafter. After this bioreactor culture period, higher tissue metabolic activity was found compared to that of non-bioreacted static controls. More insulin- and glucagon-positive structures, and extensive intact endothelial structures were observed compared to non-bioreacted static cultures. H&E staining revealed more intact tissue compared to static cultures. (C) 2017 American Institute of Chemical Engineers