Restoration of β-cell mass by inducing proliferation of residual β-cells represents an attractive therapeutic approach for diabetes treatment. Native and dispersed primary human islets undergo rapid loss of viability and function ex vivo, complicating their use in proliferation studies. Here, we establish a novel method for evaluating compound effects on β-cell proliferation and count using reaggregated primary human islet microtissues (MTs) , of homogeneous size and architecture and that maintain stable viability and function for 4 weeks in culture. We utilized this platform to evaluate the effects of short- and long-term harmine treatment on β-cell proliferation and function in a dose-dependent manner. Following harmine treatment and EdU incorporation, islet MTs were stained in 3D for DAPI (nuclear marker) , NKX6.1 (β-cell marker) , and EdU (proliferation marker) , imaged on a high-content confocal microscope, and subjected to automated 3D analysis of numbers of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, assessment of insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity was performed for a comprehensive overview of islet MT function and viability. We observed that 4 days of harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, whereas fold-stimulation of secretion peaked at intermediate doses. Interestingly, 15-day harmine treatment led to a general reduction in proliferative effects as well as altered dose-dependent trends. The methodology presented here thus constitutes a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction in parallel with a variety of functional parameters, in a representative and standardized 3D human islet microtissue model.
A.C.Title: None. B.Yesildag: None. M.Karsai: None. J.Mir-coll: None. O.Yavas grining: None. C.Rufer: None. S.Sonntag: None. F.Forschler: None. S.Jawurek: None. T.Klein: Employee; Boehringer Ingelheim International GmbH.