Insulin Secretion from Clonal MIN6 Sublines Depends on the Dimensionality of Culturing
Date:
Authors
Matthew K. Daddysman, Martha H. Renn, Alan L. Hutchison, Toan Huynh, Natalia Tamarina, Louis H. Philipson, Aaron R. Dinner, Norbert F. Scherer
Abstract
The mouse insulinoma-6 (MIN6) cell line is widely used in diabetes research. However, we found that MIN6 cells have heterogeneous responses to glucose stimulation. To address this problem, We used flow cytometry to isolate individual cells from which we established genetically pure clonal sublines of MIN6. We screened these clonal cell lines for their responsiveness to glucose stimulation monitoring insulin secretion and calcium ion uptake. Our measurements of insulin secretion indicate that we have produced a 3-fold more responsive subline (over basal conditions), which also has intense calcium oscillations following glucose stimulation. Others of our clonal sublines are nonsignificantly responsive for insulin secretion or calcium oscillations. Most importantly, unique behaviors are only present when the cells are cultured in 3D islet-like structures (Figure). RNA-seq analysis on these sublines reveals epigenetic expression differences in over 4,000 genes between the sublines at a 99% confidence interval. The number of differentially expressed genes increases to over 6,000 when 2D vs. 3D growth is considered. These epigenetic differences are present in genes throughout the glucose stimulated insulin secretion pathways. Our results demonstrate that the widely used MIN6 cell line is heterogeneous and should be used with circumspection.