Abstract
β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.
Highlights
The islets of Langerhans are three-dimensional (3-D) structures which contain insulin-producing b-cells
The formation of 3-D b cell aggregates, or pseudoislets (PIs), is useful for the study of b cell biology. b-cells in PIs show improved function as measured by increased insulin production and improved glucose-stimulated insulin secretion (GSIS) [4,5,6,7,8,9,10]. These effects are mediated in part by the formation of a 3-D configuration which enhances b-cell – cell contact [5,9], increases calcium signaling [11], and preserves extracellular matrix (ECM) proteins [12]
To examine the effects of islet-derived endothelial cells (iECs) on b-cells, we mixed MS1 iECs (6*105) together with bTC3 insulinoma cells (2*105). bTC3 cultured in the presence of MS1 cells formed cell clusters as soon as 24 h, which spontaneously detached by 72 h forming free-floating PIs (Figure 1A)
Summary
The islets of Langerhans are three-dimensional (3-D) structures which contain insulin-producing b-cells. B-cells in PIs show improved function as measured by increased insulin production and improved glucose-stimulated insulin secretion (GSIS) [4,5,6,7,8,9,10]. These effects are mediated in part by the formation of a 3-D configuration which enhances b-cell – cell contact [5,9], increases calcium signaling [11], and preserves extracellular matrix (ECM) proteins [12]. Current methods for induction of PIs include the use of mechanical manipulations such as stirred cell suspension cultures [8], culturing of b-cells on gelatin coated plates [4], and hanging drop cell cultures [13]
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