Pancreatic β-cells are responsible for production of insulin in response to an increase in glucose within the bloodstream. A failure of this process as in type I diabetes has devastating consequences for the body. The β-cells have also proven difficult to culture in vitro, exhibiting slow proliferation and loss of glucose-induced insulin secretion. Peptide based hydrogels have previously been used successfully to support a wide range of cell types. In this study, peptide hydrogels that are formed by the co-assembly of two components, fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) and Fmoc-serine (Fmoc- S) were used. These were previously shown to support culture of bovine chondrocytes and human dermal fibroblasts but have yet to be tested with β-cells. The hydrogels have a tuneable stiffness, a factor demonstrated to be important for cell survival. A range of Fmoc-FF/S hydrogels of varying stiffness values were developed to test compatibility with the β-cells. The hydrogels were assessed in terms of cell viability and ability of the cells to retain their glucose sensitive insulin secretion. First hydrogels of peptide concentrations 6 to 30 mM and stiffness values 0.5 to 23 kPa were generated.MIN6 cells cultured within these hydrogels in a 3D like manner showed the Fmoc-FF/S hydrogel of 25 mM peptide concentration and 14.4 kPa was able to sustain the greatest number of MIN6 cells (5.5x104) and showed the greatest glucose-induced insulin production at 15 ng/ml in low glucose (5 mM) and 24 ng/ml in high glucose (25 mM) conditions. The cells within this hydrogel not only retained their ability to secrete insulin in response to a change in glucose levels (5 to 25 mM) but were able to do so in a time appropriate manner, increasing from base level after 15 mins when glucose was increased from 5 to 25 mM, peaking at 90 mins and then returning to base level values when the glucose concentration was lowered again to 5 mM, similar to the physiological response, where β-cells respond depending on the concentration of glucose. This work shows for the first time that the Fmoc-FF/S system is compatible with the β-cell type and the importance of tailoring matrix stiffness, already established to be an important factor in cell fate, viability and functionality in a range of other cell lines, in order to best preserve the glucose-induced insulin secretion functionality of the MIN6 cells.
|Date of Award||13 May 2016|
- University Of Strathclyde
|Supervisor||Rein Ulijn (Supervisor) & Craig Jamieson (Supervisor)|