The islets of Langerhans are multicellular aggregates within the pancreas containing beta cells that sense changes in blood glucose level and respond by secreting insulin. The cells within the islets communicate with each other through gap junctions. The 3D structure of the islets is crucial for their metabolic function. Artificial reconstruction of isolated beta cells into 3D cell aggregates has led to the generation of Pseudoislets (PIs) which mimic islets of Langerhans and can produce insulin. However, the lifespan of PIs is generally a few days in culture after which, central cell death occurs. The overall aim of this project was to improve PI viability and biofunction through development of two new techniques. The first technique being developed was a series of coating solutions consisting of Pluronic F127 (F127) and gelatin type A (Gelatin), which systematically altered the substrate properties of cell culture plates where PIs were formed by suspension culture. The second technique was generation of gelatin beads, functioning as microchannels inside of PIs, and drug carriers when loading with anti-inflammation agents to reduce inflammation and central necrosis. The beta cell line, BRIN-BD11, was used to form PIs. Coating solutions with different ratios of gelatin and F127 (90%, 95%, 98% and 100% of Gelatin in the mixture) have been designed. Surfaces coated with these coating solutions generated different ATR-FTIR spectra, contact angle (CA) values and protein adsorption capacities and the changes were dependent on the original substrate chemistry. The coating of suspension culture plates with gelatin alone failed to generate suspended PIs, while coating with F127 alone resulted in the generation of large, irregular PIs. On the other hand, coating suspension culture plates with a combination of gelatin and F127 solutions formed suspended PIs of differing sizes. The lower the percentage of gelatin in the mixture for the coating, the higher the size of PIs. The higher the F127 content, the more hydrophilic the surface was, which led to lower CA values of the substrate. Coating solutions containing 95-98% Gelatin produced homogenous and smaller PIs than on F127 coating. PIs formed on 98% Gelatin coated plates had larger size but more homogenous morphology (268 ± 10 μm) than PIs produced on ULA plate (235 ± 30 μm). These larger PIs exhibited comparable cell viability, the same level of insulin expression in GSIS and Western blotting assays and higher gene expression of key beta cell markers than smaller PIs formed on ULA plates. Furthermore the larger PIs expressed more of the gap junction protein, connexin 36, in comparison to PIs generated on ULA plates, revealing the optimised configuration of cells in these PIs. Altogether, regulating the hydrophicility of suspension culture plates by F127 and gelatin ratio in the coating solution changed the physical properties of PI and altered the expression of key beta cell markers. Gelatin beads (GBs) of 30-40 μm sizes were produced by Water/Oil emulsion techniques. The degree of swelling and stability of GBs was controlled by crosslinking with 5% glutaraldehyde (GA) vapour for 6 hours. The incorporation of 40 μm GBs into PIs (25-50 GB/per PI) significantly increased the proliferation and cellular viability of the PIs for both large and small sized PIs. Loading of anti-inflammatory cytokines, IL-10 and anti-IL-1β into GBs allowed for sustained release of these agents over time. Incorporation of IL-10 and anti-IL-1β loaded GBs to PIs revealed a synergetic effect in the improvement of the proliferation/viability of PIs regardless of PIs size (large or small). LDH release from the PIs was decreased dramatically and insulin release was increased significantly. Thus GBs played active roles as microchannels and drug carriers which improved the viability and function of PIs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:745339 |
| Date | January 2018 |
| Creators | Elttayef, Ajile Abdulhusein Jassim |
| Publisher | Keele University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.keele.ac.uk/5000/ |
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