| Diabetes mellitus currently affects about 350 million people worldwide, and the World Health Organization (WHO) predicts that death due to the complications of diabetes will likely increase by more than 50%over the next 10 years. Type I diabetes mellitus, which represents approximately 10%of all diabetes cases, requires daily insulin injections. This treatment, however, is demanding for the patient, can be painful, and does not eliminate the risk of diabetic complications. Islet transplantation is a promising treatment for type I diabetes mellitus that achieves an insulin-independent, constant normoglycemic state and avoids diabetic complications. While islets are biologically active substances, so a successful transplantation depends on optimizing islet viability in vitro, in culture after isolation from the pancreas, and during transportation. So the aim of this paper was to establish a new islet purification process to improve the purity of islets for implantation, and Alginate gels (ALGs) encapsulation technology was used to improve the islet purity. The main work of this dissertation is summarized as follows:Firstly, a new islet purification process comprising a hypertonic-hypotonic treatment step to improve the purity of islets was established. Glucose transporter-2 (Glut-2) locates on islet cell membrane and exocrine cells without Glut-2 transporter. This transporter plays a key role in the extracellular glucose-sensing mechanism. And the Plackett-Burman method was used to determine which factors had a significant influence on the purity of islets obtained after the hypertonic-hypotonic treatment step. The hypertonic solution concentration and the incubation time were both found to have a significant effect on islet purity. The purity of islets obtained using the modified purification process was significantly higher than that of islets obtained by density gradient alone. Importantly, good cell viability and normal insulin secretion ability of islets were maintained following the modified purification. The new purification process allows isolation of islets with improved purity and does not compromise the viability or function of the islets.Secondly, a method was developed that uses alginate encapsulation to protect islets from mechanical damage during shipment. We tested several variables for their impact on islet viability during transportation and used the significant variable to build a response surface methodology (RSM) model by Box-Behnken design method. This model can be used to predict islet survival rate and can serve as a guide for optimizing the transportation method of islets and increasing the success rate of the transplant procedure.Finally, in this paper, a new engineered islet was established. Some researchers found that islet single cells can reaggregate islet cell clusters, so ALGs was used to encapsulate islet cells to form artificial islets after dispersion islets into single cells. The shape of the islet cell clusters was similar to native islets, and the size of the islet cell clusters was limited to a maximum diameter of 100 μm. By limiting the diameter of this engineered islet cell clusters, cell viability was nearly able to 100%significantly improvement over native islets. Importantly, islet cell clusters were express the genes of islets, including Isl-1, GCG and INS-1, and insulin secretion-ability was maintained in vitro.In this study, the hypertonic-hypotonic treatment was effectively improved the purity of islets; ALG bead was an efficient microenvironment culture system, which might provide a useful platform for protecting islets from mechanical damage during shipment and establishing a new engineering islet to maintain high viability of islets, and promote the development of islets transplantation. |
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