Presentation Title

Establishing a Long-Term Cell Culture System for Research and Transplant in Diabetes

Faculty Mentor

Keiko Omori, Yoko Mullen

Start Date

18-11-2017 11:00 AM

End Date

18-11-2017 11:15 AM

Location

9-273

Session

Bio Sciences 3

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Diabetes is a set of metabolic disorders characterized by the inability to maintain constant levels of glucose in the blood. Islet transplantation is a treatment option for selected patients with Type 1 diabetes which involves transplanting isolated pancreatic islets containing β-cells from brain-dead donors. In the current procedure, isolated islets can only be cultured for few days without losing function and viability. Establishment of a long-term islet culture system would allow us to evaluate the long-term effect of drug on human islet function and viability in vitro towards development of new treatments. We hypothesized that encapsulation of human islets in polysaccharide 3D hydrogel would improve the function and viability compared to islets cultured without 3D hydrogel. We also hypothesized that addition of a cytokine blocker, Etanercept, to 3D hydrogel would prevent cytokine-mediated islet death and dysfunction. Isolated human islets for research use were cultured with or without hydrogel for up to 4 weeks. Islet function was assessed by glucose stimulated insulin secretion in a static incubation assay followed by insulin ELISA. Islet viability was assessed by propidium iodide staining at 2 and 4 weeks in culture. The results showed 3D hydrogel prevented islet aggregation, maintained islet mass and viability throughout 4 weeks of culture, but did not preserve function after 2 weeks. Addition of Etanercept to 3D hydrogel increased both insulin release and viability despite addition of cytokines. Our findings suggest the potential use of 3D hydrogel for long-term islet culture; however, optimization is necessary to maintain islet function.

Summary of research results to be presented

Long-term cell culture of pancreatic islets has previously faced problems such as central cell necrosis, cell aggregation, loss of micro-structure, and degradation of function as incubation time increases due to lack of vasculature and 3D support. Previous research has indicated that hydrogel conditions mimicking the environment of the pancreas increases viability and function of islets throughout cell culture. β-cell interaction with extracellular matrix-derived peptides, such as Arginylglycylaspartic acid (RGD: Arg-Gly-Asp), has been shown to preserve viability and reduce apoptosis. Antioxidant and oxygen treatment have been shown to mitigate the effects of hypoxia in cell culture. This project utilized a combination of these various treatments to establish a better cell culture system. The results of this study revealed that polysaccharide 3D hydrogel maintained viability of islets throughout 4 weeks of culture, but was not able to preserve function after 2 weeks. Hydrogel was able to maintain islet mass and prevent cell aggregation. A large variation between data sets was seen in the measurement of insulin release, most likely due to donor to donor islet characteristic difference, but viability was consistent between the sets. No improvement was observed by the addition of RGD to 3D hydrogel culture. Addition of Etanercept, a TNF-α blocker, to 3D hydrogel showed increased average insulin release, stimulation index, and viability at 2 weeks of culture despite addition of cytokines indicating the effectiveness of functionalized 3D hydrogel with TNF-α blocker preventing cytokine-mediated islet death over two weeks.

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Nov 18th, 11:00 AM Nov 18th, 11:15 AM

Establishing a Long-Term Cell Culture System for Research and Transplant in Diabetes

9-273

Diabetes is a set of metabolic disorders characterized by the inability to maintain constant levels of glucose in the blood. Islet transplantation is a treatment option for selected patients with Type 1 diabetes which involves transplanting isolated pancreatic islets containing β-cells from brain-dead donors. In the current procedure, isolated islets can only be cultured for few days without losing function and viability. Establishment of a long-term islet culture system would allow us to evaluate the long-term effect of drug on human islet function and viability in vitro towards development of new treatments. We hypothesized that encapsulation of human islets in polysaccharide 3D hydrogel would improve the function and viability compared to islets cultured without 3D hydrogel. We also hypothesized that addition of a cytokine blocker, Etanercept, to 3D hydrogel would prevent cytokine-mediated islet death and dysfunction. Isolated human islets for research use were cultured with or without hydrogel for up to 4 weeks. Islet function was assessed by glucose stimulated insulin secretion in a static incubation assay followed by insulin ELISA. Islet viability was assessed by propidium iodide staining at 2 and 4 weeks in culture. The results showed 3D hydrogel prevented islet aggregation, maintained islet mass and viability throughout 4 weeks of culture, but did not preserve function after 2 weeks. Addition of Etanercept to 3D hydrogel increased both insulin release and viability despite addition of cytokines. Our findings suggest the potential use of 3D hydrogel for long-term islet culture; however, optimization is necessary to maintain islet function.