کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
4256148 | 1284511 | 2013 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Optimal Formation of Genetically Modified and Functional Pancreatic Islet Spheroids by Using Hanging-Drop Strategy Optimal Formation of Genetically Modified and Functional Pancreatic Islet Spheroids by Using Hanging-Drop Strategy](/preview/png/4256148.png)
BackgroundRejection and hypoxia are important factors causing islet loss at an early stage after pancreatic islet transplantation. Recently, islets have been dissociated into single cells for reaggregation into so-called islet spheroids. Herein, we used a hanging-drop strategy to form islet spheroids to achieve functional equivalence to intact islets.MethodsTo obtain single islet cells, we dissociated islets with trypsin-EDTA digestion for 10 minutes. To obtain spheroids, we dropped various numbers of single cells (125, 250, or 500 cells/30 μL drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO2 at 37°C for 7 days. The aggregated spheroids in the droplets were harvested for further culture.ResultsThe size of the aggregated islet spheroids depended on the number of single cells (125–500 cells/30 μL droplet). Their morphology was similar to that of intact islets without any cellular damage. When treated with various concentrations of glucose to evaluate responsiveness, their glucose-mediated stimulation index value was similar to that of intact islets, an observation that was attributed to strong cell-to-cell interactions in islet spheroids. However, islet spheroids aggregated in general culture dishes showed abnormal glucose responsiveness owing to weak cell-to-cell interactions. Cell-to-cell interactions in islet spheroids were confirmed with an anti–connexin-36 monoclonal antibody. Finally, nonviral poly(ethylene imine)–mediated interleukin-10 cytokine gene delivered beforehand into dissociated single cells before formation of islet spheroids increased the gene transfection efficacy and interleukin-10 secretion from islet spheroids >4-fold compared with intact islets.ConclusionThese results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.
Journal: Transplantation Proceedings - Volume 45, Issue 2, March 2013, Pages 605–610