Optimal Formation of Genetically Modified and Functional Pancreatic Islet Spheroids by Using Hanging-Drop Strategy
- Authors
- Kim, Ha Jun; Alam, Zahid; Hwang, Jin Wook; Hwang, Yong Hwa; Kim, Mu-joong; Yoon, Sangtae; Byun, Youngro; Lee, Du-Yeong
- Issue Date
- Mar-2013
- Publisher
- ELSEVIER SCIENCE INC
- Citation
- TRANSPLANTATION PROCEEDINGS, v.45, no.2, pp.605 - 610
- Indexed
- SCIE
SCOPUS
- Journal Title
- TRANSPLANTATION PROCEEDINGS
- Volume
- 45
- Number
- 2
- Start Page
- 605
- End Page
- 610
- URI
- https://scholarworks.bwise.kr/hanyang/handle/2021.sw.hanyang/163267
- DOI
- 10.1016/j.transproceed.2012.11.014
- ISSN
- 0041-1345
- Abstract
- Background. Rejection 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.
Methods. To 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 mu L drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO2 at 37 degrees C for 7 days. The aggregated spheroids in the droplets were harvested for further culture.
Results. The size of the aggregated islet spheroids depended on the number of single cells (125-500 cells/30 mu L 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.
Conclusion. These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.
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