Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy

H. J. Kim, Z. Alam, J. W. Hwang, Y. H. Hwang, M. J. Kim, S. Yoon, Y. Byun, D. Y. Lee

Research output: Contribution to journalArticlepeer-review

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 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. Results: The 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. Conclusion: These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.

Original languageEnglish (US)
Pages (from-to)605-610
Number of pages6
JournalTransplantation Proceedings
Volume45
Issue number2
DOIs
StatePublished - Mar 2013
Externally publishedYes

ASJC Scopus subject areas

  • Surgery
  • Transplantation

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