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 journalArticle

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

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Islets of Langerhans
Cell Communication
Glucose
Interleukin-10
Cell Count
Islets of Langerhans Transplantation
Edetic Acid
Trypsin
Genes
Transfection
Digestion
Air
Monoclonal Antibodies
Observation
Cytokines

ASJC Scopus subject areas

  • Surgery
  • Transplantation

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Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy. / Kim, H. J.; Alam, Z.; Hwang, J. W.; Hwang, Y. H.; Kim, M. J.; Yoon, S.; Byun, Y.; Lee, D. Y.

In: Transplantation Proceedings, Vol. 45, No. 2, 03.2013, p. 605-610.

Research output: Contribution to journalArticle

Kim, H. J. ; Alam, Z. ; Hwang, J. W. ; Hwang, Y. H. ; Kim, M. J. ; Yoon, S. ; Byun, Y. ; Lee, D. Y. / Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy. In: Transplantation Proceedings. 2013 ; Vol. 45, No. 2. pp. 605-610.
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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.",
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AB - 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.

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