title: Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy.
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% CO(2) 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.
  
  
  CONCLUSIONS
  These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.
authors.affiliation: Department of Bioengineering, College of Engineering, and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Republic of Korea.
authors.forename: H J
authors.initials: HJ
authors.lastname: Kim
authors.forename: Z
authors.initials: Z
authors.lastname: Alam
authors.forename: J W
authors.initials: JW
authors.lastname: Hwang
authors.forename: Y H
authors.initials: YH
authors.lastname: Hwang
authors.forename: M J
authors.initials: MJ
authors.lastname: Kim
authors.forename: S
authors.initials: S
authors.lastname: Yoon
authors.forename: Y
authors.initials: Y
authors.lastname: Byun
authors.forename: D Y
authors.initials: DY
authors.lastname: Lee
mesh_terms: D000818:Animals; D002449:Cell Aggregation; D002450:Cell Communication; D018929:Cell Culture Techniques; D048430:Cell Shape; D048429:Cell Size; D017630:Connexins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D018874:Spheroids, Cellular; D013997:Time Factors; D014162:Transfection; D000097004:Gap Junction delta-2 Protein
chemical_list: D017630:Connexins; D005947:Glucose; D016753:Interleukin-10; D000097004:Gap Junction delta-2 Protein; C556062:IL10 protein, mouse