Viral supernatants (20 μl) were added to 200 000 MSC cultured in 25 cm2 flasks in MSC medium supplemented with 8 μg/ml Polybrene (Sigma-Aldrich Inc, Saint Louis, MO, USA). Cells were incubated for 24 h at 37 °C, 5 % CO2 and the media was replaced the following day. Transduction efficiency was analyzed for GFP expression using FACScanto II (BD Biosciences, San Diego, CA).
Human islets of Langerhans were manually picked and used one to three days post islet isolation. Islets were incubated with blue cell tracker (1-3 uM Cell Tracker, Molecular Probes, Eugene, OR) in 5cm Sterilin dishes (Sterilin Ltd, New Port, UK) in islet culture medium (see above), 1 h, 37 °C, followed by change of medium and 1h incubation in 37 °C. For creation of composite MSC-islets, we followed earlier established protocols for coating of the islets [13 , 37 (link)]. In short, approximately 200 islets were added to 5 mL polystyrene tubes (Sarstedt, Numbrecht, Germany) together with 185 000 GFP/luciferase-transduced MSC followed by careful mixing every 30 min during 2 h at room temperature (RT). Islets and composite MSC-islets were thereafter cultured in 5 cm Sterilin dishes with MSC complete medium (see above), 37 °C, overnight.
Human islets of Langerhans were manually picked and used one to three days post islet isolation. Islets were incubated with blue cell tracker (1-3 uM Cell Tracker, Molecular Probes, Eugene, OR) in 5cm Sterilin dishes (Sterilin Ltd, New Port, UK) in islet culture medium (see above), 1 h, 37 °C, followed by change of medium and 1h incubation in 37 °C. For creation of composite MSC-islets, we followed earlier established protocols for coating of the islets [13 , 37 (link)]. In short, approximately 200 islets were added to 5 mL polystyrene tubes (Sarstedt, Numbrecht, Germany) together with 185 000 GFP/luciferase-transduced MSC followed by careful mixing every 30 min during 2 h at room temperature (RT). Islets and composite MSC-islets were thereafter cultured in 5 cm Sterilin dishes with MSC complete medium (see above), 37 °C, overnight.
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