Background: The intensity of IBMIR (Instant Blood-Mediated Inflammatory Reaction) depends on the amount of tissue factor (TF) molecules on the surface of pancreatic islet cells in time of transplantation to the portal vein. At the same time, TF is an important growth factor stimulating islet graft revascularization. In order to reduce the intensity of IBMIR and improve the early function of the graft, we tested the possibility of the temporary inhibition of TF synthesis in islet cells using RNA interference (specific siRNA).
Methods: Male Brown Norway rats (250-270g) served both as pancreatic islet (PI) donors and recipients. Diabetes was induced by a single injection of streptozotocin (50 mg/kg, i.p.) and verified by three consecutively measured glycemias over 18 mmol/l. PI were isolated using collagenase digestion according to standard protocol. After overnight cultivation PIs were transfected with anti-TF siRNA (s130189, ThermoFisher Scientific, USA) using lipofection (RNAiMAX, 100nM) or microporation (200nM, Neon, 2 pulses, 950 V, 20 ms). After 24 hours, the treated PIs (microporation n=6, lipofection n=6) were transplanted to portal vein of diabetic animals in marginal dose (2 PI/g). The volume of hypoperfused liver tissue was quantified 2 hours after transplantation of 1000 PIs into the portal vein using magnetic resonance imaging (Fig. 1).

Results: The microporation of siRNA reduced the amount of mRNA by 76/54% and protein by 100/24% for 24/48 hours. The lipofection of siRNA reduced the amount of mRNA by 77/65% and protein by 100/40% for 24/48 hours. PIs normalized glycemias in all recipients transplanted with lipofected PIs and in none with microporated PIs. The transplantation of 1000 PIs caused impairment of perfusion in 28% of liver tissue. If islets were transfected with either a specific siRNA using microporation or lipofection, the total volume of hypoperfused tissue declined to 11% or 0%, respectively. 
Conclusions: AntiTF-siRNA transfected by microporation efficently reduced the amount of TF for 24 hours but did not improve the function of transplanted PIs. AntiTF-siRNA transfected by lipofection efficiently reduced amount of TF for 24 hours and significantly improve the function of transplanted PIs. Microporation likely caused the Off-target effect in PI graft.