A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans.
OBJECTIVE
Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets.


METHODS
We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion.


RESULTS
In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure.


CONCLUSIONS
These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.
Barbu
A R
AR
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. Andreea.Barbu@medcellbiol.uu.se
Bodin
B
B
Welsh
M
M
Jansson
L
L
Welsh
N
N
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D004533:Egtazic Acid / Q000494:pharmacology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007515:Islets of Langerhans / Q000187:drug effects / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D049452:Green Fluorescent Proteins; D004533:Egtazic Acid