Noble gene transduction into pancreatic beta-cells by singularizing islet cells with low doses of recombinant adenoviral vector.
Adenovirus-mediated gene transduction into the intact islets has thus far been limited to the surface cells of islets. We evaluated the efficiency of gene delivery by singularization of islets, followed by self-reorganization into islet-like masses. Adenovirus-mediated gene transduction was performed on dispersed islet cells, obtained by two-step digestion of collagenase and ethylene glycol tetraacetic acid/dispase. Good self-reorganization of islet cells in culture was observed until 120 h in islet cells of a control group, a group with a multiplicity of infection (MOI) of 1, and a group with an MOI of 5, with their sizes of 66.7 +/-14.17, 64.0 +/- 15.14, and 60.8 +/- 23.71 microm, respectively. No significant difference in spontaneous reaggregation capability among the islet cell masses was noticed. However, fragmentation of the reaggregated islet mass was observed in the groups with an MOI of 10 and 50 at 72 and 48 h, respectively. The gene transduction rates at an MOI of 0.5, 1, and 5 into islet cells were 56.1 +/- 1.43, 97.6 +/- 0.92, and 100 +/- 0.00%, respectively. The insulin stimulation indices of the reaggregated islet mass at an MOI of 0.5 and 1 were preserved to the level of a nontransduced islet mass; those at an MOI greater than 5 were significantly low. Efficient adenovirus-mediated gene transduction into islet/beta-cells was achieved by adding a process of dispersion of islets into single cells prior to gene transduction without losing the characteristic ability of islet cells to form a functional islet mass in culture.
Tsukiyama
Shusaku
S
First Department of Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan. tukiyama@med.hokudai.ac.jp
Matsushita
Michiaki
M
Matsumoto
Shuichiro
S
Morita
Tsunehiko
T
Tsuruga
Yosuke
Y
Takahashi
Tohru
T
Kasai
Hironori
H
Kamachi
Hirofumi
H
Todo
Satoru
S
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods; D048430:Cell Shape; D002478:Cells, Cultured; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000201:enzymology / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D001616:beta-Galactosidase; D005947:Glucose