A reverse transfection technology to genetically engineer adult stem cells.
A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.
Okazaki
Arimichi
A
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Jo
Jun-Ichiro
J
Tabata
Yasuhiko
Y
D053687:Adult Stem Cells / Q000166:cytology / Q000502:physiology*; D000818:Animals; D001709:Biotechnology / Q000379:methods; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D004337:Drug Carriers / Q000737:chemistry*; D005818:Genetic Engineering / Q000379:methods*; D005936:Glucans / Q000737:chemistry*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013096:Spermine / Q000737:chemistry*; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection / Q000379:methods*
D020099:Coated Materials, Biocompatible; D004337:Drug Carriers; D005936:Glucans; D013096:Spermine; C009109:pullulan; D004247:DNA