title: Ex vivo gene delivery to hepatocytes: techniques, challenges, and underlying mechanisms.
abstract: Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.
authors.affiliation: Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114, USA.
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.forename: Erkin
authors.initials: E
authors.lastname: Seker
authors.forename: Monica
authors.initials: M
authors.lastname: Casali
authors.forename: Fangjing
authors.initials: F
authors.lastname: Wang
authors.forename: Shyam Sundhar
authors.initials: SS
authors.lastname: Bale
authors.forename: Gavrielle M
authors.initials: GM
authors.lastname: Price
authors.forename: Martin L
authors.initials: ML
authors.lastname: Yarmush
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008055:Lipids; D060328:Magnetic Phenomena; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection / Q000379:methods*; D014508:Urea / Q000378:metabolism
chemical_list: D000418:Albumins; C411955:FuGene; D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins; D014508:Urea