title: Lentivirus-Based Stable Gene Delivery into Intestinal Organoids.
abstract: Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.
authors.affiliation: Division of Molecular Carcinogenesis, Chiba Cancer Center Research Institute, 666-2 Nitona-cho, Chuo-ku, Chiba-shi, Chiba, 260-8717, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Maru
authors.affiliation: Division of Molecular Carcinogenesis, Chiba Cancer Center Research Institute, 666-2 Nitona-cho, Chuo-ku, Chiba-shi, Chiba, 260-8717, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Orihashi
authors.affiliation: Division of Molecular Carcinogenesis, Chiba Cancer Center Research Institute, 666-2 Nitona-cho, Chuo-ku, Chiba-shi, Chiba, 260-8717, Japan. yhippo@chiba-cc.jp.|Division of Animal Studies, National Cancer Center Research Institute, Tokyo, 104-0045, Japan. yhippo@chiba-cc.jp.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hippo
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007422:Intestines / Q000166:cytology* / Q000821:virology; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D009924:Organ Culture Techniques; D009940:Organoids / Q000166:cytology* / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods*
keywords: 3D culture; Matrigel; Organoids; Stem cells; Tumorigenesis; Viral transduction