title: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells.
abstract: In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.
authors: Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Miami, Miller School of Medicine; nschmid@med.miami.edu.
authors: Nathalie
authors: N
authors: Baumlin-Schmid
authors: Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Miami, Miller School of Medicine.
authors: Matthias
authors: M
authors: Salathe
authors: Department of Cell Biology, University of Miami, Miller School of Medicine.
authors: Nevis L
authors: NL
authors: Fregien
mesh_terms: D001980:Bronchi / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D004847:Epithelial Cells / Q000821:virology*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000254:growth & development*; D014161:Transduction, Genetic; D014776:Virus Cultivation / Q000379:methods*