title: Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.
abstract: Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).
authors.affiliation: EUFETS AG, Idar Oberstein, 55743, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kühlcke
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.forename: Andrea
authors.initials: A
authors.lastname: Schilz
authors.forename: Sonja
authors.initials: S
authors.lastname: Loges
authors.forename: Carsten
authors.initials: C
authors.lastname: Lindemann
authors.forename: Francis
authors.initials: F
authors.lastname: Ayuk
authors.forename: Friederike
authors.initials: F
authors.lastname: Lehmann
authors.forename: Norbert
authors.initials: N
authors.lastname: Stute
authors.forename: Axel A
authors.initials: AA
authors.lastname: Fauser
authors.forename: Axel R
authors.initials: AR
authors.lastname: Zander
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Eckert
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D002498:Centrifugation; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*