title: Highly efficient large-scale lentiviral vector concentration by tandem tangential flow filtration.
abstract: Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3h with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm(2) surface area and producing ∼560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>10(10)TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell (iPSC) generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real-time PCR assay is described for lentiviral vector titer and copy number determination.
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, USA.
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Cooper
authors.forename: Sanjeet
authors.initials: S
authors.lastname: Patel
authors.forename: Shantha
authors.initials: S
authors.lastname: Senadheera
authors.forename: Kathrin
authors.initials: K
authors.lastname: Plath
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hollis
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002498:Centrifugation; D005347:Fibroblasts / Q000378:metabolism; D005374:Filtration / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D019073:HT29 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34