multilayered culture systems corning cellstacks  (Corning Life Sciences)

Journal: BioProcess international

Article Title: Production of CGMP-Grade Lentiviral Vectors

doi:

Figure Lengend Snippet: Optimizing the upstream lentivirus manufacturing process: HEK 293 or 293T cell lines were plated at a concentration of 1.25 × 105 cells/cm2 in 10-layer CellStack trays (a) or at 1 × 105 cells/cm2 in T-75 flasks (b–d). Two days later, cells were transfected with three helper plasmids (pCgp, pCMV-Rev2, and pCMV-G) and a lentiviral construct expressing EGFP in a ratio of 20:13:5:20 respectively with 0.8 µg DNA/cm2. After a three-hour incubation, transfection reagents were removed and replaced by fresh media containing 2% serum (a–d) or either 0%, 2%, or 10% serum (b) with (a–d) or without (c) sodium butyrate. Crude supernatant was harvested 72 hours later (a–c) or either two, three, or four days later (d). For flasks with supernatant removed on day 2, an equivalent volume of media was added back to incubate until day 3 (sample day 2–3) (d). Supernatant from each sample was used to transduce HT-1080 cells, and GFP expression was measured by FACS analysis to assess viral titer. Panel a shows the mean ± standard deviation (SD) of four experiments; panels b–d show the mean ± SD of three experiments.

Article Snippet: Open in a separate window Figure 5 A semiclosed system for transfection of GMP -grade lentiviral product; Corning CellStack systems, media bags, transfection bottles, and the other components are parts of a manifold connected by disposable tubing and connectors.

Techniques: Concentration Assay, Transfection, Construct, Expressing, Incubation, Transduction, Standard Deviation

Journal: BioProcess international

Article Title: Production of CGMP-Grade Lentiviral Vectors

doi:

Figure Lengend Snippet: A semiclosed system for transfection of GMP -grade lentiviral product; Corning CellStack systems, media bags, transfection bottles, and the other components are parts of a manifold connected by disposable tubing and connectors. Following formation of DNA precipitates, existing media are removed by connecting the 10-layer trays to a manifold for transfection (a). The transfection medium is removed after incubation by a peristaltic pump using a second tubing manifold (b). On day 3 after transfection, supernatant containing secreted lentiviral vectors is collected by a third manifold (c). Sterile samples of crude supernatant and cells are collected for QC assays.

Article Snippet: Open in a separate window Figure 5 A semiclosed system for transfection of GMP -grade lentiviral product; Corning CellStack systems, media bags, transfection bottles, and the other components are parts of a manifold connected by disposable tubing and connectors.

Techniques: Transfection, Incubation, Sterility

Journal: BioProcess international

Article Title: Production of CGMP-Grade Lentiviral Vectors

doi:

Figure Lengend Snippet: Size analysis of residual host DNA present in a CGMP lentiviral product performed as described in the text

Article Snippet: Open in a separate window Figure 5 A semiclosed system for transfection of GMP -grade lentiviral product; Corning CellStack systems, media bags, transfection bottles, and the other components are parts of a manifold connected by disposable tubing and connectors.

Techniques: