Animal Cell Biotechnology: Methods and Protocols (Methods in by Ralf Pörtner

By Ralf Pörtner

Animal cellphone Biotechnology: tools and Protocols, moment version constitutes a entire handbook of cutting-edge and new thoughts for constructing mammalian phone traces for construction of biopharmaceuticals, and for optimizing severe parameters for mobile tradition contemplating the total cascade from lab to ultimate construction. the quantity is split into 5 elements that mirror the techniques required for various phases of creation. partially I, simple thoughts for institution of creation phone traces are addressed, particularly transduction thoughts, cells for gene treatment and antibody construction. half II addresses easy cultivation innovations, corresponding to microcarrier tradition and encapsulation.

Part III covers telephone characterization and research, together with circulate cytometric functions, NMR-based innovations, and biochemical and cytometric suggestions. half IV info cultivation ideas, reminiscent of disposable bioreactors, hole fiber phone tradition, mounted mattress reactors, and configuration of bioreactors. half V covers downstream options resembling membrane filtration ideas, whereas half VI describes specified purposes, together with retroviral vectors.

Animal cellphone Biotechnology: tools and Protocols, moment variation offers a compendium of options for scientists in business and learn laboratories that use mammalian cells for biotechnology purposes.

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3. Incubate for 5 min on ice and then spin down at top speed for 5 min. 4. Prepare 9 wells of a 96-well assay plate with 100 RL of 2x SEAP assay buffer, prewarm at 37°C. 5. Transfer 80 RL culture supernatant to wells containing 2x SEAP buffer. 6. Add 20 RL of substrate solution to each well. 7. Quantify absorbance at 405 nm for up to 1 h in a microplate reader (Tecan) (see Fig. 3). • Subtract values of the blank from sample values. 5 cm. 4. Notes 1. To perform transfections we recommend using ethanol or iso-propanol-precipitated DNA for sterility reasons.

1999) A high-efficiency Cre/loxP-based system for construction of adenoviral vectors. Hum. Gene Ther. 10, 2667–2672. 24. Kotin, R. , Samulski, R. , et al. (1990) Site-specific integration by adeno-associated virus. Proc. Natl. Sci. USA 87, 2211–2225. 25. Samulski, R. , et al. (1991) Targeted integration of adenoassociated virus (AAV) into human chromosome 19. EMBO J. 10, 3941–3950. 26. , Yant, S. , Storm, T. , and Kay, M. A. (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.

Suspension cultures are more appealing for large-scale production because of the ease of operation and scale-up. C6® cells can be grown in stirred tank bioreactors, and, depending on the mode of operation, cell densities are in excess of 5 × 106 cells/mL at scales from 3 to 20 L (42,56,57). Typical titers are in the range of 1–6 × 1010 virus particles (VPs) per mL. A recent study using the HeLaS3 human tumor cell line for propagating recombinant adenoviruses demonstrated yields of 6 × 1011 VP/mL and showed that this productivity could be maintained at pilot (70 L) scales (58).

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