细胞中轨道波浪振荡方式培养细胞检测方案(培养箱)

Reference' ZHAW Zurich University of Applied Sciences， School of Life Sciences and Facility Management， Institute of Biotechnology， Campus Gruental， CH-8820 Wadenswil， Switzerland Iris Poggendorfl， Lidija Lisica1， Nadia Wohlwend1，Johanna Olownia²， Regine Eibl Introduction Orbitally shaken， single-use bags represent a new method for the productionof seed cultures or protein production on the lab scale. Using this new technology， successful cultivation of the cell lines CHO XM 111(Chinese Hamster Ovary) and Sf-21 (Sf-9) (Spodoptera frugipedra) was accom-plished.Also， scale-up of the CHO cell cultivation from 1 L to 10 L working vol-ume is possible using orbital-shaken， single-use bags. For these cell culturesa serum- and protein-free chemically defined media was used， which needssophisticated system efforts for the cell growth， but is a requirement for theproduction of therapeutic products. CHO XM 111 fed-batch cultivation in 2 L shaker bag vs 2 L Cultibag 2 L-shaker bag viability -o- 2L-Cultibag viability22 L-shaker bag cell conc. -A- 2L-Cultibag cell conc. CHO XM 111 fed-batch cultivation scale-up from 1 L to 10 L 2 L-shaker bag viability 20 L-shaker bag viability-22 L-shaker bag cell conc. - 20 L-shaker bag cell conc. New Option for Incubator Shaker：Cell cultivation with orbital waves in flexiblesingle-use shaker bags up to 10 L Zurich Un aty Details The CHO and Sf-9 cell lines are frequently used in biotechnology. For this ex-perimental work， the CHO XM 111 clone was used. This clone was transformedby the group of Professor Dr. M. Fussenegger at the ETH Zurich with an expres-sion vector which codes for the gene of the recombinant protein SEAP (Secret-ed Alkaline Phosphatase) and controlled by tetracycline using the promoter Ph-CMV-1. The Sf-9 is a cell clone of the cell line Sf-21. These insect cells， developedfrom ovaries of the moth species Spodoptera frugipedra， are well known forvirus infection and production of recombinant proteins. The Multitron Cell for shaker bag was used with a 50 mm shaker stroke andaeration with maximal 2 vvm air and CO，(0-10% only for CHO cells). The cul-tivation of CHO XM 111 were done at 37°C， pH 7.2 and shaker speed between30 and 40 rpm. For the cultivation of Sf-9 cells a temperature of 27C， shakerspeed between 25 and 35 rpm and pH 6 were used. The oxygen transfer wasdetermined by amperometric method. Result The fed-batch cultivation strategy was used in chemical defined media： ·CHO(protein- and serum-free)： HP-1 (Cell Culture Technologies， Invitrogen) with 2.5 mL L- Tetracyclin and 10mLL-1 Pluronic F-68 ·Sf-9 (serum-free)： Sf-900 II SFM (Cell Culture Technologies) Cell growth results in maximal cell densities for ·CHO XM 111 about 3x106viable cells/ml with >98% viability · Sf-21 (Sf-9)> 1x10' viable cells/ml with >90% viability (data not shown) An Oxygen Transfer Coefficient (k a) about 31 h-with 2 vvm could be achieved. Oxygen Transfer Gassing WV Shaker speed ka Saturation [L min-1] [L] [rpm] [h-] 2 1 40 30.34 lyes 2 1 40 30.96 no Orbitally shaken, single-use bags represent a new method for the productionof seed cultures or protein production on the lab scale.Using this new technology, successful cultivation of the cell lines CHO XM 111(Chinese Hamster Ovary) and Sf-21 (Sf-9) (Spodoptera frugipedra) was accomplished.Also, scale-up of the CHO cell cultivation from 1 L to 10 L working volumeis possible using orbital-shaken, single-use bags. For these cell culturesa serum- and protein-free chemically defined media was used, which needssophisticated system efforts for the cell growth, but is a requirement for theproduction of therapeutic products.