BelloCell-500DP Technical Report II

Transcription

1 BelloCell-500DP Technical Report II HEK293A cell culture with Description BelloCell-300 Cell Culture System Technical Information (1) CHO cells culture batch culture Material Protocol Results BelloCell-500DP contains dissolvable cell culture matrixes BioNOC D, which provides an alternative choice from regular BelloCell-500 and BelloCell-500P bottles specially designed to harvest cells without requiring any trypsin or enzymatic treatment. This is especially useful for seed preparation or non-secreted products, such as membrane protein, non-secreted protein, or viruses, or any biological products that are sensitive to enzymatic treatment. In this study, the culture and cell harvest of HEK-293A cells with a BelloCell-500DP bottle is illustrated. Of 95% immobilizatioin efficiency was achieved within 3 hours seeding period in BelloCell-500DP bottle. Final 1.591x10 9 viable cells were harvested from one single BelloCell-500DP bottles with 89% viability within 11 days culture. This study shows that the BelloCell-500DP bottle can be applied to culture HEK-293A cells and harvest efficiently without requiring any enzymatic treatment. This technical sheet provides a general protocol for users to start up their culture. This study is only an example, and not an optimum condition. User has to determine the factors to the optimization of the culture. Material Device Cell Line/Product Medium Seed BelloCell-500DP HEK-293A DMEM/10%FCS cells/bottle Protocol *Detail protocol is in Step-by-Step Operation Instruction Inoculum preparation Inoculate HEK-293A cells in 4 x T150 flasks with each of 3.0 x 10 6 cells in 30 ml DMEM/10%FCS pre-warmed culture medium. Incubate in 37 C 5% CO 2 incubator for 3 days. Collect cells by centrifugation and resuspend with 30 ml culture medium in a 50 ml centrifuge tube. These cells were ready for BelloCell-500DP bottles inoculation. Culture preparation Pre-warm DMEM%FCS culture medium in 37 water bath. Take out one BelloCell-500DP bottle aseptically and place in a biosafety cabinet. Open the cap and add 470 ml culture medium plus 2.5 ml Reagent A (Cell Culture Additive) in the bottle. Place the bottle in BelloStage and start compression at the rate of UP: 2.0 mm/s, Down: 2.0 mm/s without holding time for at least 3 mins. Inoculation Bring the BelloCell-500DP bottle in the biosafety cabinet. Open the bottle. Dispense 30 ml prepared inoculums on top of the matrixes and bring to BelloStage Page 1

2 immediately. Fix the bottles on BelloStage 3000 console in CO 2 incubator with 37, and 5%CO 2 and start the compression immediately. Immobilization Set up immobilization parameters on the BelloStage control box and start the controller by pressing START button. The inoculation parameters are set as below: Rising rate Top holding time Down rate Bottom holding time 2.0 mm/s 20 sec 2.0 mm/s 0 sec Culture After 3 hours, sampling the medium to measure the un-attached free cells and determine if it should be turn to culture phase. If the seeding rate achieve above 90%, reset the parameters to culture parameters as below: Up rate Top holding time Down rate Bottom holding time 1.5 mm/s 0 sec 1.5 mm/s 1 min 30 sec Recirculation Connect the bottle with reservoir containing 2.2 L culture medium (plus 11 ml Reagent A) 72 hours after seeding and start recirculation. Start with 1000 ml/day with 24 cycle/day and increase to 1999 ml/day when the ph drop below 7.0. Sequentially adjust the CO 2 concentration in the incubator as the ph of medium goes down. Around 7 days culture will allow the cell growth to reach plateau. The setup parameters are only for reference. It does not necessary to be optimum parameters. Matrix Degradation and Cell Mass Harvest After culture finished, discard the culture medium. Prepare sterile 500 ml Reagent B (Matrix Degradation Solution) by filtration. Pour the Reagent B directly into the BelloCell-500DP bottle. Compress the bellows and swirl the bottle until the matrixes are degraded except the netting. Harvest the cells from the solution by centrifugation. The solution can be reloaded into the bottle after centrifugation and wash off the cells from the bottle repeatedly with at least 3 times. Most of the cells are fomed in clusters. Further treatment will be needed for individual cells. Cell Dissociation Follow the protocol in the BelloCell-500DP Step-by-Step Operation section VII for cell dissociation and obtaining individual cells. Page 2

3 Note: Reagent A must be added during cell culture phase. 5 ml Reagent A in every 1 L culture medium. Culture medium must contain serum. Result Table 1. Culture Record Culture Time (Hour) ph Glucose (g/l) GUR(mg/hr) Feeding rate (ml) Note CO2=5%; NaHCO3=2.2g/L BelloCell Reservoir BelloCell Reservoir BelloCell Reservoir start recirculation Exp. terminated Table.2 The final harvested cells Cell Line Seeding Culture Viability Viable cell Total cell no. Medium cell no. Day no. (live+dead) consumption HEK-293A 1.5E8 11 day 90.8% 1.59E9 1.75E9 2.7 L Page 3

4 Cell stability test 4.5x10 6 Control BioNOC-D 4.0x x x10 6 Cell number 2.5x x x x x st 2nd Generations 3rd Cells harvested from the BelloCell-500DP were re-loaded and cultured in T-flask, It was then performed with three passages and measure the cell density with the control group. Data shows that the cells, after growth and harvest from BelloCell-500DP, did not show any difference comparing with the cells grow in T-flask originally. Morphology comparison between control and HEK-293A from BelloCell-500DP Control: T-flask Cell from BioNOC D Cells harvested from the BelloCell-500DP were re-loaded and cultured in T-flask. The photo was taken after 3 days culture(160x photographed). It indicates that the cell morphology did not alter after cultivated in the BelloCell-500DP bottle with the dissolvable BioNOC D matrixes. Page 4

5 Cell Morphology in BioNOC D (SEM 300x) Please contact Cesco Bioengineering Technical Support for any questions or comments info@cescobio.com.tw Page 5