BelloCell-500 Technical Report IX

Transcription

1 Cultivation of BHK-1 cells Description BelloCell-5 provides a powerful tool to achieve high cell density and high BelloCell-3 Cell Culture System productivity of target bioproducts in a cell culture because it has a unique feature of offering Technical Information (1) CHO cells culture batch culture high oxygen transfer and low shear stress culture environment. Users can easily collect highly Material concentrated cells, virus or secreted products from one 5 ml BelloCell-5 bottle. In this study, the application of BelloCell-5 for growth of BHK-1 cells is illustrated Protocol cells/bottle was seeded and obtain a total of cells counted by crystal violet dye nuclei count method at hours, with a total 9 folds increase of cell population. Glucose Results concentration in the culture medium was monitored and kept above 1. g/l. This technical sheet provides a general protocol for users to start up their culture. However, the optimum condition of each cell culture for each case may require the users to determine. Material Device Cell Line Medium Seed BelloCell-5 BHK-1 DMEM/1%FBS, glucose.5g/l, glutamine mmol/l, NaHCO3. g/l cells/bottle Protocol *Detail protocol is in General Instruction Manual Inoculum preparation Prepare two T-15 flasks. Seed with cells total. Culture at 37, 5% CO for total 3 days. Harvest cells by standard trypsinization protocol. Prepare suspend cells, and concentrate cells in 5 ml culture medium. Inoculation Pre-warm DMEM/1%FBS medium in 37 water bath. Take out one BelloCell-5 bottle aseptically and place in a biosafety cabinet. Open the cap and add 5 ml culture medium in the bottle. Dispense 5 ml media containing suspend cells that has been prepared previously on top of the matrix of BelloCell-5. Bring the bottle and lock up on the BelloStage console immediately in incubator at 37, 5% CO and start the run immediately. Avoid swirling or shaking the bottle before start compression. Page 1

2 Immobilization Set up operation 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. mm/s secs. mm/s sec Culture After 3.5 hours, switch the parameters to culture parameters. The culture control parameters are set as below: Up rate Top holding time Down rate Bottom holding time 1.5 mm/s sec 1.5 mm/s 1 min 3 secs Monitor the residual glucose concentration and the color of medium in order to predict the time to change culture medium. The setup parameters are only for reference. It does not necessary to be optimum parameters. Cell Harvest The cell harvest was followed according to the protocol on CD manual Results Glucose and Lactate profile 7 5 Glucose Lactate Change medium Glucose, Lactate (g/l) Page

3 Glutamine and Ammonia profile 7 Glutamine Ammonia Glutamine, Ammonia (mmol/l) Culture Age (hours) Glucose uptake rate and Lacate production rate profile 1 1 GUR LPR GUR, LPR (mg/hr) Page 3

4 Cell grow curve by crystal violet dye nuclei count method x1 9 5x1 9 Total Cell (cells/bottle) x1 9 3x1 9 x1 9 1x Culture Age (hours) PH/CO ph ph CO CO Conc. in Incubator (%) Page

5 Cell Harvest.x x1 9 1.x1 9 9 Total Cell Mass (cells) 1.x1 9 1.x1 9 1.x1 9 8.x1 8.x1 8 Viability Harvest alive cells dead cells 8 7 Viability (%).x1 8.x1 8. PBS-EDTA nd wash 5 First wash Trypsin-EDTA treatment Second wash 3rd+th wash The growth of BHK-1 cells in BelloCell-5 is very fast, it requires only 8 days to have nearly 5 folds increase of cell population. The maximum cell density in BelloCell-5 system for BHK1 cells is around cells/bottle. PH is out of control during late phase of culture. We suggest to control the initial glucose concentration to below 3 g/l in order to control the ph within range. For cell harvest, most cells (9%) could be collected before 3 rd time of wash with 95% viability. Summary Seed Inoculum volume Medium volume Medium cells/bottle 5 ml/bottle 5 ml/bottle DMEM/1%FCS Total culture age Total medium Total medium Final cell density consumed replenish frequency (nuclei count) hrs 5 ml cells/bottle Please contact Cesco Bioengineering Technical support for any questions or comments info@cescobio.com.tw