This method can help answer some of the important questions in the field of Bio process manufacturing regarding aspects of cell line characterization and mediate and process optimization. Advantages of this technique are that it provides greater control in shake flasks and allows automation and the performance of large numbers of small scale studies for quick data generation. Demonstrating the procedure will be, Sai Rashmika Velugula and Casey Kohnhorst, research fellows in my laboratory.
Begin by immersing a stock vile of three times 10 to the seventh show cells per milliliter of freezing medium in a 37 degree Celsius water bath. When only a small sliver of ice remains, gently pipet the rapidly thawed cell suspension a few times and transfer one milliliter of cells into a sterile 125 milliliter vented shake flask containing 29 milliliters of OptiCHO medium. Place the flask in a cell culture incubator at 37 degrees Celsius and 8%CO2 with shaking it 130 RPM.
Subculturing the cells after 72 hours in 100 milliliters of fresh 37 degrees Celsius medium in a 125 milliliter spinner flask for another 72 hour incubation at 37 degrees Celsius and 8%CO2 with stirring at 70 RPM. On the third day of subculture, add fresh pre-warmed medium to the spinner flask to maintain the cells at an at least 90%viability for a final 24 hours of culture. The next morning, click on the remote desktop icon and click connect.
When the remote desktop is connected, open the cell counter software and install a new Reagent Pak. Then, empty the Trypan Blue waste and prime the system. Next, minimize the remote connection and open the micro bio reactor software using an existing experiment as a template to create a new experiment.
Before starting a run, define the plates used during the run in the mimic section of the software and place 12 sterile culture vessels in each culture station. Place autoclaved clamp plates on top of the vessels and place the stir plates on top of the clamp plates making sure that each pin is securely inserted. Then, secure the clamp plates with the screws and nobs provided.
To start up the system scan the barcode provided with each culture vessel. The system will initialize and check for the presence of the appropriate vessels. Set the temperature control to 37 degrees Celsius and the stirring to 1, 000 RPM and turn on the dissolved oxygen PH monitor.
Next, execute the medium charging program. When all of the medium has been loaded, 35 microliters of EX-CELL Antifoam will be added from the Antifoam plate to the culture vessels. After 30 minutes begin recording the dissolved oxygen and PH within the culture media, allowing the dissolved oxygen to reach a setpoint of 50%After the dissolved oxygen equilibration, turn on the background base additions to attain a PH setpoint of 7.1 for all of the culture vessels.
The next morning, execute the paused PH step and transfer the entire contents of the spinner flask into a sterile 250 milliliter conical tube for centrifugation. Resuspend the pellet in enough fresh medium that the final density will be one times 10 to the sixth CHO cells per milliliter after adding the inoculum to the culture vessels and add the suspended cells into the appropriate wells of a sterile lidded 24 well plate. Add three milliliters of inoculum to each well and place the inoculum plate onto the designated desk inside the hood.
Then, let the culture vessels equilibrate for at least an hour and initiate the five EX-CELL count step in the program. For daily nutrient and metabolite analysis on day two place the sample tube holder plates on their designated decks and load open microcentrifuge tubes into the appropriate holders. Then, place the samples in the analyzer tray, to perform the nutrient analysis.
To terminate the run first turn off the temperature control followed by the agitation. Stop the dissolved oxygen PH control and background base additions, all of the other controls, and the system monitor. Unscrew the clamp and stir plates, remove the culture vessels, and screw the drying plates into the culture station.
Then, execute the two hour drying cycle on the program;clicking stop in the bioreactor software once the drying cycle has finished. To harvest the cells transfer the cell culture fluid from the reactor vessels into corresponding conical tubes for centrifugation and filter the supernatants through sterile 0.22 micrometer PVDH filters. Then, transfer one milliliter of each sterile cell culture supernatant into individual 1.5 milliliter microcentrifuge tubes for negative 20 degrees Celsius storage until titer analysis.
To measure the IgG titers of the samples, first turn on the protein aid biosensor system. After the lamp has warmed for at least one hour, allow the samples to equilibrate to room temperature and presoak one protein aid tip per sample in fresh cell medium for at least 30 minutes. Then, set the plate temperature to 26 degrees Celsius.
Load the samples into the system, and run the assay using the default high sensitivity assay with regeneration in the data acquisition software. Using the integrated cell counter, the average viable cell densities and viabilities can be obtained daily for all culture conditions. Using the nutrient analyzer we can also obtain the nutrient and byproduct profiles demonstrating the feasibility of monitoring these attributes in the microbio reactor system.
Further, the total productivity of the cell cultures under the various culture conditions of interest can be quantified using the protein aid biosensor system as demonstrated. While attempting this procedure, it is important to remember that the protocol only works if the programming is done correctly;ensuring timely execution of protocol steps with minimal errors. Following this procedure other methods like:liquid chromatography, mass spectrometry, and multi-angle light scattering can be performed to answer additional questions regarding the physical and chemical properties of the product manufactured.
