Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

The overall goal of this procedure is to grow large quantities of mammalian cells. This is accomplished by first calibrating the pH and dissolved oxygen or do probes, and then autoclaving the pH, do probes and the temperature probe sleeve. The second step is to insert the probes and the sleeve into the vessel and to position the vessel on the bioreactor and to connect the exhaust, filter, the gas lines, and the temperature probe.

The next step is to connect the DO and pH cables and to aseptically connect the medium and micro carriers which are pumped into the bioreactor. The final step is to start the controllers to ensure the bioreactor achieves the desired impeller speed, temperature, and oxygen saturation. Ultimately, after inoculation and computer monitoring, the culture can be processed for extraction and purification of the desired product.

The main advantage of this technique over existing methods like the reusable stir tank bioreactor, is that the air wheel bioreactor is easy to use and minimizes chances for microbial contamination. Let's get started. To begin the protocol log in into the hello screen.

Next, inspect the pH sensor and confirm the sensor tip is filled with electrolyte solution. Obtain one beaker with pH four solution, another beaker with pH seven solution and a wash bottle with distilled water. Then connect the pH cable to the pH sensor.

Navigate to the actions tab on the hello interface, select calibrate. Then select the PHA tab and two point calibration. Enter the buffer temperature into the calibration solution temperature field.

Place the sensor in the pH four solution to calibrate Enter the value in the zero field. Once the graph stabilizes, push, calibrate and rinse the pH sensor with distilled water. Repeat the calibration for pH seven and save the settings.

Enter the buffer value in the span field and wait for the graft to stabilize. To begin dissolved oxygen or do calibration. Ensure the DO sensor has been connected to the system for several hours and has been polarized within the calibrate tab, select DOA and two point calibration.

Disconnect the DO sensor. Enter zero in the zero tab, and wait until the graph stabilizes. Before selecting calibrate one, reconnect the do sensor and enter 100 in the span field.

When the graph stabilizes, click calibrate two. Then save the settings and close the program. After calibration, place the sensors and the thermal well into autoclave pouches and autoclave them for 30 minutes at 121 degrees Celsius and 15 PSI.

After the autoclave has finished, sanitize the autoclave pouches with 70%isopropyl alcohol and transfer the pouches to a biological safety cabinet or BSC. Sanitize and introduce the reagent vessel into the BSC. Remove the inner packaging and inspect the vessel and tubing for damage.

Next, install the DO sensor in the front left port of the reagent vessel and the thermal well in the back left port. Install the pH sensor into the right front port to install. Open the sensor cap and thread the sensor lightly into the port on the top of the vessel.

Then transfer the vessel out of the BSC. Ensure that the pH and DO sensor cables are outside of the vessel sleeve and nothing is located within the sleeve. Hold the vessel so you are facing the front and slide the vessel feet first into the sleeve.

Ensure that the bottom of the vessel is resting on the heaters. Then carefully fit the temperature sensor into the vessel thermal.Well. Next, remove the tubing sets from the bag.

Match the color coding on the tubing to the corresponding connectors and pumps on the bioreactor control unit. Route the media addition and sampling lines behind the DO sensor and the thermal. Well insert the tubing into the color coded pump on the left side of the bioreactor.

Connect the main and micro gas lines. Press the main gas line connector into its gas outlet to install the main gas line. Then twist the micro gas line connector clockwise into its gas outlet To install the micro gas line.

To install the filter oven tubing, open the filter oven and secure the exhaust filter tubing to the uan. Make sure the tubing goes through the two hooks to the filter and out of the oven. Route the bottom line through the hook at the bottom of the filter oven and close the door.

Then connect the cables to the DO and pH sensors. After installation is complete, form a sterile connection using lure fittings between an unused medium edition line and the medium bottle bag source. Navigate to the actions tab on the Hello interface.

Click control pumps, select media pump, and click the slider to turn on the media pump. Then add 1.5 to 3.0 liters of media, and click the slider to turn the pump off. The system is now ready to pump previously prepared micro carriers into the reactor.

These can be added using an addition line. Attach the MICROCAR bottle to the appropriate addition line using a sterile lure lock. Turn on the addition pump within the control pump screen.

After addition of the micro carriers turn off the pump using the slider, navigate back to the main screen on the hello interface, select actions and then advanced. Select the appropriate controller to edit the settings. Set the controllers to auto and enter the set points.

After setting the controllers, turn on the air pump. Look within the reagent vessel to ensure that the air wheel is rotating. Ensure the DO sensor is fully polarized and that the do value has stabilized.

Then calibrate the do sensor form, a sterile connection between an unused medium addition line identified by one orange band and the cell bottle bag source using lure fittings. Next, install the silicone section of the tubing into the media pump. Make sure the tubing clamp is open and that the branched tubing clamp is closed.

Navigate to the control pump tab and then turn the media pump on. Add the cells and turn the media pump off. After inoculation, navigate to the actions tab and select take sample.

Ensure that the sampling tubing is in the sampling pump and manipulate the sampling stop cock according to the onscreen instructions. Remove the syringe with the sample for cell counting and other downstream applications perform cell counts. Finally, when the cells have reached the desired density of 1.2 times 10 to the six cells per milliliter, a septically add the virus inoculum to a 20 milliliter syringe.

Connect the syringe to a spare addition port on the reactor. Use the control pump screen to turn on the addition pump and add the virus inoculum. The majority of the A 5 49 cells adhered to the micro carriers within two hours.

After 12 hours, the cells demonstrated signs of flattening and spreading on the microcar surface. By 24 hours, micro carriers displayed an even distribution of cells. Microcar colonization happens quickly as demonstrated here.

The percentage of microcar colonization by a 54 9 cells is 75%by 24 hours. This graph shows the number of viable a 54 9 cells in culture pre and post infection with adenovirus. The cells continue to grow exponentially to 1 million cells per milliliter after 48 hours.

After watching this video, you should have a good understanding of how to assemble and operate an air wheel bioreactor to grow mammalian cells at high density for cellular or protein production. Thank you for watching and best of luck with your experiments.