The overall goal of this procedure is to demonstrate the correct closed system use of the Corning Hypers stack vessels. This is accomplished by first inoculating and filling the vessels with media via the gravity feed method. Next, the cell suspension is isolated in each of the layers and the cells are incubated.
Following incubation, the spent media is removed. The final step of the procedure is to harvest the cells from the hypers stack vessels. Ultimately, gas diffusion results are obtained to show the ability of cells to access oxygen during growth to co fluency when the hypers stack closed system is used.
The main advantage of hypers stack vessel with gas permeable material is that it provides increased surface area for cell growth. By eliminating the head space in the vessel, my colleague p pardo will now demonstrate a closed system protocol for using the hypers stack vessel. The hypers stack vessel is composed of a stat, which is the individual cell culture compartment that is made up of the top plate and gas permeable film.
The cells are cultured within this compartment. The liquid manifold connects each of the ette layers together within a module. Modules are connected with tubing to form vessels in multiples of 12 layers.
The manifold allows the user to make one fluid manipulation to the entire vessel. The air manifold also connects the stat layers together, but is used to displace air from the vessel when fluid additions occur and contains a fill line for use during filling between each. Stat layer is an open air space called the tracheal space, allowing gas exchange to occur through each layer's gas permeable film.
The liquid handling tubing is connected to the liquid manifold and is a customizable component used to make all closed system fluid manipulations to release excess air while maintaining sterility. The vent tubing is used. The vent tubing is connected to the air manifold and contains an air filter.
Connected to the liquid handling tubing is the chase tubing and filter, which are used to evacuate the fluid from the liquid handling tubing after filling the vessel. Also used in this protocol is the Corning Stack manipulator or CSM, which is a handling device to assist in placing the vessels in the correct positions during use. In this demonstration, closed system cell culture is demonstrated for a gas permeable 36 layer vessel.
Using A CSM, however, details for performing this procedure on a 12 layer vessel using a filling wedge can be found in the accompanying written protocol. Begin by preparing 3.9 liters of bagged media prior to inoculating the media tube. Wet the serum bag to the media bag and mix thoroughly to result in the desired serum concentration.
Clamp off approximately 300 milliliters of media in the bag using large bag clamps. This will ensure all inoculated cells are used during filling, and none are remaining in the media bag. Place the media on a bag stand to inoculate the media.
Fill a syringe attached to tubing with cell suspension. Attach the syringe containing cell suspension to the media bag by tube welding. Three over 16 inch tubing from the syringe to the bag.
Inject the cell suspension into the media bag and mix well. The inoculated media bag can be attached to the vessel's three over eight inch liquid handling tubing by tube welding or using multipurpose connectors. Today we will use tube welding.
Close the clamps on the liquid handling and chase tubes of the vessel. Place the 36 layer vessel into A CSM in the load position. Hook the vent filter tube into the holding clamp on the CSM.
Tighten the lid and move the CSM to the fill position to place the air filter in the highest position to prevent wetting during fill operation. The 10 degree angle also permits equilibration of the liquid to the layers during filling clear air from the fill line. By positioning the media bag at the same level as the vessel keeping the chase tube clamp closed.
Open the liquid handling tube clamp and the media bag clamp to allow fluid to enter the vessel. Using the bag stand, raise the media bag to help cell suspension flow into the vessel. Fill the vessel until all of the inoculated media enters the vessel.
The top portion of the media bag should still contain 300 milliliters of media. Remove the clamp from the media bag to continue filling the vessel as liquid approaches the upper air manifold. Slow down the fill rate by lowering the media bag to prevent over filling.
Slowly bring the liquid level to the fill line and clump the liquid handling tubing. Bring the vessel in the CSM to the isolation position placing both sets of manifolds in the highest position. Lower the media bag below the height of the vessel.
With the chase filter held in an upright position, open the chase tube clamp. This will empty or chase the media in the liquid handling tube back into the media bag. Once the tubing is empty, close the clamp on the media bag tubing.
Keeping the chase tube filter in an upright position. Open the clamp on the liquid handling tube of the vessel to allow any remaining liquid to enter and equilibrate in the vessel. Wait one to two minutes for this to occur.
Next, turn the vessel in the CSM so that the manifolds are on the left. Lower the vessel to the load position on the CSM. Close the clamps on both the chase and liquid handling tubing.
The media bag may now be removed from the vessel, or it may remain attached for use later in the harvest procedure. To store the attached media bag, roll the media bag and place it under the retaining bands in the storage tray of the vessel. Finally, move the vessel to the incubator When carrying the vessel, take care to keep any liquid from entering the air vent filter.
This is accomplished by tipping the manifold end slightly upward. To begin the harvest procedure tube, weld the cells Association solution and quench bags together To form the harvesting bag assembly, ensure all tubing clamps are closed. Remove the vessel from the incubator and place it in the CSM in the load position.
Release the media bag from under the retaining bands and hang it on the bag. Stand next, tighten the lid to secure the vessel. Hook the vent tubing into the holding clamp and move the CSM to the fill position.
Making sure the media bag on the bag stand is hanging lower than the vessel. Open the clamp on the media bag tubing and the vessel's liquid handling tubing to allow media to flow into the attached bag. Once the vessel is about three quarters empty, change the CSM settings to the final empty position.
When the vessel and liquid handling tubing are empty, lift the tubing to chase the media past the clamp on the spent media bag tubing. Then close both the clamp on the liquid handling tubing and the clamp on the spent media bag tubing. Replace the spent media bag with the harvesting bag assembly by tube welding.
Raise the height of the cell dissociating bag assembly above the height of the vessel using the bag stand. Place the CSM in the fill position. Open the cell dissociation solution tube clamp and the harvest bag assembly Clamp and transfer the solution into the vessel.
When the transfer is complete, close the clamps on the liquid handling tubing and the cell dissociation solution tubing. Next, bring the CSM to the load position while holding the horizontal positioning lever open. Gently rock the vessel side to side using the wheel, distributing the solution over the cell layers.
The vessel may occasionally be returned to the fill position during rocking to maintain equal distribution of the solution. Once the solution has been adequately distributed, release the lever on the CSM to lock in the fill position. Leave the vessel in this position during the required dissociation time for the cells.
Use turbidity as a guide to determine cell detachment. Once detached, move the CSM to the load position. Open the quench bag clamp and the liquid handling tubing clamp to allow the quench media to enter the vessel.
Close the clamp on the liquid handling tubing. Once the transfer is complete, return the CSM to the load position and hold the horizontal positioning lever to rock the vessel side to side. Using the bag stand, lower the position of the harvest bag assembly below the level of the vessel.
Adjust the CSM to the empty position. Open the vessel liquid handling tube Clamp and transfer the cell solution back into the quench bag. Return the CSM to the load position and disconnect the harvest bag assembly from the vessel using a tube sealer.
Now the cell solution is ready for processing in traditional cell growth systems. With Headspace, the oxygen in the media is depleted at an average of 50%over three days. In culture, this figure shows the decrease in milligrams per liter of oxygen at the level of the cells over time in the same growth system, the oxygen gradient through a media height of three millimeters is almost 50%greater at the media to Headspace junction than it is at the cell layer.
The percentage of oxygen in the tracheal space of the hypers stack during cell growth is shown. The figure shows that over 96 hours, the percentage of oxygen between the layers which supplies each gas permeable film bottom remains constant. This demonstrates the ability of the cells to access oxygen during growth to co fluency.
After watching this video, you should have a good understanding of how to fill and harvest Corning Hypers stack vessel in the closed system.
