Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

This technique allows us to study the effects of simulated microgravity on immune cells to help us better understand how aspects of the space environment affect human physiology on the molecular level. This technique is economically feasible and is relatively easy to set up and maintain as compared to sending cell culture to space or other methods of subjecting cell culture to simulated microgravity on earth. It is recommended to go slowly while filling the vessels in order to avoid overflow and spillage.

After initial bubble removal, ensure that the vessels are completely full by using the syringes. This will minimize the chances of large bubble formation throughout the treatment. Begin by taking the culture vessels out of the plastic packaging.

Label each vessel on the rim according to the cell type or line being used whether it's in the control or treatment and any other relevant information. Stabilize the vessel and open the fill port carefully without touching the fill port cap peg or the O-ring. Place the cap on an ethanol pad with the peg or O-ring facing up while the vessel is filled.

Do not remove the caps from the syringe ports. Load the vessel with 10 mL of the appropriate complete media for the cell culture using a sterile serological pipet, and carefully place the cap on the fill port. Ensure that the syringe port caps and fill port are securely closed.

Then, place the filled vessels in an incubator while performing the subsequent steps to prime the vessels for cell culture. Retrieve the media primed vessels from the incubator. For a flask control, load a T25 suspension culture flask with 10 mL of cell culture from the stock prepared above.

Add 10 mL of stock cell culture to a separate 15 mL conical tube, which will be used to load the syringes. Unscrew the syringe port caps to remove them from the vessel and ensure the stock stopcocks are in the open position. Stabilize the vessel and open the fill port carefully without touching the fill port cap peg or the O-ring.

Place the cap on an ethanol pad with the peg or O-ring facing up while the vessel is being filled. Ensure that the stopcocks are in the open position. The vessel can be emptied by pouring the media from the vessel into a waste container using a serological pipette.

Alternatively, aspirate the media using a sterile glass past your pipette attached to a vacuum system. While using a pipette or the vacuum system, be sure not to touch the oxygenation membrane as this could damage it, and render the vessel unusable. Tightly close the 50 mL conical tube containing the prepared stock cell culture and gently invert it a few times to mix the contents thoroughly.

Draw up 10 mL of cell culture stock from the 50 mL tube with a fresh sterile serological pipet. Pick up the vessel and tilt it, so that the fill port is towards the top. Then carefully dispense the cell culture stock into the vessel through the fill port.

Fill the vessel right to the tip of the fill port, while tilting the vessel back down to avoid spilling. Be careful not to touch the oxygenation membrane with the pipette as it is very fragile. Once the vessel is loaded, carefully place the fill port cap back on without touching the O-ring.

Remove the first 3 mL syringe from its package and pump it a few times to loosen it. Then attach the syringe to one of the syringe ports, ensuring that the syringe is fully depressed. Tightly close the 15 mL conical tube containing 10 mL of aliquot cell culture, and gently invert it a few times to mix the contents thoroughly.

Similarly, remove the second 3 mL syringe from its package and pump it a few times. Then carefully semi submerged this syringe in the cell culture from the 15 mL conical tube and draw up some culture. Minimize air bubbles by entirely dispensing the syringe back into the tube and drawing up 3 mL of culture.

Attach the filled syringe to the remaining syringe port, and carefully sanitize the syringes and around the fill port with an ethanol pad. Do not get ethanol on the oxygenation membrane. Repeat the process for the remaining vessels.

If there's a slightly insufficient cell culture left in the 50 mL conical tube for the last vessel, recover the remaining amount from the 15 mL conical tube used to load the syringes. Ensure that the syringe port stopcocks are closed to limit the migration of cells and bubbles from the syringes into the vessels. Flip the vessel upside down and hit it side a few times to collect the initial bubbles.

Quickly flip the vessel back to face upward, and then on a slight angle facing away, so that the bubbles all float to the upward side of the vessel. Maneuver the bubbles under the port with the empty syringe, and watch them start to enter up into the port. Open both of the syringe port stopcocks.

Gently strike the vessel to encourage the bubbles to float up into the empty syringe. Slowly suck up the larger bubbles with the empty syringe while carefully depressing the full syringe to maintain the pressure in the vessel, so that the oxygenation membrane does not burst. Repeat this process a few times to ensure all bubbles are removed.

When the bubbles have been effectively removed, close the syringe port stopcocks. Keep the syringes on during treatment to allow subsequent bubble removal, ensuring that the volume in both syringes is approximately equal. Carefully wipe down the surface of the rotating base and ribbon cable with 70%ethanol.

Ensure that the ribbon cable is attached to the power supply. Place the rotating base in the incubator, and attach the ribbon cable to the base. Place the power supply near, but outside the incubator.

Attach the SMG treatment vessel by lining up the vessel threads to the rotating peg and gently turning the rotating peg on the base counterclockwise. Ensure the vessel is attached securely. Maintain the incubator at approximately 100%humidity by filling the water tray with autoclave RO water.

Adjust the incubator's rotation speed to match the sedimentation velocity of the cells, such that the cells do not fall through the media. On the first day of setting up treatment, check for bubble formation every few hours as described in the text manuscript. Remove the bubbles at least once a day going forward until the end of the desired treatment length.

Once the planned treatment length is elapsed, stop the rotation and disassemble the apparatus. Remove the treatment vessel by gently holding the vessel stationary while turning the rotating peg of the device in a clockwise direction. Bring the control flask, control, and treated vessel into a sterile biological safety cabinet.

Take each vessel excluding the control flask and flip it, so it faces downward. Then gently strike it to bring all the cells into suspension. Then turn the vessel on its side with the fill port toward the bottom, and strike it again to guide the cells toward the fill port for efficient aspiration.

Handle each vessel individually. Unscrew the syringes from the two ports and dispense them into the biohazard waste. Open the stopcocks on the syringe ports.

Carefully remove the fill port cap and draw up the contents using a sterile 10 mL serological pipette, tilting the vessel as it is emptied. Dispense the contents of all the control and treatment groups into individually labeled 15 mL conical tubes. Close each tube and gently invert them a few times to ensure they're properly mixed.

Use the preferred method for determining the concentration and viability of the resulting cell culture to prepare for subsequent experimental assays. The NK-92 cell line starting viability and seating densities were compared to the resulting end viability and end concentrations. After a 72-hour SMG treatment, two instances of negative and positive outcomes were compared.

For comparison, the optimal concentration range of the NK-92 cell line used was between 0.3 to 1.2 million cells per milliliter with a doubling time of around two to three days. Proliferation in the SMG treatment group was approximately equal across the control and treatment groups. These parameters were essential for the downstream experimental success and the resulting cells from the two control groups performed similarly in downstream experimental assays.

It is crucial to monitor and account for bubble formation throughout the course of the treatment, especially in the cell culture being subjected to simulated microgravity. Large bubble formation can cause disruption to the fluid dynamics within the vessel, and this will essentially negate the simulated microgravity condition.