This work is supported by the Canadian Space Agency (CSA), research grant (17ILSRA3, Immuno Profile). Authors would like to acknowledge and thank Dr. Roxanne Fournier (University of Toronto), Dr. Randal Gregg (Lincoln Memorial University), and Preteesh Mylabathula (University of Arizona) for their help with the initial troubleshooting of this protocol.

NOTE: The following steps should be completed inside a sterile biological safety cabinet.
1. Preparation of vessels for cell culture
<ol>
	<li>Take the culture vessels out of the plastic packaging. Label each vessel on the rim according to what cell type/line is being used, whether it is the control (1G) or treatment (SMG), and any other relevant information.</li>
	<li>Stabilize the vessel and open the fill port carefully, without touching the fill port cap peg/O-ring. Place...

<ol>
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R., Dipert, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physical+basis+of+gravity+stimulus+nullification+by+clinostat+rotation.">The physical basis of gravity stimulus nullification by clinostat rotation.</a> Plant Physiology. 47 (6), 756-764 (1971).</li><li>Hammond, T. G., Hammond, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+suspension+culture:+the+rotating-wall+vessel.">Optimized suspension culture: the rotating-wall vessel.</a> American Journal of Physiology-Renal Physiology. 281 (1), 12-25 (2001).</li><li>Crabb&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+proteomic+responses+of+Pseudomonas+aeruginosa+PAO1+to+spaceflight+conditions+involve+Hfq+regulation+and+reveal+a+role+for+oxygen.">Transcriptional and proteomic responses of Pseudomonas aeruginosa PAO1 to spaceflight conditions involve Hfq regulation and reveal a role for oxygen.</a> Applied and Environmental Microbiology. 77 (4), 1221-1230 (2011).</li><li>Ulbrich, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+simulated+and+real+microgravity+on+bone+cells+and+mesenchymal+stem+cells.">The impact of simulated and real microgravity on bone cells and mesenchymal stem cells.</a> BioMed Research International. 2014, 1-15 (2014).</li><li>Martinez, E. M., Yoshida, M. C., Candelario, T. L. T., Hughes-Fulford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+and+simulated+microgravity+cause+a+significant+reduction+of+key+gene+expression+in+early+T-cell+activation.">Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 308 (6), 480-488 (2015).</li><li>Jong, J., Maki, G., Klingemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+cell+line+(NK-92)+with+phenotypical+and+functional+characteristics+of+activated+natural+killer+cells.">Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.</a> Leukemia. 8 (4), 652-658 (1994).</li><li>Williams, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+NK-92+cells+for+refractory+hematological+malignancies+relapsing+after+autologous+hematopoietic+cell.">A phase I trial of NK-92 cells for refractory hematological malignancies relapsing after autologous hematopoietic cell.</a> Oncotarget. 8 (51), 89256-89268 (2017).</li><li>Cryopreservation of mammalian cell lines video protocol. Abcam Available from: <a target="_blank" href="https://www.abcam.com/protocols/cryopreservation-of-mammalian-cell-lines-video-protocol">https://www.abcam.com/protocols/cryopreservation-of-mammalian-cell-lines-video-protocol</a> (2022)</li><li>Counting cells using a hemocytometer. Abcam Available from: <a target="_blank" href="https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer">https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer</a> (2022)</li><li>Mylabathula, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+microgravity+disarms+human+NK-cells+and+inhibits+anti-tumor+cytotoxicity+in+vitro.">Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro.</a> Acta Astronautica. 174, 32-40 (2020).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+simulated+microgravity+on+primary+human+NK+cells.">Effects of simulated microgravity on primary human NK cells.</a> Astrobiology. 13 (8), 703-714 (2013).</li><li>Shao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+the+effect+of+simulated+microgravity+on+the+cytotoxicity+of+NK+cells+following+the+DNA+methylation+of+NKG2D+and+the+expression+of+DAP10.">Mechanisms of the effect of simulated microgravity on the cytotoxicity of NK cells following the DNA methylation of NKG2D and the expression of DAP10.</a> Microgravity Science and Technology. 33 (1), 6 (2021).</li><li>Castro, S. L., Nelman-Gonzalez, M., Nickerson, C. A., Ott, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+attachment-independent+biofilm+formation+and+repression+of+hfq+expression+by+low-fluid-shear+culture+of+Staphylococcus+aureus.">Induction of attachment-independent biofilm formation and repression of hfq expression by low-fluid-shear culture of Staphylococcus aureus.</a> Applied and Environmental Microbiology. 77 (18), 6368-6378 (2011).</li><li>Phelan, M. A., Gianforcaro, A. L., Gerstenhaber, J. A., Lelkes, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air+bubble-isolating+rotating+wall+vessel+bioreactor+for+improved+spheroid/organoid+formation.">An air bubble-isolating rotating wall vessel bioreactor for improved spheroid/organoid formation.</a> Tissue Engineering Part C: Methods. 25 (8), 479-488 (2019).</li></ol>

As humanity prepares for longer space missions to the Moon and Mars, more research needs to be conducted to mitigate serious health risks in astronauts. One major aspect of the space environment that impacts human physiology is microgravity. Here, a cell culture method has been described for subjecting lymphocytes to SMG using a commercially available rotary cell culture system.
This protocol contains a few critical steps that may need to be optimized depending on the cell type or line that is...

Spaceflight has been shown to impact many aspects of human physiology, including the immune system. Many studies have demonstrated evidence of immune dysregulation as a result of spaceflight in vivo and exposure to simulated microgravity (SMG) in vitro1,2,3,4. One major aspect of the space environment that impacts human physiology is microgravity. Microgravity refers to the &#34;weightlessness&#34; experienced due to low gravitational forces in the space environment5. As humanity prepares for longer space missions to the Moon and Mars, more research needs to be conducted to mitigate serious health risks in astronauts.
Real microgravity conditions for scientific research can be achieved in space onboard the International Space Station (ISS) or in nanosatellites launched into orbit; however, these options can be incredibly costly and complex to orchestrate. Given the current limitations of conducting biological research in space, several options exist for inducing real microgravity and SMG on Earth. Large-scale operations exist that can produce short periods of real microgravity on Earth, including drop towers, parabolic flight, and sounding rockets. However, these methods are not overly suitable for studying the effects of microgravity on biological systems, largely due to their short periods of microgravity treatment (i.e., seconds to 20 min). These methods are discussed in greater detail elsewhere5,6. Options that are suitable for biological cell culture include small-scale devices such as 2D clinostats or rotating wall vessel (RWV) devices and 3D clinostats or random positioning machines (RPM). These devices can be set up inside cell culture incubators maintained at 37 &#176;C and 5% CO2, and they rotate the cell culture either on a horizontal axis (2D) or on two perpendicular axes (3D)5. However, it is important to emphasize that these culturing methods produce SMG as opposed to real microgravity, which is most feasibly attained in space for biological research contexts.
The goal of the current paper is to outline the steps for subjecting lymphocytes to SMG using a commercially available RWV device (Table of Materials), which falls under the 2D clinostat classification. While there is a general protocol available from the manufacturer for operating this device, the current article aims to cover the troubleshooting and optimization steps in more detail. This article also covers the theory behind how this device works to produce SMG in suspension cell culture, specifically with lymphocytes. In this context, suspension cell culture refers to cells growing freely in supplemented culture media, without adhering to any additional scaffolding. Many cell types are grown in suspension cell culture, including lymphocytes. Lymphocytes are cells of the immune system, including T, B, and Natural Killer (NK) cells, that reside in lymphoid organs and the bloodstream7.
The RWV 2D clinostat described here operates on the principle of time-averaged gravity vector nullification5,6,8,9, whereby the gravity vector is randomized through rotation of the cell culture on a horizontal axis. This is achieved by matching the rotational velocity of the culture vessel to the sedimentation velocity of the cells. As long as the rotational velocity of the culture vessel is matched well to the sedimentation velocity of the cells, the cells are maintained in free-fall and unable to sediment, as experienced in the space environment. After an initial speed-up phase, the media in the culture vessel eventually reaches &#34;solid body rotation&#34; over time. This horizontal rotation also induces laminar flow in the cell culture vessel. This creates a &#34;low shear&#34; environment, given that the shear stress induced on the cells by laminar flow is much less than that of turbulent flow. However, given that the clinostat is not a perfect system, there are some small, laminar fluid motions introduced, which inflict minimal shear stress on the cells. As such, the cells suspended in the media get dragged along by this flow during rotation. During horizontal rotation, the gravity vector acts on the cells and brings them into an oscillating trajectory, as visualized in Figure 1. Another small source of shear stress is caused by the cells &#34;falling&#34; through the media, causing laminar flow around the cells. As the culture vessel rotates on a horizontal axis, the gravity vector experienced by the cells rotates as well. Over time, this rotating gravity vector averages to approach zero; this phenomenon is called time-averaged gravity vector nullification and induces a state of SMG5,6,8,9. This device has been used to study the effects of SMG on many types of cells, some of which are covered in references10,11,12. More examples can be found on the device manufacturer&#39;s website.
This RWV device uses specialized &#34;high aspect ratio vessels&#34; (HARVs) available through the device manufacturer. These HARVs hold 10 mL of cell culture each; however, 50 mL HARVs are also available. Either 10 mL or 50 mL HARVs can be used depending on how many cells are needed to complete any downstream experimental assays, which is outlined further in the discussion section. The HARVs are made of polycarbonate and include a silicone oxygenation membrane to allow for gas exchange during cell culture. This maintains the pH of the cell media and allows for efficient cellular respiration. There is a main fill port and two capped syringe ports on the face of the vessel (Figure 2A). After loading the cell culture through the main fill port, two syringes are loaded onto the vessel to assist with bubble removal. When using the 10 mL vessels, two 3 mL syringes work well. One syringe is attached to the device empty, with the syringe completely depressed, and the other is attached filled with 3 mL of cell culture (Figure 2E). These are used in combination to remove bubbles from the vessel, which is important for maintaining the SMG treatment. In general, it is advised to set up two negative controls, which can be referred to as the &#34;Flask&#34; control and the &#34;1G&#34; control. The &#34;Flask&#34; control corresponds to cells that are grown in a standard T25 suspension cell culture flask. The 1G control corresponds to cells that are grown in the specialized 10 mL culture vessel, which is simply placed in the incubator (i.e., without being subjected to the SMG treatment). Please see the Discussion section for more details on controls.
The method described here is appropriate for any researcher looking to study the effects of SMG on lymphocytes, with a specific focus on NK cells by using the NK92 cell line13. The results from these studies may help us better understand and mitigate the adverse effects of spaceflight on the human immune system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disposible High Aspect Ratio Vessel (HARV) (10 mL)</td>
			<td>Synthecon</td>
			<td>D-410</td>
			<td>Gamma sterilized culture vessels (4/box)</td>
		</tr>
		<tr>
			<td>Luer-Lok tip syringes (3 mL)</td>
			<td>BD</td>
			<td>309657</td>
			<td>For attaching to the 10 mL HARVs</td>
		</tr>
		<tr>
			<td>NK92 Cell-line</td>
			<td>ATCC</td>
			<td>CRL-2407</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Cell Culture System (RCCS)</td>
			<td>Synthecon</td>
			<td>RCCS-4D</td>
			<td>Rotating wall vessel device; 2D clinostat</td>
		</tr>
		<tr>
			<td>Sarsedt 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>50-809-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sarsedt 50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>50-809-218</td>
			<td></td>
		</tr>
		<tr>
			<td>Sarsedt sterile serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>86.1254.001</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 suspension culture flasks</td>
			<td>Sarsedt</td>
			<td>83.3910.502</td>
			<td>For flask control</td>
		</tr>
	</tbody>
</table>

culturing lymphocytes in simulated microgravity using a rotary cell culture system

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This culturing method is considered successful if 1) the proliferation of the cells is approximately consistent across the control groups (and ideally all experimental groups), 2) the proliferation is appropriate given the seeding density, length of treatment, and the doubling time of the cell type/line, and 3) the viability of the harvested cells is 85% or higher (Table 1). Ideally, the resulting cells should be as healthy as they would be in standard cell culture, especially for use in subsequent exper...

Watch this Scientific Journal Video about Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System at JoVE.com

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

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.

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2.3K vistas.  University of Toronto. Esta técnica nos permite estudiar los efectos de la microgravedad simulada en las células inmunes para ayudarnos a comprender mejor cómo los aspectos del entorno espacial afectan la fisiología humana a nivel molecular. Esta técnica es económicamente factible y es relativamente fácil de configurar y mantener en comparación con el envío de cultivos celulares al espacio u otros métodos de someter el cultivo celular a microgravedad simulada en la tierra. Se recomienda ir lentamente mien...