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 the cap on an ethanol pad, with the peg/O-ring facing up, while the vessel is being filled. Do not remove the caps from the syringe ports. 
	NOTE: If the peg/O-ring is accidentally touched, spray the cap peg with 70% ethanol and let it sit (facing upward) for a few moments while filling the vessel. When ready to close the fill port again, pick up the cap with a fresh paper wipe to thoroughly dry the cap before placing it back on the vessel.</li>
	<li>Load the vessel with 10 mL of the appropriate complete media for the cell culture using a sterile serological pipette, and carefully place the cap back on the fill port. 
	NOTE: Here, the NK92 NK cell line was used, which was cultured in GM1 media, as previously validated in a clinical trial using NK92 cells as a cell therapy14. The culture media used depends on the cell line or cell type being cultured. Please check the supplier&#39;s website for the correct media recipe for the cell line or cell type being used.</li>
	<li>Ensure that the syringe port caps and fill port are securely closed, and place the filled vessels in a 37 &#176;C, 5% CO2 incubator while carrying out the subsequent steps to prime the vessels for cell culture. 
	&#8203;NOTE: This priming step helps to minimize large bubble formation to make expelling bubbles easier later in Step 6.</li>
</ol>
2. Preparation of stock cell culture for the SMG treatment setup
<ol>
	<li>When not in use, store the cells of interest at a temperature lower than -130 &#176;C or, ideally, in a liquid nitrogen vapor phase.</li>
	<li>At least 1 week before setting up the planned treatment, thaw the cells and culture them using the appropriate cell culture media recipe found on the supplier&#39;s website. Plan how many cells are needed to set up the treatment (details below in the subsequent steps) and ensure to start the cultures early enough to allow for sufficient proliferation and adaptation to culture.</li>
	<li>Store the cell culture in a 37 &#176;C,&#160;5% CO2 incubator. Further details on standard cell culture practice are covered elsewhere15.</li>
	<li>On the day of the planned SMG treatment setup, determine the starting concentration and viability of the initial cell culture.
	<ol>
		<li>Use the preferred method for determining the concentration of the initial cell culture (e.g., hemocytometer, ViCell, flow cytometry, etc.). Take note of the viable cell concentration (cells per mL) and viability (%) of the cells, ensuring that the viability is ideally greater than 85%. A protocol for using a hemocytometer can be found elsewhere16.</li>
	</ol>
	</li>
	<li>Determine the appropriate cell seeding density/concentration of the cell culture for SMG treatment by considering the optimal cell concentration range of the particular cell type/line, their doubling time, and the length of SMG treatment. Please refer to the Discussion section for further elaboration. 
	NOTE: From experience, the optimal concentration range for NK92 cells is between 0.3 x 106- 1.2 x 106 cells/mL. Generally, these cells have a doubling time of 48-72 h and are fed every 2-3 days as such. Given that the treatment length was 72 h, the cells were seeded at the lower end of their optimal range at 0.4-0.5 x 106 cells/mL. The supplier also recommends this seeding density when starting the initial cell culture from a fresh vial of NK92 cells.</li>
	<li>Determine the volume of stock cell culture that is needed to set up the treatment and the controls. 
	NOTE: 10 mL of cell culture is needed per experimental group. If three experimental groups are set up (i.e., one treatment: &#34;SMG&#34;, and two negative controls: &#34;Flask&#34; and &#34;1G&#34;), another 10 mL of cell culture is sufficient to aliquot into a 15 mL tube used to load the HARV syringes. This is 40 mL in total, which may fluctuate depending on the specific experimental design.</li>
	<li>Determine the number of cells needed to set up the treatment.
	<ol>
		<li>Calculate the total number of cells needed by multiplying the chosen seeding density (cells/mL) by the total volume needed (mL). 
		NOTE: From experience with NK92, 40 mL of stock cell culture is prepared as detailed above at a seeding concentration of 0.4-0.5 x 106 cells/mL. As such, 16-20 x 106 NK92 cells are needed to complete the typical treatment setup. For the NK92 cell line, using slightly more cells is better than slightly fewer cells to maintain high viability throughout the treatment.</li>
	</ol>
	</li>
	<li>Determine the volume of initial cell culture to spin down.
	<ol>
		<li>Given that cell concentration is represented as cells/mL, divide the total number of cells (cells) needed for the experiment by the measured starting concentration of the cell culture (cells/mL). 
		NOTE: For example, if 16 x 106 cells are needed and the starting concentration of the culture is 0.8 x 106 cells/mL, 20 mL of the culture should be spun down.</li>
	</ol>
	</li>
	<li>Make the final stock cell culture.
	<ol>
		<li>Centrifuge the appropriate number of cells at 300 x g for 8 min in a 50 mL conical tube.</li>
		<li>Carefully pour off the supernatant into a waste container, and gently flick the tube to resuspend the pellet in the small leftover volume.</li>
		<li>Resuspend the cells in the appropriate volume of warmed (37 &#176;C) complete media to bring the culture to the desired seeding density. This volume was calculated in Step 2.6.</li>
		<li>Securely cap the 50 mL tube, and gently invert it a few times to thoroughly mix the cell suspension. 
		&#8203;NOTE: If more than 50 mL are needed to set up the full experiment, it is best to centrifuge the total number of cells needed in a 50 mL conical tube and resuspend this in half the volume needed. Then, mix well and make a 1:2 dilution in a separate 50 mL conical tube. For example, if 100 mL is needed at 0.4 x 106 cells/mL, spin down 40 x 106 cells in a 50 mL tube, resuspend in 50 mL of media, transfer 25 mL to another 50 mL tube, and then top up both with 25 mL of fresh media.</li>
	</ol>
	</li>
</ol>
3. Loading vessels with stock cell culture
<ol>
	<li>Retrieve the media-primed vessels from the incubator.</li>
	<li>If a &#34;Flask&#34; control is being used, load a T25 suspension culture flask with 10 mL of cell culture from the stock prepared above. Also, add 10 mL of stock cell culture to a separate 15 mL conical tube, which will be used to load the syringes.</li>
	<li>Unscrew the syringe port caps to remove them from the vessel and ensure the stopcocks are in the open position (Figure 2C).</li>
	<li>Stabilize the vessel and open the fill port carefully, without touching the fill port cap peg/O-ring. Place the cap on an ethanol pad, with the peg/O-ring facing up, while the vessel is being filled. Carefully unscrew the caps from the syringe ports.</li>
	<li>Ensure that the stopcocks are in the open position (Figure 2C). Carefully pour out the media from the vessel into a waste container, remove the media using a serological pipette, or aspirate it using a sterile glass Pasteur pipette attached to the vacuum system, without touching the oxygenation membrane.</li>
	<li>Tightly close the 50 mL conical tube that contains the stock cell culture and gently invert it a few times to thoroughly mix the contents.</li>
	<li>Draw up 10 mL of cell culture stock from the 50 mL tube with a fresh sterile serological pipette. Pick up the vessel and tilt it so that the fill port is toward the top and then carefully dispense the cell culture stock into the vessel through the fill port. 
	NOTE: Be careful and watch the volume so that the culture does not spill through the syringe ports while tilting the vessel.</li>
	<li>Aim to fill the vessel right to the top of the lip of the fill port without spilling; tilt the vessel back down as it is being filled to avoid spilling. Be careful not to touch the oxygenation membrane with the pipette as it is very fragile. 
	NOTE: The vessel filling process may take some practice; be patient and go slowly. If a bubble film forms at the opening of the fill port, this will interfere with loading the vessel. If this happens, gently hit the vessel to break the seal created by the bubble film.</li>
	<li>Once the vessel is loaded, carefully place the fill port cap back on, ensuring not to touch the peg/O-ring.</li>
	<li>Place on the syringes. 
	NOTE: There are two syringe ports on the face of the vessel (Figure 2A). One will hold an empty syringe, and the other will hold a syringe full of cell culture (Figure 2E).
	<ol>
		<li>First, attach the 3 mL empty syringe to one of the syringe ports, ensuring that the syringe is fully depressed. 
		NOTE: Pump the syringes a few times before attaching them to the vessel as they are a bit tight at first. Do this inside of the biological safety cabinet to maintain sterility.</li>
		<li>Tightly close the 15 mL conical tube with 10 mL of aliquoted cell culture and gently invert it a few times to thoroughly mix the contents.</li>
		<li>Next, carefully semi-submerge the second 3 mL syringe in the cell culture and draw up some culture. To minimize the air bubble in the top of the syringe, completely dispense this back into the tube, and then draw up 3 mL of the culture.</li>
		<li>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. Now the vessel is ready for being rid of bubbles. 
		NOTE: The second filled syringe for the second vessel is more difficult to load given the depth of the 15 mL tube. It helps to tilt the tube while filling the syringe to maintain the seal between the syringe being filled and the cell culture.</li>
		<li>Repeat Steps 3.3-3.10 for the remaining vessels. If there is a slightly insufficient amount of cell culture left in the 50 mL conical tube for the last vessel (e.g., 9.5 mL instead of 10 mL), recover the remaining amount from the 15 mL conical tube used to load the syringes. 
		NOTE:&#160;The following steps do not need to take place within a sterile biological safety cabinet, as the vessel is now a closed, sterile system.</li>
	</ol>
	</li>
</ol>
4. Removing bubbles from the vessels
NOTE: Bubbles are inevitable within this setup and must be consistently removed throughout the treatment (Figure 3). Please refer to the Discussion section for more details on this.
<ol>
	<li>Ensure that the syringe port stopcocks are in the closed position (Figure 2D) to limit the migration of cells and bubbles from the syringes into the vessels. This is especially important later during the treatment (discussed below). To collect the initial bubbles, flip the vessel upside down and hit the side of it a few times.</li>
	<li>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 (Figure 3B).</li>
	<li>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 (Figure 2C).</li>
	<li>Gently hit 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. 
	NOTE: Larger bubbles will need to be sucked up, whereas small bubbles and microbubbles can be encouraged to float up into the syringe by maneuvering them into the syringe ports and gently hitting the vessel. After a few hours of treatment, it is highly important to minimize the &#34;cross-contamination&#34; between the cells in the syringes and the cells in the culture vessel. This is because the cells in the syringes do not receive the same amount of oxygenation or exposure to SMG as the cells in the vessel do.</li>
	<li>Repeat Steps 4.2-4.4 a few times to ensure all bubbles are removed. Remove all bubbles including microbubbles from the vessels. 
	NOTE: The bubbles can be easily seen through the oxygenation membrane (back of the vessel) when the vessel is aimed at a light source (window, light, etc.). When carrying out Steps 4.2 and 4.3, look at the bubbles moving from the back of the vessel to help visualize the harder-to-see bubbles.</li>
	<li>When the bubbles have been effectively removed, close the syringe port stopcocks. Keep the syringes on during treatment to allow for subsequent bubble removal, ensuring that the volume in both syringes is approximately equal.</li>
</ol>
5. Attaching the vessel to the rotating base
<ol>
	<li>Carefully wipe down the surface of the rotating base and ribbon cable with 70% ethanol.</li>
	<li>Ensure that the included ribbon cable is attached to the power supply. Place the rotating base in the incubator and attach the ribbon cable to the base. Ensure that the power supply is kept near but outside of the incubator. Figure 4 depicts the rotating base and power supply for context.</li>
	<li>Attach the SMG treatment vessel by lining up the vessel threads to the rotating peg and gently turning the rotating peg on the base in a counterclockwise direction. Ensure the vessel is attached securely. Ensure that the incubator maintains close to 100% humidity by filling the water tray with sufficient autoclaved, reverse osmosis (RO) water.</li>
	<li>Choose an appropriate rotation speed (rpm). Adjust the rotation speed to match the sedimentation velocity of the cells, such that the cells do not &#34;fall through the media&#34; at all but end up rotating in a small orbital path. This phenomenon is visualized as a schematic diagram in Figure 1. 
	NOTE: The company recommends starting the rotation at 8-10 rpm for lymphocytes. In this case, NK92 cells were rotated at 11 rpm, as demonstrated in a previous paper17. Depending on the cell size, the rpm should be increased for larger cells and decreased for smaller cells. The same pattern applies if the cells used tend to clump during culturing. Please see the Discussion section for further details on this.</li>
</ol>
6. Treatment
<ol>
	<li>Choose an appropriate treatment length for the research application, which may depend on what parameters of cell function/physiology are being assayed. Please see the Discussion section for further details on this. 
	NOTE: In this case, NK92 cells were subjected to a 72 h SMG treatment, based on results from previous studies18,19. Bubbles will inevitably form throughout the treatment and will need to be removed. The rotation can be briefly stopped and restarted to facilitate this. See Step 7.1 for how to safely remove the SMG vessel.</li>
	<li>On the first day of setting up treatment, check for bubble formation every few hours by repeating Steps 4.2-4.6. After the first day, check the vessels as necessary (at least once per day), repeating Steps 4.2-4.6, until the end of the treatment.</li>
</ol>
7. Harvesting cells from the vessels
<ol>
	<li>Once the planned treatment length has elapsed, stop the rotation, and disassemble the apparatus. Remove the SMG treatment vessel by gently holding the vessel stationary while turning the rotating peg of the device in a clockwise direction.</li>
	<li>Bring the control flask, control 1G vessel, and treated SMG vessel into a sterile biological safety cabinet.</li>
	<li>Take each vessel (1G and SMG only; not the Flask) and flip it upside down so it faces downward and gently hit it to bring all the cells into suspension. Then, turn the vessel on its side with the fill port toward the bottom and hit the vessel again to encourage the cells toward the fill port for efficient aspiration.</li>
	<li>Handle each vessel individually. Unscrew the syringes from the two ports and dispose of them into the biohazard waste. Open the stopcocks on the syringe ports.</li>
	<li>Carefully remove the fill port cap and draw up the contents using a sterile 10 mL serological pipette, tilting the vessel as it is being emptied. Dispense the contents of the &#34;Flask&#34;, &#34;1G&#34;, and &#34;SMG&#34; groups into individually labeled 15 mL conical tubes.</li>
	<li>Close each tube and gently invert them a few times to ensure they are properly mixed. Use the preferred method for determining the concentration and viability of the resulting cell culture to prepare for use within subsequent experimental assays.</li>
</ol>

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The authors have no conflicts of interest.

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...

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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) ...

culturing-lymphocytes-simulated-microgravity-using-rotary-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

2.6K Views.  University of Toronto. 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. 

Video: Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Summary

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Chapters in this video

piggyBac Transposon System Modification of Primary Human T Cells

Culturing Microglia from the Neonatal and Adult Central Nervous System

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Assessing the Innate Sensing of HIV-1 Infected CD4⁺ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands