Name: culturing lymphocytes in simulated microgravity using a rotary cell culture system
Uploaded: 2022-08-25T12:30:00
Description: Watch this Scientific Journal Video about Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System at JoVE.com

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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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.

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

Research

JoVE Journal

Immunology and Infection

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

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&amp;#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 ...