1. Isolation of T cells with microbubbles using positive selection
NOTE: This protocol details a small-scale CD3+ positive selection approach using SAMBs.
<ol>
	<li>Incubate 3 x 108 commercially obtained PBMCs in 2.5 mL of separation buffer with biotinylated anti-CD3 (OKT3) antibody at a concentration of 25 ng of antibody per 1 million cells (25 ng/M). Gently mix by pipetting up and down, and incubate at room temperature for 10 min.</li>
	<li>Add streptavidin microbubbles (SAMBs) at a ratio of 0.5 (SAMB quantity):1 (cell quantity) according to the manufacturer&#39;s reported SAMB concentration.</li>
	<li>Mix using a commercial end-over-end (EOE) rotator at 20 rpm for 10-15 min at room temperature. Centrifuge for 5 min at 400 x g at room temperature.</li>
	<li>After centrifugation, the positively selected cells will be at the top of the suspension with the SAMBs. The remaining non-selected cells will be in the cell pellet at the bottom of the tube. Using a 9 in glass pipette, insert the tip below the bubble-cell layer to the bottom of the tube, aspirate the cell pellet and subnatant with an electronic pipette and transfer them to a new tube.</li>
</ol>
2. Co-stimulation (activation) of the positively selected T cells
<ol>
	<li>Resuspend the bubble-cell layer remaining in the original tube in 1 mL of complete T cell medium (or another desired medium).</li>
	<li>Count the cells in the subnatant with brightfield microscopy using an automated cell counter, and subtract this value from the starting cell number to determine the number of cells captured in the bubble-cell layer.</li>
	<li>Prior to this step, mix the biotinylated anti-CD28 antibody with commercial SAMBs for a minimum of 2 h to create the conjugated anti-CD28 SAMBs. Contact the SAMBs manufacturer to determine the amount of anti-CD28 antibody needed for conjugation. Add the anti-CD28 conjugated SAMBs to the bubble-cell suspension resulting from step 2.1 using a ratio of 1.5 (anti-CD28 SAMBs):1 (cells).</li>
	<li>Mix using EOE rotation for 15 min, and then adjust the total volume to 2 million cells per mL with complete T cell medium or another desired medium according to the cell number obtained in step 2.2.</li>
</ol>
3. Expansion of the co-stimulated cells in cell culture medium
<ol>
	<li>Distribute 1 mL of activated cells from step 2.4 at a concentration of 2 M/mL per well in a 24-well plate. Incubate in a humidified 5% CO2 incubator at 37 &#176;C.</li>
	<li>After 24 h, add 50 U/mL of IL-2 and 25 ng/M of soluble anti-CD3 (OKT3) to further encourage expansion, as calculated using the initial number of cells plated on day 0. Place the cell plate back into the humidified CO2 incubator, and let it incubate overnight at 37 &#176;C.</li>
</ol>
4. Optional: Transduction of activated T cells with lentivirus
NOTE: The approach used here is adapted from Prommersberger et al.10.
<ol>
	<li>Thaw the lentivirus at room temperature, and briefly mix by pipetting.</li>
	<li>Gently remove the mid-natant, 600 &#181;L from each well, without touching the daughter cells that are now at the bottom of the well or the bubble layer that has remained at the surface of the solution.</li>
	<li>Add 5 &#181;g/mL hexadimethrine bromide per well to enhance viral transduction. Add the lentiviral particles at a multiplicity of infection (MOI) of 3 (lentiviral particles per cell).</li>
	<li>Centrifuge the plate for 45 min at 800 x g and 32 &#176;C using slow acceleration and no break for deceleration. Incubate the cells for 4 h in a humidified CO2 incubator at 37 &#176;C.</li>
	<li>After 4 h, add 600 &#181;L of commercially available, fresh complete T cell medium and 50 U/mL of IL-2 to each well, and place the cell plate back into the humidified CO2 incubator at 37 &#176;C for T cell expansion.</li>
</ol>
5. Expansion of the T cells (with or without prior transduction)
<ol>
	<li>Every 2 days, remove half of the medium from the mid-natant, replace it with fresh, complete T cell medium, and add IL-2 at a concentration of 50 U/mL.</li>
	<li>Count the T cells twice per week to assess the cell density. When the cell density exceeds 2 x 106-2.5 x 106 cells/mL, transfer the cells into a bigger vessel, diluting them to 5 x 105 cells/mL.</li>
</ol>
6. Harvesting the T cells and flow cytometry
<ol>
	<li>Gently mix the contents of each well by pipetting up and down. Remove the entire contents of the well, including the microbubbles, and transfer them into a 1.5 mL tube.</li>
	<li>Wash each well with 400 &#181;L of calcium-free and magnesium-free DPBS (&#8722;/&#8722;), and transfer the solution into a 1.5 mL tube. Centrifuge at 400 x g for 5 min at room temperature.</li>
	<li>Aspirate the supernatant, and resuspend the cell pellet in 50 &#181;L of separation buffer.</li>
	<li>Stain with an activation and exhaustion antibody/staining cocktail, and incubate for 10 min at room temperature in the dark. Prepare the staining cocktails as follows- activation cocktail: AF700-CD3, PE/Dazzle-CD4, PE/Cy7-CD8, BV510-CD25, PE-CD69; exhaustion cocktail: AF700-CD3, PE/Dazzle-CD4, PE/Cy7-CD8, PE-PD-1.</li>
	<li>Add 1 mL of separation buffer, and gently mix. Centrifuge at 400 x g for 5 min at room temperature to wash out excess antibodies. Aspirate the supernatant completely.</li>
	<li>Resuspend the cell pellet in 1 mL of separation buffer, and transfer to an appropriate vessel (e.g., a FACS tube, a 96-well plate, etc.) for the flow cytometry analysis. The recommended flow cytometry analysis gating scheme is detailed in Figure 1.</li>
</ol>

<ol>
	<li>Albinger, N., Hartmann, J., Ullrich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+perspective+of+CAR-T+and+CAR-NK+cell+therapy+trials+in+Germany.">Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany.</a> Gene Therapy. 28 (9), 513-527 (2021).</li><li>Tyagarajan, S., Spencer, T., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+CAR-T+cell+manufacturing+processes+during+pivotal+clinical+trials.">Optimizing CAR-T cell manufacturing processes during pivotal clinical trials.</a> Molecular Therapy. Methods &amp; Clinical Development. 16, 136-144 (2019).</li><li>Stock, S., Schmitt, M., Sellner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+manufacturing+protocols+of+chimeric+antigen+receptor+T+cells+for+improved+anticancer+immunotherapy.">Optimizing manufacturing protocols of chimeric antigen receptor T cells for improved anticancer immunotherapy.</a> International Journal of Molecular Sciences. 20 (24), 6223 (2019).</li><li>Rohaan, M. W., Wilgenhof, S., Haanen, J. B. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cellular+therapies:+The+current+landscape.">Adoptive cellular therapies: The current landscape.</a> Virchows Archiv. 474 (4), 449-461 (2019).</li><li>Abou-El-Enein, M., 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=Scalable+manufacturing+of+CAR+T+cells+for+cancer+immunotherapy.">Scalable manufacturing of CAR T cells for cancer immunotherapy.</a> Blood Cancer Discovery. 2 (5), 408-422 (2021).</li><li>Poltorak, M. P., 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=Expamers:+A+new+technology+to+control+T+cell+activation.">Expamers: A new technology to control T cell activation.</a> Scientific Reports. 10, 17832 (2020).</li><li>Kagoya, Y., 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=Transient+stimulation+expands+superior+antitumor+T+cells+for+adoptive+therapy.">Transient stimulation expands superior antitumor T cells for adoptive therapy.</a> JCI Insight. 2 (2), 89580 (2017).</li><li>Snow, T., Roussey, J., Wegner, C., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+No.+63/326,446.">Application No. 63/326,446.</a> US Patent. , (2022).</li><li>McNaughton, B., 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=Application+No.+16/004,874.">Application No. 16/004,874.</a> US Patent. , (2018).</li><li>Prommersberger, S., Hudecek, M., Nerreter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+CAR+T+cells+produced+by+lentiviral+transduction.">Antibody-based CAR T cells produced by lentiviral transduction.</a> Current Protocols in Immunology. 128 (1), 93 (2020).</li><li>Wijewarnasuriya, D., Bebernitz, C., Lopez, A. V., Rafiq, S., Brentjens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+costimulation+leads+to+dysfunction+of+adoptively+transferred+T+cells.">Excessive costimulation leads to dysfunction of adoptively transferred T cells.</a> Cancer Immunology Research. 8 (6), 732-742 (2020).</li><li>Li, Y., Kurlander, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-CD3+and+anti-CD28-coated+beads+with+soluble+anti-CD3+for+expanding+human+T+cells:+Differing+impact+on+CD8+T+cell+phenotype+and+responsiveness+to+restimulation.">Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation.</a> Journal of Translational Medicine. 8, 104 (2010).</li></ol>

All authors, at the time of submission, are employees of Akadeum Life Sciences, which manufactures and sells microbubble separation products.

The described protocol allows for the isolation of T cells from PBMC samples and the activation of suspended T cells in culture media with microbubbles. This method relies on functionalized microbubbles whose inherent buoyancy offers a unique opportunity to introduce co-stimulatory signals to cells and activate them while they are suspended in a culture medium, thereby reducing the exposure of the expanding cells to prolonged stimulation; such overstimulation can result in the increased expression of T cell exhaustion markers and reduced therapeutic efficacy11. Stimulated T cells that are buoyantly attached to functionalized microbubbles produce untouched daughter cells that drop to the bottom of the cell culture plate for expansion, allowing a period of growth away from the buoyant stimulation factors. It has been detailed in the literature how the prolonged exposure of isolated T cells to stimulation factors-such as magnetic bead-based protocols12-can adversely impact the expansion and therapeutic efficacy 6,7,11.
As this reported protocol relies on the positive selection of CD3+ cells, it is critical to remove the subnatant below the bubble-cell layer carefully but thoroughly during the isolation phase of this protocol. This ensures that only the positively selected T cell population is further stimulated and plated. This is also an important step for determining the number of cells selected from the starting PBMC sample, which is necessary for accurate co-stimulation and plating calculations.
These microbubble protocol development activities for T cell activation and expansion leveraged a wide array of markers during flow cytometry analysis, allowing for the thorough characterization of the isolated and stimulated T cell population to assess critical T cell parameters, including activation, exhaustion, and clonal expansion. When compared to commonly used T cell isolation and stimulation technologies, such as magnetic bead-based protocols, this microbubble protocol aims to minimize the overstimulation of cells without sacrificing the expansion and corresponding effector function abilities of the isolated T cells. Future applications of this microbubble technique will include various protocols for T cell positive selection, co-stimulation, and subsequent microbubble cell cultures to meet a variety of workflow needs for cell therapy research and manufacturing communities.

More than 500 chimeric antigen receptor (CAR)-T cell therapy clinical trials are currently being conducted across the world, and four CAR-T cell therapy products are available on the market1. However, numerous CAR-T cell research and manufacturing needs still exist that must be addressed to improve the efficacy, scalability, and long-term success of these potentially curative therapies2,3,4,5. Adoptive CAR-T cell clinical research and manufacturing begins with T cell isolation from a peripheral blood sample and the subsequent stimulation, transduction, and expansion of the isolated cells. Parameters such as T cell recovery, purity, and activation/exhaustion signals require careful consideration when choosing the cell isolation and stimulation techniques for CAR-T cell research and manufacturing3,4,6. Importantly, improvement in the therapeutic persistence of CAR-T cell therapies by minimizing the biological impediments that result from the current manufacturing processes, such as T cell exhaustion, is needed to enhance the therapeutic efficacy6,7.
As an alternative to traditional cell isolation methods such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), here, buoyancy-activated cell sorting (BACS) with microbubbles for T cell isolation is demonstrated. Microbubble separation uses buoyant, hollow microspheres (microbubbles) to bind the targets and float them to the surface of fluid samples8,9. By functionalizing microbubbles with antibodies (i.e., anti-CD3), the desired T cell populations can be positively selected from peripheral blood samples. Subsequently, the use of a different population of antibody-functionalized microbubbles (i.e., anti-CD28) to co-stimulate and activate positively selected T cells in suspension is demonstrated in this work. Microbubbles offer a simple and highly tunable isolation and activation workflow that generates T cells ready for suspended cell culture and downstream applications such as genetic modification and expansion. Critically, buoyant cell activation with microbubbles promotes restrained cell stimulation to prevent excessive T cell exhaustion7.
For this study, flow cytometry was the primary tool used to analyze the isolation, activation, and transduction success of the functionalized microbubbles, as well as to provide detailed information about the specific subpopulations present during the growth and expansion phases post-transduction. In addition to flow cytometry, brightfield and fluorescence microscopy were used to confirm the cell health, morphology, and transduction success. Based on these results, the microbubble technology and protocol provide a more tunable and gentler alternative to the traditional isolation and activation methods currently in use today; in particular, microbubble-activated cells show notably lower expression of T cell exhaustion markers than that typically observed with industry-standard tools and kits.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985-023</td><td>CAS: 60-24-2</td></tr><tr><td>Biologix Multi-Well Culture Plates 24-well plates</td><td>VWR&amp;nbsp;</td><td>76081-560</td><td></td></tr><tr><td>Biotin anti-human CD28 (28.2) Antibody</td><td>Biolegend</td><td>302904</td><td></td></tr><tr><td>Biotin anti-human CD3 (OKT3) Antibody</td><td>Biolegend</td><td>317320</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermofisher</td><td>35050061</td><td></td></tr><tr><td>Human Recombinant IL2&amp;nbsp;</td><td>BioVision (vwr)</td><td>10006-122</td><td></td></tr><tr><td>Lentiviral Particle rLV.EF1.zsGreen1-9</td><td>Takara Bio</td><td>0038VCT</td><td></td></tr><tr><td>Leukopak</td><td>BioIVT</td><td>HUMANLMX100-0001129</td><td></td></tr><tr><td>Normal Human PBMCs</td><td>BioIVT</td><td>HUMANHLPB-0002562</td><td></td></tr><tr><td>Penicillin/Streptomycin 100X for tissue culture</td><td>VWR</td><td>97063-708</td><td>CAS: 8025-06-7</td></tr><tr><td>Polybrene Infection/Transfection Reagent</td><td>Millipore Sigma</td><td>TR-1003-G</td><td>CAS:28728-55-4</td></tr><tr><td>Pooled Human AB Serum Plasma Derived Heat Inactivated</td><td>Innovative Research</td><td>ISERABHI100mL</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement, HEPES</td><td>Gibco</td><td>72400047</td><td></td></tr><tr><td>Streptavidin Microbubble Kit (includes Akadeum&#039;s separation buffer)</td><td>Akadeum</td><td>11110-000</td><td></td></tr></tbody></table>

flotation-based t cell isolation, activation, and expansion from human peripheral blood mononuclear cell samples using microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

T cells were isolated from purchased PBMCs and plated for activation as described in the protocol. The negative control samples (purchased PBMCs) were not activated. These control samples were included to demonstrate the effect that the microbubble activation process had on the experimental samples as compared to the untouched and unstimulated T cell controls, ensuring that the activation markers observed were the result of the added activation factors and were not inherent to the T cells themselves. As per the experimental outline in Figure 2, the cells were seeded at 2 x 106 cells/mL in T cell medium and were either untouched/unstimulated or co-stimulated with anti-CD3 (clone OKT3) and anti-CD28 (clone 28.2) SAMBs. After 48 h of stimulation in culture, the cells were transduced with the lentiviral particle encoding for zsGreen. At 4 days and 6 days post-transduction, the cells were imaged, harvested, and surface-stained using AF700-CD3, PE/Dazzle-CD4, PE/Cy7-CD8, BV510-CD25, PE-PD-1 (or PE-CD69 depending on if the exhaustion or activation panels were used, respectively), and 7-AAD. The zsGreen transgene was detectable in the FITC channel. The flow cytometry gating approach is detailed in Figure 1. Increases in viable T cell numbers and transgene-positive T cells were observed between the control sample and the cells that received microbubble co-stimulation (Figure 3). Increased effector cell populations were also observed in the microbubble samples (Figure 4). T cells expressing increased activation and exhaustion markers were observed among the cell samples that received microbubble co-stimulation (Figure 5 and Figure 6). Cell expansion was observed between the day 4 and day 6 time points of the co-stimulated samples, indicating that the cells were active, proliferative, and passing the transgene as they expanded.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64573/64573fig01.jpg" /> 
Figure 1: Example gating scheme-untouched/negative control sample. Starting from the singlets, the population cells were gated next using SSC-A/FSC-A. The total CD3+ cells were gated out, followed by viable CD3+ gating using 7-AAD to determine the viability of the population. All the subsequent populations and calculations were determined from the viable 7-AAD(&#8722;)/CD3+(+)&#160;population, as shown using the arrows indicating the subpopulation gates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64573/64573fig02v2.jpg" /> 
Figure 2: Experimental timeline overview. The days of the protocol are noted above, and the corresponding days post-transduction (D0-D6) are used in the figures below. The cells were plated immediately following selection and activation. The control wells were generated using a microbubble negative selection protocol. The control T cells did not receive co-stimulation agents and did not undergo transduction, although they did receive IL-2 to ensure the cells were kept healthy enough to maintain reasonable viability throughout the experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64573/64573fig03.jpg" /> 
Figure 3: Assessment of viable and successfully transduced cells post-transduction. (A) Viable CD3+ cells 4 days and 6 days post-transduction. Viability was determined through flow cytometry analysis, in which the population was quantified by gating on 7-AAD(&#8722;)/CD3+(+) cells. (B) The number of cells successfully transduced with rLV.EF1.zsGreen was determined through flow cytometry, in which the viable 7-AAD(&#8722;)/CD3+(+) population was further gated into zsGreen(+) cells. All the conditions were performed in triplicate (n = 3). The data represent mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64573/64573fig04.jpg" /> 
Figure 4: Viable CD4+ and CD8+ T cells. The CD4+ and CD8+ T cell subpopulations were quantified by gating on the viable CD3+ population (CD3+ [+]/7-AAD[&#8722;]) and measuring the expression of (A)&#160;CD4+ and (B)&#160;CD8+. All the conditions were performed in triplicate (n = 3). The data represent mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64573/64573fig05.jpg" /> 
Figure 5: Viable activated T cells. The viable CD3+ population was also analyzed for specific activation markers as denoted in the figures above. (A) CD69 is an early marker of activation; (B) CD25 is a middle-to-late activation marker. The percentages above the error bars represent the percentage of viable CD3+ cells expressing the respective marker. All the conditions were performed in triplicate (n = 3). The data represent mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64573/64573fig06.jpg" /> 
Figure 6: Exhausted T cells. The viable CD3+ population was also analyzed for exhaustion (PD-1) markers. (A) The total number of 7-AAD(&#8722;)/CD3+(+)/PD-1+(+) cells at day 4 and day 6 post-transduction. (B)&#160;The percentage of PD-1+ cells. On day 4 and day 6, the activated/transduced sample population had ~25% viable CD3+/PD-1+ cells, whereas the control sample population had ~2% viable CD3+/PD-1+ cells. Of note, the starting/isolated material had &#60;~15% viable CD3+/PD-1+ cells (post-isolation/pre-culture data not shown). All conditions were performed in triplicate (n = 3). The data represent mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64573/64573fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles at JoVE.com

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

flotation-based-t-cell-isolation-activation-expansion-from-human

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.4K Views.  Akadeum Life Sciences. Het microbubbelactiverings- en uitbreidingsprotocol is belangrijk omdat het tegemoetkomt aan een grote onvervulde behoefte in celtherapieonderzoek en -productie, waardoor de persistentie en werkzaamheid van T-celtherapieën wordt verbeterd. De op drijfvermogen gebaseerde positieve selectie-, activerings- en expansietechniek van Akadeum beperkt overstimulatie en daaropvolgende T-celuitputting tijdens activerings- en uitbreidingsworkflows. Om te beginnen incubeer 3 keer 10 tot de 8e commercieel...

Video: Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Cancer Research

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Bioengineering

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Biology

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

Samenvatting

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