Name: flotation-based t cell isolation, activation, and expansion from human peripheral blood mononuclear cell samples using microbubbles
Uploaded: 2022-12-23T14:20:00
Description: 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

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 strept...

<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>

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 ma...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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&#39;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 experimen...

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 ...

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (Video) | JoVE

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...