We would like to thank the members of the Liu laboratory (Dr. Hsiang-chi Tseng, Dr. Xuening Wang, and Dr. Chih-Hsiung Chen) for their comments on the manuscripts. We would like to thank Dr. Gianpietro Dotti for the SFG vectors and Dr. Eric Long for the 721.221 cells. This work was supported in part from HL125018 (D. Liu), AI124769 (D. Liu), AI129594 (D. Liu), AI130197 (D. Liu), and Rutgers-Health Advance Funding (NIH REACH program), U01HL150852 (R. Panettieri, S. Libutti, and R. Pasqualini), S10OD025182 (D. Liu), and Rutgers University-New Jersey Medical School Startup funding for D. Liu Laboratory.

Human tissues and blood-related work in this protocol follows the guidelines of the Rutgers University Institutional Review Board (IRB)
1. NK cell expansion from liver tissues (Day 0), as shown in&#160;Figure 1.
NOTE: Initial cell number and viability are strongly correlated with the time since organ removal and the initial tissue sample amount. However, if tissues are placed in 30 mL of Hank&#39;s Balanced Salt Solution (HBSS) and kept on ice or in the fridge at 4 &#176;C overnight, NK cells can still be expanded at high purity and viability up to 24 h later.
<ol>
	<li>Identify viable tissue areas to obtain lymphocytes from tissues and sections using sterile surgical equipment.</li>
	<li>Place tissues in 30 mL of HBSS (w/o calcium or magnesium) and keep on ice until ready to prepare for isolation.</li>
	<li>Mince the tissue into &#60;0.5 cm cubes using sterile razor blades or scissors and forceps inside a biosafety cabinet.</li>
	<li>Prepare a 1x collagenase IV solution (1 mg/mL) by diluting a 10x stock in HBSS (10x Collagenase IV: 10 mg/mL or ~200 U/mL).</li>
	<li>Place the minced tissue pieces in the tissue dissociator tubes. Fill the tubes with no more than 4 g of tissue and immerse the tissue pieces in ~10 mL of 1x collagenase IV. 
	NOTE: Using DNase I is not recommended as it may slightly decrease NK viability and yield. Please refer to the Table of Materials for the specific tissue dissociator tubes used.</li>
	<li>Place the tissue dissociator tubes into a tissue dissociator and blend at 37 &#176;C to mince the tissue thoroughly. 
	NOTE: For liver tissue, this may take over 30 min. For more friable tissue, around 15 min may be sufficient. Please refer to the Table of Materials for specific tissue dissociator tubes and the tissue dissociator used.</li>
	<li>Remove the tissue dissociator tubes and triturate through 40 &#181;m nylon cell strainer using the backend of a 5 mL syringe. Collect the eluent and discard the large undigested fragments.</li>
	<li>Spin down the eluent at 400 x g for 5 min at room temperature. Aspirate the supernatant.</li>
	<li>Resuspend the cell pellets in 30% polyvinylpyrrolidone (PVP)-coated silica to remove fat cells that will otherwise contaminate the final lymphocyte fraction.
	<ol>
		<li>To prepare 1x PVP-coated silica, use 9:1 dilution of PVP-coated silica in 10x PBS. 
		NOTE: Refer to the Table of Materials for the specific PVP-coated silica used.</li>
		<li>To prepare 30% PVP-coated silica, dilute 1x PVP-coated silica with PBS/HBSS.</li>
	</ol>
	</li>
	<li>Spin down the cell pellet at 400 x g for 5 min at room temperature. Aspirate the supernatant.</li>
	<li>Resuspend the cell pellet in 9 mL of R-10 media. 
	NOTE: Refer to the Table of Materials for the composition of R-10 media</li>
	<li>Carefully layer the cell suspension over 4 mL of Ficoll or lymphocyte separation media to separate lymphocytes from red blood cells and polymorphonuclear cells.</li>
	<li>Separate the layers by centrifuging at 400 x g for 23 min at room temperature with the acceleration and brakes off or at the lowest setting. Carefully decant the upper-medium layer and harvest the interphase containing tissue-infiltrating lymphocytes.</li>
	<li>Rinse the cells with 10 mL of media and proceed to cell counting, flow cytometry, aliquoting and freezing of cells, or primary NK expansion protocol.</li>
</ol>
2. Primary NK cell expansion from PBMCs (or CB or organ tissues) (Day 0), as shown in Figure 2.
<ol>
	<li>Thaw the frozen PBMC and the frozen, irradiated feeder cells in a 37 &#176;C water bath.</li>
	<li>Wash the PBMC and 100 Gamma-irradiated (Gy-irradiated) 221-mIL-21 cells by centrifugation at 400 x g for 5 min with 10 mL of R-10 media separately.</li>
	<li>Save 1 x 106 cells of PBMC for flow cytometry. 
	NOTE: The initial NK cell purity is an important factor in calculating NK cell expansion rate.</li>
	<li>Resuspend the cells in 1 mL of R-10 media. Count the cells using Trypan Blue.</li>
	<li>Mix 5x 106 cells of PBMC with 10 x 106 cells of 100 Gy-irradiated 221-mIL-21 cells in a special 6-well plate (see Table of Materials).</li>
	<li>Add 30 mL of R-10 media supplemented with human IL-2 (200 U/mL) and human IL-15 (5 ng/mL) (see Table of Materials).</li>
	<li>Incubate the special 6-well plate at 37 &#176;C with 5% CO2.</li>
	<li>Replace the media with R-10 media supplemented with human IL-2 (200 U/mL) and human IL-15 (5 ng/mL) to maintain NK cells every 3-4 days. 
	NOTE: Maintain less than 20 x 106 cells per well for further expansion at each media change. For the best viability, ensure that the total cell number in each well does not exceed 100 x 106 cells.</li>
	<li>Record the total cell number, viability and perform flow cytometry every 3-4 days to calculate the NK cell expansion rate.</li>
</ol>
3. Attachment of 293T cells (Day 2), as shown in Figure 2.
<ol>
	<li>Split 1.8 x 106 293T cells in 11 mL of D-10 media per treated 100 mm plate. 
	NOTE: Refer to the Table of Materials for the composition of D-10 media</li>
	<li>Incubate 293T cells at 37 &#176;C with 5% CO2.</li>
</ol>
4. Retrovirus transfection (Day 3)
<ol>
	<li>In a 1.7 mL tube, mix 470 &#181;L of reduced serum media with 30 &#181;L of transfection reagent. 
	NOTE: Refer to the Table of Materials for the specific reduced serum media and transfection reagent used.</li>
	<li>In a separate 1.7 mL tube, add 2.5 &#181;g of pRDF plasmid, 3.75 &#181;g of Pegpam3 plasmid, and 2.5 &#181;g of CAR construct in SFG vector into the reduced serum media so that the final volume is 500 &#181;L.</li>
	<li>Mix the solutions in steps 4.1 and 4.2 dropwise.</li>
	<li>Incubate the tube at room temperature for 15 min.</li>
	<li>Add 1 mL of the mixture from step 4.4 to 293T cell plate on Day 1 in a dropwise manner.</li>
	<li>Incubate the plate(s) at 37 &#176;C with 5% CO2 for 48-72 h.</li>
</ol>
5. Retronectin plate-coating (Day 3)
<ol>
	<li>Dilute retronectin protein with Phosphate Buffered Saline (PBS) to a final concentration of 50-100 &#181;g/mL.</li>
	<li>Add 500 &#181;L of the diluted retronectin into each well of an untreated 24-well plate (5 wells per CAR construct). Seal the plate using parafilm and incubate the plate at 4 &#176;C overnight.</li>
</ol>
6. Transduction (Day 4)
<ol>
	<li>Centrifuge the retronectin plate at 2103 x g for 30 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Block each well of the 24-well plate with 1 mL of R-10 medium.</li>
	<li>Incubate the plate at 37 &#176;C with 5% CO2 for 1 h.</li>
	<li>Pre-warm the centrifuge to 32 &#176;C while the retronectin plate is being blocked.</li>
	<li>Collect the retrovirus supernatant by filtering the transfected 293T cells using a 0.45 &#181;m filter.</li>
	<li>Aliquot 2 mL of the filtered retrovirus supernatant into each well.</li>
	<li>Centrifuge the 24-well plate at 2103 x g for 2 h at 32 &#176;C.</li>
	<li>During plate centrifugation, collect the expanded PBNK cells from Day 0 and count the cells using Trypan Blue. 
	NOTE: Continue expanding PBNK cells by adding R-10 media supplemented with IL-2 (200 U/mL) and IL-15 (5 ng/mL).</li>
	<li>Dilute the expanded PBNK cells with R-10 media supplemented with IL-2 (200 U/mL) and IL-15 (5 ng/mL) to 2.5 x 105-5 x 105 cells/mL (0.5 x 106-1 x 106 cells per well). 
	NOTE: Record the total cell number, viability, and save 5 x 105 expanded PBNK cells for flow cytometry as these values are important in determining the NK cell expansion rate.</li>
	<li>After centrifugation, partially aspirate the retrovirus supernatant from each well. 
	NOTE: Do not completely aspirate, i.e., leave approximately 100 &#181;L of retrovirus supernatant per well, as this will decrease the transduction efficiency.</li>
	<li>Aliquot 2 mL of the diluted expanded PBNK cells from step 6.8 to each well.</li>
	<li>Centrifuge the plate at 600 x g for 10 min at 32 &#176;C. Incubate the plate at 37 &#176;C with 5% CO2 for 48-72 h. 
	&#8203;NOTE: Do not parafilm the plate.</li>
</ol>
7. CAR-NK cells collection (Day 6 or 7), as shown in Figure 2.
<ol>
	<li>Gently collect the cells from the 24-well plate and transfer the cells to a 50 mL centrifuge tube 
	NOTE: Try not to generate bubbles, as this will result in a decrease in cell viability.</li>
	<li>Centrifuge the tube at 400 x g for 5 min.</li>
	<li>Resuspend the pellet with 1 mL of R-10 media and count the cells using Trypan Blue. 
	NOTE: Save 5 x 105 cells for flow cytometry to determine the transduction efficiency.</li>
	<li>Transfer the resuspended cells to a special 6-well plate containing 30 mL of R-10 media supplemented with IL-2 (200 U/mL) and IL-15 (5 ng/mL).</li>
	<li>Incubate the special 6-well plate at 37 &#176;C with 5% CO2.</li>
	<li>Replace R-10 media supplemented with IL-2 (200 U/mL) and IL-15 (5 ng/mL) to maintain NK cells every 3 - 4 days. 
	NOTE: Maintain less than 20 x 106 cells per well for further expansion at each change. For the best viability, ensure that the total cell number in each well does not exceed 100 x 106 cells.</li>
	<li>Record the total cell number, viability, and perform flow cytometry every 3-4 days to calculate the NK cell expansion rate.</li>
	<li>Use the cells for appropriate in vitro or in vivo assays. 
	NOTE: The ex vivo expanded PBNK, and CAR-NK cells can be cultured in a 37 &#176;C incubator for approximately 4 weeks.</li>
	<li>Examine the NK cell number and purity at day 7, day 11, day 14, day 18, and day 21 by flow cytometry.</li>
</ol>

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O., 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=Membrane+bound+IL-21+based+NK+cell+feeder+cells+drive+robust+expansion+and+metabolic+activation+of+NK+cells.">Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells.</a> Scientific Reports. 9 (1), 14916 (2019).</li><li>Denman, C. J., 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=Membrane-bound+IL-21+promotes+sustained+ex+vivo+proliferation+of+human+natural+killer+cells.">Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.</a> PLoS One. 7 (1), 30264 (2012).</li><li>Yang, 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=Superior+expansion+and+cytotoxicity+of+human+primary+NK+and+CAR-NK+cells+from+various+sources+via+enriched+metabolic+pathways.">Superior expansion and cytotoxicity of human primary NK and CAR-NK cells from various sources via enriched metabolic pathways.</a> Molecular Therapy. Methods &amp; Clinical Development. 18, 428-445 (2020).</li><li>Liu, S., 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=NK+cell-based+cancer+immunotherapy:+from+basic+biology+to+clinical+development.">NK cell-based cancer immunotherapy: from basic biology to clinical development.</a> Journal of Hematology &amp; Oncology. 14 (1), 7 (2021).</li><li>Gong, J. H., Maki, G., Klingemann, H. G. <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>Tam, Y. K., 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=Characterization+of+genetically+altered,+interleukin+2-independent+natural+killer+cell+lines+suitable+for+adoptive+cellular+immunotherapy.">Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy.</a> Human Gene Therapy. 10 (8), 1359-1373 (1999).</li><li>Robertson, M. J., 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=Characterization+of+a+cell+line,+NKL,+derived+from+an+aggressive+human+natural+killer+cell+leukemia.">Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia.</a> Experimental Hematology. 24 (3), 406-415 (1996).</li><li>Hu, Y., Tian, Z. G., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+(CAR)-transduced+natural+killer+cells+in+tumor+immunotherapy.">Chimeric antigen receptor (CAR)-transduced natural killer cells in tumor immunotherapy.</a> Acta Pharmacologica Sinica. 39 (2), 167-176 (2018).</li><li>Tseng, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+anti-CD147+chimeric+antigen+receptors+targeting+hepatocellular+carcinoma.">Efficacy of anti-CD147 chimeric antigen receptors targeting hepatocellular carcinoma.</a> Nature Communications. 11 (1), 4810 (2020).</li><li>Easom, N. J. 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The authors declare no competing interests.

Most of the current CAR-NK products in clinical trials utilize NK cell lines17, such as NK-92, a cell line isolated from a non-Hodgkin&#39;s lymphoma patient18, NK-92MI, IL-2 independent NK-92 cell line19, and NKL, isolated from a large granular lymphocyte patient20, as these cell lines are easily proliferative for &#39;off-the-shelf&#39; products. However, these cell lines, e.g., NK-92 cells, have marginal clinical efficacies and in vivo expansion, as they require irradiation prior to infusion, thus limiting their proliferation and cytotoxicity in vivo21. Given these reasons, various strategies are currently being explored to expand primary NK cells from several sources, including peripheral blood, CB, bone marrow (BM), human embryonic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and tumor tissues21,22,23. For instance,&#160;NK cells can be expanded ex vivo using interleukins including IL-15, IL-18, and IL-21. Lymphoblastoid cell lines such as K562 cells or Epstein-Barr Virus-transformed lymphoblastoid cell lines such as 721.221 cells, are also used for NK cell expansion16. However, the aforementioned strategies often generate insufficient number of NK cells for an adoptive transfer of CAR-NK immunotherapy22,24. To help solve the problem, the study here shows a protocol for an ex vivo NK cell expansion using a genetically modified EBV-transformed cell line, 221-mIL-21 feeder cells.
The expansion methodology using 221-mIL-21 feeder cells shown in this protocol is optimized to expand NK cells with an expansion rate of at least 10 to 100-fold higher than other leukemia cell lines, including HL-60 and OCl-AML3 expressing membrane IL-21, K562, and K652-mIL21 expressing OX40 ligand22,24,25. The CAR expression is also evaluated for approximately 2 weeks ex vivo. More significantly, the 221-mIL-21 feeder cell expansion strategy can be applied to expand NK cells from various sources, including PBMCs, CB, and solid organs such as the liver, without an initial NK enrichment step. Although the 221-mIL-21 feeder system is not as donor-dependent as the aforementioned feeder cell lines, it is not entirely independent of donors. On average, the 221-mIL-21 expansion system can achieve 90% of NK cell purity with a high NK cell number, with approximately &#60;5% of T cell contamination on day 14 post-expansion. Therefore, to eliminate the possibilities of T cell contamination, it is necessary to isolate NK cells from obtained samples prior to the ex vivo expansion or use a CD3+ selection system to eliminate T cells after an ex vivo expansion.
One of the criticisms in using an NK cell expansion system is that the feeder cells may not have been fully eradicated after the expansion or prior to a transfusion, which may possess significant regulatory concerns; therefore, complete eradication of feeder cells before a transfusion is crucial. However, recent CAR-NK clinical trials in which K562-mIL21-4-1BBL feeder cells were used for the ex vivo CBNK cell expansion24,25 showed no concerning complications. Furthermore, our preliminary data showed a gradual decrease of the irradiated 221-mIL-21 population as the expansion progressed (data not shown). However, more extensive studies are required for this expansion method to be implemented in a clinical setting. Collectively, the 221-mIL-21 expansion system helps solve the challenge of expanding primary CAR-NK cells, and therefore will significantly contribute to the broader use of CAR-NK cell-based immunotherapy in the near future.

Natural killer (NK) cells are an important subset of lymphocytes that express CD56 and lack expression of the T cell marker, CD31,2. Conventional NK cells are classified as innate immune cells responsible for immunosurveillance of virally infected cells and cancerous cells. Unlike T cells, NK cells recognize infected or malignant cells using CD16 or other activating receptors and do not require the formation of a complex between antigen peptides and major histocompatibility complex (MHC) class I molecules3,4. Recent clinical investigations using chimeric antigen receptor (CAR)-NK cells to treat relapsed or refractory CD19-positive cancers (non-Hodgkin&#39;s lymphoma or chronic lymphocytic leukemia [CLL])showed the outstanding safety advantages of CAR-NK cells5. For instance, CAR-NK cell infusion is associated with minimal or negligible graft versus host disease (GVHD), neurotoxicity, cardiotoxicity, and cytokine release syndrome (CRS)6,7,8,9,10. However, conventional methods to expand human NK cells showed exhaustive phenotypes with strong fratricidal killing and telomere shortage, which presents a major challenge in obtaining an adequate number of functional NK cells for adoptive immunotherapy11.
To overcome these challenges, a method was developed to expand primary NK cells directly from unfractionated peripheral blood mononuclear cells (PBMCs) or cord blood (CB) using an irradiated and genetically engineered 721.221 (hereinafter, 221) cell line, a human B-lymphoblastoid cell line with low expression of MHC class I molecules3. Previous studies showed the importance of IL-21 in NK cell expansion; therefore, a genetically engineered membrane-bound IL-21 expressing a version of the 721.221 cell line (starting now, 221-mIL-21) was developed11,12,13,14,15. The results showed that 221-mIL-21 feeder-cell-expanded primary NK cells were expanded to an average of &#62;40,000-fold with persistent high NK cell purity for approximately 2-3 weeks. Additional information regarding the application of this protocol can be found in Yang et al.16.
This protocol aims to demonstrate the step-by-step procedure of the novel expansion of PBNK, CBNK, tissue-derived NK, and CAR-NK cells ex vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm surface treated sterile tissue culture dishes</td>
			<td>VWR</td>
			<td>10062-880</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>6-well G-REX plate</td>
			<td>Wilson Wolf</td>
			<td>80240M</td>
			<td>For NK cell expansion</td>
		</tr>
		<tr>
			<td>AF647-conjugated AffiniPure F(ab&#39;)2 fragment goat anti-human IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-606-088</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>CAR construct in SFG vector</td>
			<td>Generated in Dr. Dongfang Liu&#39;s lab</td>
			<td></td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C4-22-1G</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>Cryopreserve media 
			Ingredient: Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Cryopreserve media 
			Ingredient: Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2050</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>D-10 media 
			Ingredient: DMEM</td>
			<td>VWR</td>
			<td>45000-304</td>
			<td></td>
		</tr>
		<tr>
			<td>D-10 media 
			Ingredient: Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>D-10 media 
			Ingredient: Penicillin Streptomycin</td>
			<td>VWR</td>
			<td>45000-652</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>FastFlow &#38; Low Binding Millipore Express PES Membrane</td>
			<td>Millex</td>
			<td>SLHPR33RB</td>
			<td>For transduction</td>
		</tr>
		<tr>
			<td>Genejuice transfection reagent</td>
			<td>VWR</td>
			<td>80611-356</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>gentleMACS C-tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>gentleMACS Octo</td>
			<td>Miltenyi Biotec</td>
			<td>Quote</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS - w/o calcium or magnesium)</td>
			<td>ThermoFisher</td>
			<td>14170120</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>Human IL-15</td>
			<td>Peprotech</td>
			<td>200-15</td>
			<td>For NK cell expansion</td>
		</tr>
		<tr>
			<td>Human IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td>For NK cell expansion</td>
		</tr>
		<tr>
			<td>Irradiated 221-mIL21 feeder cells</td>
			<td>Generated in Dr. Dongfang Liu&#39;s lab</td>
			<td></td>
			<td>Frozen in cryopreserve media</td>
		</tr>
		<tr>
			<td>Lymphocyte Separation Media</td>
			<td>Corning</td>
			<td>25-072-CV</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>OPTI-MEM</td>
			<td>ThermoFisher</td>
			<td>31895</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>PE anti-human CD3 clone HIT3a</td>
			<td>Biolegend</td>
			<td>300308</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-human CD56 (NCAM) clone 5.1H11</td>
			<td>BioLegend</td>
			<td>362509</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>Pegpam3 plasmid</td>
			<td>Generated in Dr. Dongfang Liu&#39;s lab</td>
			<td></td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17089101</td>
			<td>For TILs isolation</td>
		</tr>
		<tr>
			<td>Peripheral blood mononuclear cells (PBMCs)</td>
			<td>New York Blood Center</td>
			<td></td>
			<td>Isolated from plasma of healthy donors, frozen in cryopreserve media</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>For transduction</td>
		</tr>
		<tr>
			<td>pRDF plasmid</td>
			<td>Generated in Dr. Dongfang Liu&#39;s lab</td>
			<td></td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>R-10 media 
			Ingredients: RPMI 1640</td>
			<td>VWR</td>
			<td>45000-404</td>
			<td></td>
		</tr>
		<tr>
			<td>R-10 media 
			Ingredient: L-glutamine</td>
			<td>VWR</td>
			<td>45000-304</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>R-10 media 
			Ingredient: Penicillin Streptomycin</td>
			<td>VWR</td>
			<td>45000-652</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>R-10 media 
			Ingredient: Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Generated in Dr. Dongfang Liu&#39;s lab</td>
			<td>home-made</td>
			<td>For transduction</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25-900-Cl</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Untreated 24-well plate</td>
			<td>Fisher Scientific</td>
			<td>13-690-071</td>
			<td>For transduction</td>
		</tr>
	</tbody>
</table>

natural killer (nk) and car-nk cell expansion method using membrane bound-il-21-modified b cell line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

A schematic workflow of tissue-infiltrating NK cell isolation and PBNK cell expansion using the 221-mIL-21 feeder cell methodology is shown in Figure 1 and Figure 2. Expanded PBNK cells were collected every 3 or 4 days for flow cytometry to determine the NK cell purity by staining cells with anti-human CD56 and anti-human CD3. The experiment was repeated using two different donors to show the reproducibility of the expansion system (Figure 3). PBNK cells expanded by 221-mIL-21 were shown to expand nearly 5 &#215; 104 folds (Figure 3A). Furthermore, the NK cell purity was highly maintained, around 85% throughout the 21-day expansion (Figure 3B). Using the 221-mIL-21 feeder cell expansion system, the NK cell purity consistently ranged between 85%-95%, independent of the donors (data not shown). To demonstrate the robustness of the 221-mIL-21 expansion system, PBMCs were stained for anti-CD56 and anti-CD3 prior to the expansion, which showed a cell purity of 7.09% for NK cells and a high percentage of T cells (Figure 4A). PBMCs were cocultured with 221-mIL-21 to expand NK cells; the NK purity was checked prior to CAR-NK transduction on Day 4 (Figure 4A). CAR-NK cells were collected and stained for anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab&#39;)2, which showed a high NK cell population (86.9% on Day 7) and a high CAR transduction efficiency of approximately 70% (Figure 4). Higher transduction efficiencies (up to 95%) were also observed using the retrovirus packaging system. Altogether, these data show that the 221-mIL-21 feeder cells could successfully expand NK cells and preserve the NK cell purity ex vivo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62336/62336fig01.jpg" /> 
Figure 1: Diagram of NK cell expansion from solid human organ samples. Briefly, the obtained human liver samples are minced into small cubes for mechanical digestion. Dissociated cells are then isolated using PVP-coated silica and Lymphocyte Separation Media. Further, the NK cells are expanded using the expansion protocol described in Figure 2. <a href="https://www.jove.com/files/ftp_upload/62336/62336fig01large.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/62336/62336fig02.jpg" /> 
Figure 2: Schematic workflow of CAR-NK cell generation from PBMCs. Briefly, 221-mIL21 feeder cells were irradiated at 100 Gy prior to coculturing with PBMCs supplemented with IL-2 and IL-15 on Day 0. In parallel, 293T cells were transfected with the retrovirus packaging system to produce CAR retrovirus that was then transduced into the expanded PBNK cells in the presence of IL-2 and IL-15. Primary CAR-NK cells were harvested on Day 7 and continued expansion for 21 days. This figure has been modified from Yang et al.16. <a href="https://www.jove.com/files/ftp_upload/62336/62336fig02large.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/62336/62336fig03.jpg" /> 
Figure 3: Dynamic time-lapsed expansion of PBNK cells. (A) Fold expansion of PBNK during a 22- day time course. Cells were stained with anti-CD56 and anti-CD3 at indicated days for flow cytometry. The total number of NK cells was determined by multiplying NK cell purity to the total number of cells. Expansion rate was generated as follows: (Number of NK cells)Tn/(Number of NK cells)T0, where Number of NK cells = (percentage of NK cell purity)&#160;&#215; (total number of cells), T0 is the number of NK cells at time day 0, and Tn is the number of NK cells at time day n. (B) NK cell purity during a 22-day time course. The NK cell expansion was repeated two times with two different donors. Error bars represent &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/62336/62336fig03large.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/62336/62336fig04.jpg" /> 
Figure 4: Representative flow cytometric analysis of CAR-NK cells. (A) Representative dot plots showing the dynamic time-lapse of NK cell purity of the CAR-NK cells during an 18-day course. Flow cytometry analysis was assessed by staining the cells with anti-CD56 and anti-CD3 at indicated timepoints. Day 0 indicates pre-expansion of PBNK. Day 4 indicates post-expansion of PBNK and pre-transduction of CAR-NK cells. Day 7 indicates the post-transduction of CAR-NK cells. (B) Representative dot plots showing the transduction efficiency of CAR-NK cells using the retrovirus packaging system. Cells were stained with anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab&#39;)2 for flow cytometry. (C) Representative dot plots showing the CAR expression in various subsets, including CD56+CD3-, CD56-CD3+, CD56+CD3+, and CD56-CD3- on Day 18. Cells were stained with anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab&#39;)2 (indicating CAR expression) for flow cytometry. <a href="https://www.jove.com/files/ftp_upload/62336/62336fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line at JoVE.com

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

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Cancer Research

5.3K Views.  Rutgers-New Jersey Medical School. Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Video: Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...