This study was supported by China Medical University Hospital (DMR-Cell-1809).

The clinical protocol was performed and approved in accordance with the guidelines of the Institutional Review Board of the China Medical University and Hospital Research Ethics Committee. Peripheral blood specimens were harvested from healthy volunteers with their informed consent.
1. Preparation of materials
<ol>
	<li>Store reagents, antibodies, and chemicals as shown in the Material Safety Data Sheet (MSDS). Dissolve the drugs or cytokines in solvents as stock solutions and then aliquot f...

<ol>
	<li>Cappuzzello, E., 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=Cytokines+for+the+induction+of+antitumor+effectors:+The+paradigm+of+Cytokine-Induced+Killer+(CIK)+cells.">Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells.</a> Cytokine &amp; Growth Factor Reviews. 36, 99-105 (2017).</li><li>Schmidt-Wolf, R. 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=Propagation+of+large+numbers+of+T+cells+with+natural+killer+cell+markers.">Propagation of large numbers of T cells with natural killer cell markers.</a> British Journal of Haematology. 87 (3), 453-458 (1994).</li><li>Grainger, 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=Wnt+Signaling+in+Hematological+Malignancies.">Wnt Signaling in Hematological Malignancies.</a> Progress in Molecular Biology and Translational Science. 153, 321-341 (2018).</li><li>Dai, 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=Implication+of+combined+PD-L1/PD-1+blockade+with+cytokine-induced+killer+cells+as+a+synergistic+immunotherapy+for+gastrointestinal+cancer.">Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer.</a> Oncotarget. 7 (9), 10332-10344 (2016).</li><li>Schmidt-Wolf, I. G., 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=Use+of+a+SCID+mouse/human+lymphoma+model+to+evaluate+cytokine-induced+killer+cells+with+potent+antitumor+cell+activity.">Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity.</a> TheJournal of Experimental Medicine. 174 (1), 139-149 (1991).</li><li>Introna, 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=Rapid+and+massive+expansion+of+cord+blood-derived+cytokine-induced+killer+cells:+an+innovative+proposal+for+the+treatment+of+leukemia+relapse+after+cord+blood+transplantation.">Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.</a> Bone Marrow Transplantation. 38 (9), 621-627 (2006).</li><li>Schmeel, L. 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=Cytokine-induced+killer+(CIK)+cells+in+cancer+immunotherapy:+report+of+the+international+registry+on+CIK+cells+(IRCC).">Cytokine-induced killer (CIK) cells in cancer immunotherapy: report of the international registry on CIK cells (IRCC).</a> Journal of Cancer Research and Clinical Oncology. 141 (5), 839-849 (2015).</li><li>Rutella, 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=Adoptive+immunotherapy+with+cytokine-induced+killer+cells+generated+with+a+new+good+manufacturing+practice-grade+protocol.">Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol.</a> Cytotherapy. 14 (7), 841-850 (2012).</li><li>Nausch, N., 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=NKG2D+ligands+in+tumor+immunity.">NKG2D ligands in tumor immunity.</a> Oncogene. 27 (45), 5944-5958 (2008).</li><li>Gammaitoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+activity+of+cytokine-induced+killer+cells+against+autologous+metastatic+melanoma+including+cells+with+stemness+features.">Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features.</a> Clinical Cancer Research. 19 (16), 4347-4358 (2013).</li><li>Rettinger, E., 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=The+cytotoxic+potential+of+interleukin-15-stimulated+cytokine-induced+killer+cells+against+leukemia+cells.">The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells.</a> Cytotherapy. 14 (1), 91-103 (2012).</li><li>Narayan, R., 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=Donor-derived+cytokine-induced+killer+cell+infusion+as+consolidation+after+nonmyeloablative+allogeneic+transplantation+for+myeloid+neoplasms.">Donor-derived cytokine-induced killer cell infusion as consolidation after nonmyeloablative allogeneic transplantation for myeloid neoplasms.</a> Biology of Blood and Marrow Transplantation. 19, 1083 (2019).</li><li>Castiglia, 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=Cytokines+induced+killer+cells+produced+in+good+manufacturing+practices+conditions:+identification+of+the+most+advantageous+and+safest+expansion+method+in+terms+of+viability,+cellular+growth+and+identity.">Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.</a> Journal of Translational Medicine. 16 (1), 237 (2018).</li><li>Bonanno, G., 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=Thymoglobulin,+interferon-&#947;+and+interleukin-2+efficiently+expand+cytokine-induced+killer+(CIK)+cells+in+clinical-grade+cultures.">Thymoglobulin, interferon-&#947; and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures.</a> Journal of Translational Medicine. 8, 129 (2010).</li><li>Iudicone, 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=Interleukin-15+enhances+cytokine+induced+killer+(CIK)+cytotoxic+potential+against+epithelial+cancer+cell+lines+via+an+innate+pathway.">Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway.</a> Human Immunology. 77 (12), 1239-1247 (2016).</li><li>Liu, 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=Phenotypic+characterization+and+anticancer+capacity+of+CD8++cytokine-induced+killer+cells+after+antigen-induced+expansion.">Phenotypic characterization and anticancer capacity of CD8+ cytokine-induced killer cells after antigen-induced expansion.</a> PLoS One. 12 (4), 0175704 (2017).</li><li>Chen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-induced+killer+cells+as+a+feasible+adoptive+immunotherapy+for+the+treatment+of+lung+cancer.">Cytokine-induced killer cells as a feasible adoptive immunotherapy for the treatment of lung cancer.</a> Cell Death &amp; Disease. 9 (3), 366 (2018).</li><li>Tario, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell+proliferation+by+dye+dilution:+considerations+for+probe+selection.">Monitoring cell proliferation by dye dilution: considerations for probe selection.</a> Methods in Molecular Biology. 1678, 249-299 (2018).</li><li>Last'ovicka, 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=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular Immunology. 256 (1-2), 79-85 (2009).</li><li>Yoshida, T., 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+natural+killer+cells+in+tamarins:+a+technical+basis+for+studies+of+innate+immunity.">Characterization of natural killer cells in tamarins: a technical basis for studies of innate immunity.</a> Frontiers in Microbiology. 1, 1-9 (2010).</li></ol>

The described method is a fast, convenient, and reliable protocol for the isolation and expansion of cytotoxic cytokine-induced killer (CIK) T cells from whole blood samples of healthy donors. It also shows the cytotoxic effect of CIK against leukemia (K562) and ovarian cancer cells (OC-3) using a flow cytometry setup and tracking (CS &#38; T) system. CIK cells can be induced and expanded in good manufactory practices (GMP) conditions by using GMP-grade cytokines and serum-free medium for further clinical infusion<sup cl...

Cytotoxic T lymphocytes are a specific immune effector cell population that mediates immune responses against cancer. Several effector cell populations including lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, &#947;&#948; T cells, and cytokine-induced killer (CIK) cells have been developed for adoptive T cell therapy (ACT) purposes1. There is a growing interest in CIK cells, because they represent a mixture of cytokine-induced cytotoxic cell populations expanded from autologous peripheral blood mononuclear cells (PBMCs)2.
The uncontrolled growth of lymphoid progenitor cells, myeloblasts, and lymphoblasts leads to three main types of blood cancers (i.e., leukemia, lymphoma, and myeloma), solid tumors, including carcinomas (e.g., lung cancer, gastric cancer, cervical cancer), and sarcomas, among other cancers3. CIK cells are a mixture of cell populations that exhibit a wide range of major histocompatibility complex (MHC)-unrestricted antitumor activity and thus hold promise for the treatment of hematological and advanced tumors4,5,6,7. CIK cells comprise a combination of cells, including T cells (CD3+CD56&#8722;), NK-T cells (CD3+CD56+), and NK cells (CD3&#8211;CD56+). Optimization of the CIK induction protocol by use of a fixed schedule for the addition of IFN-&#947;, anti-CD3 antibody, and IL-2, results in the expansion of CIK cells8. The cytotoxic capability of CIK cells against cancer cells mainly depends on the engagement of NK group 2 member D (a member of the C-type lectin-like receptor family) NKG2D ligands on tumor cells, and on perforin-mediated pathways9. The results of a preclinical study revealed that IL-15-stimulated CIK cells induced potent cytotoxicity against primary and acute myeloid leukemia cell lines in vitro and exhibited a lower alloreactivity against normal PBMCs and fibroblasts9. Recently, the outcome of one-time healthy donor-derived CIK (1&#8239;x&#8239;108/kg CD3+ cells) infusion as consolidation following nonmyeloablative allogeneic transplantation for myeloid neoplasms treatment in a phase II clinical study was published10.
In the present study, we developed an optimized cell culture formula composed of IFN-&#947;, IL-1&#945;, anti-CD3 antibody, and IL-2 added to the hematopoietic cell medium to increase CIK production, and investigated the cytotoxic effect of CIK cells against human chronic myeloid leukemia (K562) cells and ovarian cancer (OC-3) cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-Amino Actinomycin D</td>
			<td>BD</td>
			<td>559925</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD56 antibody</td>
			<td>BD</td>
			<td>555518</td>
			<td>B159</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometer</td>
			<td>BD</td>
			<td>338962</td>
			<td></td>
			<td>SN: R33896202856</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>BD</td>
			<td>565082</td>
			<td></td>
			<td>Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>SIGMA</td>
			<td>G8644</td>
			<td></td>
			<td>For K562 cell culture. Add 12.5 mL to 500 mL of complete medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/F12</td>
			<td>HyClone</td>
			<td>SH30023.02</td>
			<td></td>
			<td>Basal medium for OC-3 cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
			<td>For K562 and OC-3 cell culture. Complete medium contains 10% of FBS</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare Life Sciences</td>
			<td>71101700-EK</td>
			<td></td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD3 antibody</td>
			<td>BD</td>
			<td>555332</td>
			<td>UCHT1</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555748</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-CD3 mAb</td>
			<td>TaKaRa</td>
			<td>T210</td>
			<td>OKT3</td>
			<td>Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
			<td>Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Proleukin</td>
			<td>NOVARTIS</td>
			<td></td>
			<td></td>
			<td>Reconstitution of Proleukin Powder (22x106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interferon-gamma</td>
			<td>CellGenix</td>
			<td>1425-050</td>
			<td></td>
			<td>Reconstitution of rh IFN-g (5x105 IU/50 &#181;g) with 200 &#181;L of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-1 alpha</td>
			<td>PEPROTECH</td>
			<td>200-01A</td>
			<td></td>
			<td>Reconstitution rh IL-1&#945; (10 &#181;g) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
			<td>Basal medium for K562 cell culture. Storage stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sigma 3-18K Centrifuge</td>
			<td>Sigma</td>
			<td>10295</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
			<td>Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature</td>
		</tr>
		<tr>
			<td>X-VIVO 15 medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
			<td>Basal medium for PBMC and CIK cells. Storage at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

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.

The purpose of the present protocol is to isolate and expand cytokine-induced killer (CIK) T cells from peripheral blood monocytes and evaluate the cytotoxic effect of CIK against hematological malignancy and solid cancer cells, respectively. The induction of CIK was identified by the CD3/CD56 recognition. Figure 1A shows the protocol for CIK induction and expansion. The representative results of the gating strategy for analyzing the subpopulation of CD3+CD56...

Watch this Scientific Journal Video about Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment at JoVE.com

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

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 ...

We optimized a highly qualified and clinically applicable method for the expansion of PBMC-derived cytokine-induced killer T cells and for evaluation of their cytotoxicity against cancers. The advantage of this technique is to provide a standard operating procedure for technicians and clinicians to quantify the potency of cytokine-induced killer cells in both scientific research and clinical evaluation. Begin this procedure with a preparation of CIK cells as described in the text protocol, then wash the CIK cells with 10 milliliters of sterile PBS.Centrifuge for 10 minutes at 300 times G and 18 to 20 degrees Celsius. Aspirate the supernatant and resuspend the cells with five milliliters of PBS. Count the cell numbers and test cell viability using the trypan blue exclusion assay.Aliquot the CIK cells into six steril 1.5 milliliter tubes at a density of approximately 5 to one million cells per milliliter of PBS. Label and treat the cells as detailed in the text protocol. Gently mix the CIK cells with the antibodies by gently pipetting up and down at least three times with a one milliliter steril pipette.Incubate the cells for 15 minutes at room temperature in the dark. Centrifuge the tubes for 10 minute at 300 times G and 18 to 20 degrees Celsius. Following centrifugation, aspirate the supernatant and resuspend the cell pellet with one milliliter of PBS, then gently pipette the cells up and down at least three times with a one milliliter sterile pipette.Transfer the cell suspension to a sterile five milliliter polystyrene round bottom tube with a cell strainer cap by gently pipetting through the cap, then place the tubes on the carousel in order. Open the flow cytometry analysis software and create an experimental folder, then click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol.To create a scatter gating system for the CIK cell populations, first select tube one and click on the dot plot button to create an FSCA SSCA plot. Draw a rectangle gate over the entire cell population with an FSCA threshold greater than 5000 to exclude cell debris. Select the FSCA FSCH parameter for the new dot plot and draw a polygon gate around all single cells.Select the count versus FITC-conjugated CD3 and count versus APC-conjugated CD56 parameter for the new histogram plot. Select the FITC-conjugated CD3 versus APC-conjugated CD56 parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Record the data from 20, 000 single cells in each specimen.Click the load sample button to analyze the blank control sample first. Identify the whole CIK cell population by using the CD56 and CD3 channel parameters. Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.To perform the cytotoxic assay, coculture CIK and human chronic myeloid leukemia K562 cells by adding one milliliter of K562 cells to each well in a six-well plate at a density of 5 million cells per milliliter, then add one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Place the plate in the incubator for 24 hours.Now, coculture CIK and ovarian OC-3 cells by adding one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Put the plate in the incubator for 24 hours.Following incubation, harvest the CIK K562 cell suspension directly into a 15 milliliter sterile tube. To harvest both the suspension and adherence cells from the CIK OC-3 groups, first transfer the cell suspension to a 15 milliliter sterile tube. Wash the well with one milliliter of steril PBS, collect the PBS, and add it to the tube, then add 5 milliliters of cell dissociation enzyme solution and incubate for five minutes at 37 degrees Celsius.Add one milliliter of the solution from the same tube to the corresponding well, and gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Collect all the cells in the same tube. Centrifuge at 300 times G for 10 minutes.Aspirate the supernatant and resuspend the cells in one milliliter of sterile PBS. Pellet the cells at 300 times G for 10 minutes, then aspirate the supernatant and resuspend the cells in 100 microliters of sterile PBS. Add five microliters of 7-AAD dye to the cell suspension.Gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Incubate for 10 minutes and leave in the dark before analysis. To perform the cytolytic capability assay, mix the cell suspension and repeat the preparation for flow cytometry.Click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol. To create a scatter gating system for the cytolytic assay, first select tube one and click on the dot plot button to create an FSCA SSCA plot.Draw a rectangle gate over all of events with an FSCA threshold greater than 50, 000 to exclude cell debris. Select the SSCA CFSE parameter for the new dot plot, then select the 7-AAD CFSE parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Click the load sample button to analyze the blank control sample first.Adjust the voltage of SSCA and FSCA. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from greater than 20, 000 CFSE positive cells in each specimen.Open the files containing the statistical values of each individual specimen to analyze the nonviable cell populations, and export the data into analysis files. The representative results of the gating strategy for analyzing the subpopulation of CD-3 positive CD56 positive T-cells from healthy donors are shown with the statistical analysis of the CIK proportion from three individuals. The CD-3 positive CD56 positive cell proportion significantly increased after 14 days of expansion.This is evident through the 65%for the original PBMC which increased to 27.4%for the CIK cells harvested on day 14. In this culture system, the CIK cells yielded about half a hundred fold changes compared to the original number of PBMCs. K562 cells were stained with a nonfluorescent dye CFSE, which is cleaved by intracellular esterases within viable cells and becomes a highly fluorescent dye.The size and granularity of the CIK and CFSE-positive cells are illustrated. The CFSE stained K562 cells were co-treated with CIK cells at a ratio of zero to one, five to one, and 10 to one. These cells were stained with 7-AAD dye as a viability probe for dead cell exclusion.The 7-AAD positive cells of CFSE positive K562 cells were all evaluated. The CFSE-stained OC-3 cells were co-treated with CIK cells at a ratio of 10 to one. The obvious cytotoxicity of CIK against OC-3 cells is demonstrated here following 24 hours of incubation.CIK T-cells has been reported to exert significant cytotoxic effects against cancer cells and reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. Immunotherapy is a promising treatment for a number of cancers. CIK can be efficiently expanded in vitro by incubation of patient-derived PMBCs in GMP-grade condition for further autologous infusion.Following this protocol, we could execute the immune cellular therapy in a GTP-or GMP-grade facility for further clinical cancer treatment.

isolation-expansion-cytotoxic-cytokine-induced-killer-t-cells-for

Research

JoVE Journal

Cancer Research

10.6K visualizações.  Wan Fang Hospital. Otimizamos um método altamente qualificado e clinicamente aplicável para a expansão de células T assassinas induzidas por citocinas derivadas do PBMC e para avaliação de sua citotoxicidade contra cânceres. A vantagem desta técnica é fornecer um procedimento operacional padrão para técnicos e médicos quantificarem a potência das células assassinas induzidas por citocinas tanto na pesquisa científica quanto na avaliação clínica. Inicie este procedimento com uma preparação de...