Name: a novel feeder-free system for mass production of murine natural killer cells in vitro
Uploaded: 2018-01-09T14:00:00
Description: Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

This study was supported by the Research Grants Council of Hong Kong (GRF 468513, CUHK3/CRF/12R) and the Innovation and Technology Fund of Hong Kong (ITS/227/15, ITS InP/164/16, ITS-InP/242/16), Direct Grant for Research-CUHK (2016.035), and Hong Kong Scholar Program.
H.-Y.L. designed and supervised all experiments and contributed to manuscript preparation. P.M.-K.T. performed experiments, analyzed data and contributed to manuscript preparation. P.C.-T. T., J.Y.-F.C., J.S.-C.,H., Q.-M.W., and G.-Y.L. collected animal samples and participated in animal experiments. J.S., X.-R.H., and K.-F.T. contributed to manuscript preparation.

The protocol for obtaining and differentiating bone marrow-derived NK cells (BM-NK) is based on previously published methods20,21,22. All procedures with mice have been approved by the Animal Ethics Experimental Committee (AEEC) at the Chinese University of Hong Kong.
1. Preparation of OP9 Conditional Medium
<ol>
	<li>Culture the murine stroma cell line OP9 in alpha-MEM containing 20% FBS, 100 U/mL ...

<ol>
	<li>Schreiber, R. D., Old, L. J., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunoediting:+integrating+immunity's+roles+in+cancer+suppression+and+promotion.">Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion.</a> Science. 331 (6024), 1565-1570 (2011).</li><li>Junttila, M. R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+tumour+micro-environment+heterogeneity+on+therapeutic+response.">Influence of tumour micro-environment heterogeneity on therapeutic response.</a> Nature. 501 (7467), 346-354 (2013).</li><li>Rusakiewicz, 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=Immune+infiltrates+are+prognostic+factors+in+localized+gastrointestinal+stromal+tumors.">Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors.</a> Cancer Res. 73 (12), 3499-3510 (2013).</li><li>Mamessier, 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=Human+breast+cancer+cells+enhance+self+tolerance+by+promoting+evasion+from+NK+cell+antitumor+immunity.">Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.</a> J Clin Invest. 121 (9), 3609-3622 (2011).</li><li>Stern, 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=Pre-emptive+immunotherapy+with+purified+natural+killer+cells+after+haploidentical+SCT:+a+prospective+phase+II+study+in+two+centers.">Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers.</a> Bone Marrow Transplant. 48 (3), 433-438 (2013).</li><li>Miller, J. 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=Successful+adoptive+transfer+and+in+vivo+expansion+of+human+haploidentical+NK+cells+in+patients+with+cancer.">Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.</a> Blood. 105 (8), 3051-3057 (2005).</li><li>Rubnitz, J. 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=NKAML:+a+pilot+study+to+determine+the+safety+and+feasibility+of+haploidentical+natural+killer+cell+transplantation+in+childhood+acute+myeloid+leukemia.">NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia.</a> J Clin Oncol. 28 (6), 955-959 (2010).</li><li>Curti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+transfer+of+alloreactive+haploidentical+KIR+ligand-mismatched+natural+killer+cells+after+infusion+in+elderly+high+risk+acute+myeloid+leukemia+patients.">Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients.</a> Blood. 118 (12), 3273-3279 (2011).</li><li>Bachanova, V., 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=Clearance+of+acute+myeloid+leukemia+by+haploidentical+natural+killer+cells+is+improved+using+IL-2+diphtheria+toxin+fusion+protein.">Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein.</a> Blood. 123 (25), 3855-3863 (2014).</li><li>Stringaris, 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=Leukemia-induced+phenotypic+and+functional+defects+in+natural+killer+cells+predict+failure+to+achieve+remission+in+acute+myeloid+leukemia.">Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia.</a> Haematologica. 99 (5), 836-847 (2014).</li><li>Rouce, R. H., 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+TGF-&#946;/SMAD+pathway+is+an+important+mechanism+for+NK+cell+immune+evasion+in+childhood+B-acute+lymphoblastic+leukemia.">The TGF-&#946;/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia.</a> Leukemia. 30 (4), 800-811 (2016).</li><li>Derynck, R., Akhurst, R. J., Balmain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+in+tumor+suppression+and+cancer+progression.">TGF-&#946; signaling in tumor suppression and cancer progression.</a> Nature Genet. 29 (2), 117-129 (2001).</li><li>Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+cancer.">TGFbeta in cancer.</a> Cell. 134 (2), 215-230 (2008).</li><li>Ikushima, H., Miyazono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+signalling:+a+complex+web+in+cancer+progression.">TGFbeta signalling: a complex web in cancer progression.</a> Nat Rev Cancer. 10 (6), 415-424 (2010).</li><li>Pickup, M., Novitskiy, S., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+TGF&#946;+in+the+tumour+microenvironment.">The roles of TGF&#946; in the tumour microenvironment.</a> Nat Rev Cancer. 13 (11), 788-799 (2013).</li><li>Rook, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+transforming+growth+factor+beta+on+the+functions+of+natural+killer+cells:+depressed+cytolytic+activity+and+blunting+of+interferon+responsiveness.">Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness.</a> J Immunol. 136 (10), 3916-3920 (1986).</li><li>Bellone, G., Aste-Amezaga, M., Trinchieri, G., Rodeck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+NK+cell+functions+by+TGF-beta+1.">Regulation of NK cell functions by TGF-beta 1.</a> J Immunol. 155 (3), 1066-1073 (1995).</li><li>Trotta, 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=TGF-&#946;+utilizes+SMAD3+to+inhibit+CD16-mediated+IFN-&#947;+production+and+antibody-dependent+cellular+cytotoxicity+in+human+NK+cells.">TGF-&#946; utilizes SMAD3 to inhibit CD16-mediated IFN-&#947; production and antibody-dependent cellular cytotoxicity in human NK cells.</a> J Immunol. 181 (6), 3784-3792 (2008).</li><li>Shull, M. 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=Targeted+disruption+of+the+mouse+transforming+growth+factor-&#946;1+gene+results+in+multifocal+inflammatory+disease.">Targeted disruption of the mouse transforming growth factor-&#946;1 gene results in multifocal inflammatory disease.</a> Nature. 359 (6397), 693-699 (1992).</li><li>Chen, T. J., Kotecha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytobank:+providing+an+analytics+platform+for+community+cytometry+data+analysis+and+collaboration.">Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.</a> Curr Top Microbiol Immunol. 377, 127-157 (2014).</li><li>Williams, N. 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=Differentiation+of+NK1.1+,+Ly49++NK+cells+from+flt3++multipotent+marrow+progenitor+cells.">Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells.</a> J Immunol. 163 (5), 2648-2656 (1999).</li><li>Fathman, J. W., 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=Identification+of+the+earliest+natural+killer+cell-committed+progenitor+in+murine+bone+marrow.">Identification of the earliest natural killer cell-committed progenitor in murine bone marrow.</a> Blood. 118 (20), 5439-5447 (2011).</li><li>Tang, P. 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=Smad3+promotes+cancer+progression+by+inhibiting+E4BP4-mediated+NK+cell+development.">Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development.</a> Nat Commun. 6 (8), 14677 (2017).</li></ol>

In the present work, we have described a novel method for producing bone marrow-derived murine NK cells in vitro. The cell feeder in the original system21,22 is successfully replaced by the conditional medium of OP9 cells, which largely increased the stability of the differentiation system. In addition, the system can produce high quantity and purity of mature NK cells for in vitro as well as in vivo assays, which can facilitate the mec...

Cancer progression is largely dependent on the tumor microenvironment1,2, including host-derived immunocytes, e.g., NK cells. Several studies demonstrated that intratumoral NK cells are negatively correlated with the tumor progression3,4. In addition, clinical studies showed that NK cell adoptive therapy is a possible strategy for cancer5,6,7,8,9. NK cell-based cancer immunotherapy was recently suggested as a therapeutic option for solid tumors, but challenges exist due to the secretion of immunosuppressive cytokines and downregulation of activating ligands in the microenvironment of solid tumors10,11. Transforming growth factor-&#946; (TGF-&#946;) has been suggested to play a suppressive role in carcinogenesis, but paradoxically cancer cells also produce TGF-&#946;1 to support the tumor development12,13,14,15. TGF-&#946; signaling can suppress the cytolytic activity of NK cells via down-regulating interferon responsiveness and CD16-mediated interferon-gamma (IFN-&#947;) production in vitro16,17,18.
Although disruption of TGF-&#946; signaling in the tumor microenvironment may be a possible way for eliminating cancers, completely blocking TGF-&#946; signaling will cause autoimmune diseases due to its anti-inflammatory function, as evidenced by the development of adverse side effects including systemic inflammation, cardiovascular defects, and autoimmunity in mouse models19. Thus, understanding the working mechanism of TGF-&#946;-mediated immunosuppression will lead to the identification of an accessible therapeutic target for treating cancer.
To elucidate the molecular events necessary for NK cell development, Williams et al. established an in vitro system for differentiating murine bone marrow hematopoietic stem cells into NK cells20. This system largely facilitates the mechanistic study of NK cell development, including the identification of novel progenitors of NK cells21. However, the bone marrow progenitors should be cultured in the system with supporting OP9 stromal cells as a feeder layer20,21, and this heterogeneous cell population largely limits the further application of gene-disrupting tools (e.g., siRNA-mediated gene silencing) specifically applied to the differentiating NK cells.
Here, we describe a feeder-free system that has been developed by further modifying the in vitro system of Williams et al20. In our system, the OP9 stromal feeder cells are not required, and instead OP9 conditional medium is used without affecting the differentiation of NK cells in vitro, and this recently lead us to uncover that TGF-&#946; is able to promote cancer progression via suppressing E4bp4-dependent NK cell development in the tumor microenvironment22. This novel system successfully provides a background-free method for elucidating the molecular mechanism of NK cell development under specific conditions (e.g., high TGF- &#946;1, siRNA-mediated gene silencing, etc.) in vitro.

<table border="0" cellpadding="0" cellspacing="0" style="width:1113px;" width="1113">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM &#945;, no nucleosides</td>
			<td>Gibco</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine SCF</td>
			<td>PEPROTECH</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-2</td>
			<td>PEPROTECH</td>
			<td>212-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
		</tr>
	</tbody>
</table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Representative results are obtained following the described protocol. Total bone marrow suspension cells were cultivated under the feeder-free differentiation system for 11 days; significant increase in proliferation rate was observed by day 7 compared with the number of total cells on day 0 (Figure 1A). Mature NK cells with high nuclear to cytoplasmic ratio and granule-rich cytoplasm morphology were found by day 6 in the system (<strong clas...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

The overall goal of this novel feeder-free system is to provide a method to mass produce high-purity natural killer cells for in vitro as well as in vivo assays. This method can help answer the key questions in innate immunology, including natural killer cell development and cancer immunity. The main advantage of this technique is that this novel feeder-free system can largely increase the purity of the produced natural killer cells.That significantly facilitates the following assays in vitro and in vivo. The indication of this technique extends to the natural killer immunity because mass production of murine natural killer cells can be produced from the bone marrow of cancer patients for analysis and modification for personal immunotherapy. Generally, individuals new to this method will struggle because the consumed medium should be collected from the OP9 cell undergoing load phase growth.Facial demonstration of this method is critical as the bone marrow instruction steps are difficult to learn, because the bones need to be sterile with ethanol with optimal time to preserve the viability. To begin this procedure, collect the OP9 cells from a culture flask. Add FBS to stop trypsin digestion.Collect the OP9 cells, and centrifuge at 465 times gravity for five minutes. After this, re-suspend the cell pellet. Using a hemocytometer, count the cells.Then, seed two times 10-to-the-six cells in 15 milliliters of alpha-mem medium in a 75 centimeter-square culture flask, and incubate for 48 hours. Once the cells are 80%confluent, wash the cells with 10 millimeters of phosphate-buffered saline and add 20 milliliters of plain alpha-mem medium without antibiotics and fetal bovine serum to the cells. Incubate for 24 hours.The next day, collect the OP9-conditioned media by removing the cell debris with a filter and preserve at four degrees Celsius. Inject overdose of 100 milligrams per kilogram of pentobarbitone intraperitoneal to euthanize a 12-week-old mouse. After confirming lack of breathing, excise the femur bones with a surgical knife.Remove the remaining muscles by gently scratching with a blade. Transfer the bones in a 50 milliliter centrifuge tube filled with chilled, sterile alpha-mem medium, and keep in a biosafety cabinet. Then, expel the alpha-mem medium, and wash the bones with 70%ethanol for 30 seconds.Again, wash the bones with ice-cold sterile phosphate-buffered saline twice to remove any remaining ethanol. After washing, transfer the bones to a mortar containing five milliliters of ice-cold phosphate-buffered saline and fragmentize the bones with a pestle. Swirl the pestle gently to release the bone marrow cells into the phosphate-buffered saline.Transfer the phosphate-buffered saline containing bone marrow cells in a new 50 milliliter centrifuge tube. Then, centrifuge the tube at 465 times gravity for five minutes at four degrees Celsius. After centrifugation, discard the supernatant.Extract until the bone turns white in color to get maximum yield. Next, re-suspend the pellet with 18 milliliters of sterile distilled water, and wait for 30 seconds to remove the red blood cells. Then, add two milliliters of 10X ice-cold phosphate-buffered saline to stop the reaction.Use a 70 micrometer cell strainer to remove the lysed red blood cells. Then, centrifuge the tubes at 465 times gravity for five minutes at four degrees Celsius. Discard the supernatant and re-suspend the pellet in 20 milliliters of ice-cold phosphate-buffered saline.Centrifuge the cells one more time. Next, transfer the cell pellet in a 100 milliliter sterile petri dish containing 10 milliliters of OP9 conditional medium, supplemented with additional reagents. Transfer the petri dish in an incubator at 37 degrees Celsius with 5%carbon dioxide for two hours.Remove all unattached cells and seed in a new culture dish containing OP9 conditional medium, and place in an incubator at 37 degrees Celsius for five minutes. On the fourth day, replace the culture medium with OP9 conditional medium, supplemented with 20%fetal bovine serum and 2, 000 units per milliliter of murine interleukin 2. Keep changing the culture medium every three days until the seventh day to obtain the mature natural killer cells.Follow the product manual to premix the small interfering RNA or non-sense control with the transfection reagent at zero, fourth and seventh day of changing the medium until the experimental endpoint. Add 50 nanomolar of small interfering RNA, or non-sense mixture, to the unattached cells during medium refreshment. Collect the differentiating cells at an appropriate timepoint.Centrifuge the cells at 465 times gravity for five minutes. Discard the supernatant and wash the cell pellet with ice-cold phosphate-buffered saline. After washing the fixed cells with ice-cold phosphate-buffered saline, re-suspend the cells in 100 microliters of flow cytometry staining buffer containing SY3 conjugated antibodies.After this, re-suspend the samples in 300 microliters of phosphate-buffered saline for fax analysis. A representative growth curve demonstrating the proliferation rate of the bone marrow cells undergoing differentiation into natural killer cells in feeder-free system. The graph shows a significant increase in the proliferation rate of the cells by Day Seven.A representative bright field image showing differentiated natural killer cells in a novel feeder-free system. Mature natural killer cells exhibited high nuclear to cytoplasmic ratio with a granular cytoplasmic morphology. A representative bar plot showing the effect of small interfering RNA on the bone marrow cells undergoing differentiation into natural killer cells on Day Six.The plot shows significant reduction on the E4bp4 mRNA levels and small interfering RNA treated natural killer cells compared to the control group. A representative flow cytometric data demonstrating the effect of silenced E4bp4 on the differentiation of bone marrow derived natural killer cells in a TGF beta-1 Smad3-dependent manner. Data shows knockdown of E4bp4 mRNA vastly decreases in mature natural killer cell production on Day Six in comparison to the non-sense control.Small interfering RNA mediated decreased production of natural killer cells is shown to be rescued by suppressing TGF beta-1 Smad3 with one micromolar SIS3 Smad3 inhibitor in comparison to the small interfering RNA transfected group. Once mastered, this study can be done in two hours if it's performed properly. Following this procedure allows genetic modifications such as gene lockdown and over-expression can be conducted in order to answer additional questions like the regulation mechanisms for natural killer cell activity as well as the development.The first I had the idea for this method when I was expanding the natural killer cells in the old system. The presence of the fetal cells decreased the load and efficiency of siRNA to the interest gene. After watching this video, you should have a good understanding of how to produce massive high-quality natural killer cells from this local system in vitro.Don't forget that working with RNA extraction reagents can be extremely hazardous, and precautions should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...