这项研究得到香港研究资助局 (GRF 468513、CUHK3/CRF/12R) 及香港创新科技基金 (ITS/227/15、其 InP/164/16、ITS-InP/242/16)、中大 (2016.035) 及香港学者的直接资助。程序.
/设计和监督所有的实验, 并为手稿的编写做出贡献。康普茶进行了实验, 分析了数据并为稿件的编写做出了贡献。t。彼莱, 俱乐部, M.W., 和 G.-/收集动物标本, 并参与动物实验。右侧和 K-f.t 为手稿的编写做出了贡献。

获得和区分骨髓源性 nk 细胞的协议是基于以前发布的方法20,21,22。所有与老鼠有关的程序已获香港中文大学动物伦理实验委员会 (AEEC) 批准。
1. OP9 条件培养基的制备
<ol><li>培养小鼠基质细胞系 OP9 在α-记忆中含有 20% FBS, 100 U/毫升青霉素 G 和100µg/毫升链霉素, 在37° c 空气湿润大气/CO2 (95%: 5%)。<ol>
<li>用例计数单元格, 然后种子 2 x 106 OP9 细胞到每75厘米2培养瓶与15毫升培养基。文化为 48 h。</li>
		<li>当养殖达到80% 汇流时, 用10毫升 PBS 洗涤烧瓶, 并在每瓶中加入20毫升的普通α记忆培养基 (不含 FBS 和抗生素)。</li>
	</ol>
</li>
	<li>收集 OP9 条件培养基从培养烧瓶在24小时, 清理细胞碎片与0.25 µm 砜 (PES) 过滤器和保留在4° c (使用在2星期之内)。</li>
</ol>2. 分离小鼠骨髓细胞
<ol><li>安乐12周大的 C57BL/6J 鼠, pentobarbitone 过量 (100 毫克/千克, 腹腔注射)。确认死亡的缺乏呼吸, 然后收获股骨骨与手术刀切割的交界处之间的骨骼和删除剩余的肌肉与刀片的温柔划伤。</li>
	<li>将骨头放在一个50毫升的离心管中, 其中含有冷冻无菌的α记忆体, 然后在生物安全柜中执行以下步骤。</li>
	<li>丢弃α-记忆介质, 用70% 乙醇在三十年代冲洗骨头。</li>
	<li>用 ice-cold 消毒的 PBS 两次清洗骨头, 清理剩下的乙醇。</li>
	<li>将骨骼转移到含有5毫升 ice-cold PBS 的灰浆中, 用杵轻轻 fragmentize 骨头 (不要将其粉碎)。</li>
	<li>通过旋转杵来轻轻搅动骨骼, 将骨髓细胞释放到 PBS 中。将含有骨髓细胞的 PBS 收集到新的50毫升离心管中。</li>
	<li>重复步骤 2.6, 四次, 直到骨骼碎片变成纯白色的颜色。</li>
	<li>离心管在 465 x g 为5分钟在4° c, 然后放弃上清。</li>
	<li>重18毫升无菌蒸馏水, 并让它的立场三十年代, 以消除红血球。</li>
	<li>添加2毫升的 ice-cold 10X PBS 停止反应, 然后过滤的混合物通过一个70µm 细胞过滤器, 以消除裂解红血细胞。</li>
	<li>离心管在 465 x g 为5分钟在4° c。用20毫升的 ice-cold PBS 重颗粒。</li>
	<li>第二次重复步骤2.11 来清洗细胞。</li>
	<li>将细胞小球转移到100毫米的无菌培养皿中, 10 毫升的 OP9 条件培养基从步骤1.2 补充 20% FBS 和混合 0.5 ng/毫升小鼠 IL-7, 30 ng/毫升老鼠, 和 100 U/毫升鼠 flt3L。</li>
	<li>孵育盘在37° c 与 5% CO2为 2 h. 将未连接的单元格转移到新的区域性容器中, 其密度为 5 x 105可生存单元/mL (计数单元格例与台盼蓝排除) 与步骤2.13 中的相同介质公式, 并在37° c 下孵育 5% CO2。</li>
	<li>4天, 将培养条件转变为 OP9 条件培养基, 辅以 20% FBS 和2000小鼠 IL-2。</li>
	<li>每3天刷新培养基;成熟的 NK 细胞可在7天获得, 并符合4节的规定。</li>
</ol>3. siRNA 介导的分化 NK 细胞基因沉默
<ol><li>根据产品说明书将 siRNA 或无意义的控制 (NC) 与转染剂混合。</li>
	<li>从0天起, 在任何时间点执行步骤3.1。</li>
	<li>将 50 nM 的 siRNA 或 NC 混合物添加到步骤2.14 中的单元格。</li>
	<li>重复步骤3.1 在每个中等茶点 (例如, 天 0, 4 和 7), 直到实验结束点。</li>
</ol>4. 应用流式细胞仪分析 NK 细胞分化
<ol><li>在适当的时间点, 收集的悬浮分化细胞和洗涤与 ice-cold PBS。</li>
	<li>根据产品说明书将细胞固定缓冲液固定。</li>
	<li>用 ice-cold PBS 冲洗固定细胞, 然后在100µL 流式细胞术染色缓冲液中重 1 x 106细胞, 其中含有 Cy3-conjugated anti-mouse NKp46 和 PE 共轭 anti-mouse CD244 抗体, 稀释率为1:100。</li>
	<li>在室温下对样品进行2小时的染色。</li>
	<li>用 PBS 的300µL 重染色样品, 然后获取 Cytobank 平台20 (http://cytobank.org/) 的数据并进行分析。</li>
</ol>

<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>

作者没有什么可透露的。

在目前的工作中, 我们描述了一种新的方法生产骨髓源性小鼠 NK 细胞体外。原始系统21、22中的单元格进纸器被 OP9 单元的条件介质成功替换, 这极大地提高了微分系统的稳定性。此外, 该系统还能为体外的成熟 nk 细胞产生高纯度的增殖和纯化, 并能促进人体疾病中 nk 细胞的机械研究和转化研究. 
所描述的方法已?...

癌症进展主要依赖于肿瘤微环境1,2, 包括主机派生的免疫,, NK 细胞。几项研究表明, 肿瘤的 NK 细胞呈负相关的癌症进展3,4。此外, 临床研究表明 NK 细胞过继疗法是一种可能的治疗癌症的策略5,6,7,8,9。NK 细胞癌免疫治疗是最近被认为是一种治疗选择的固体肿瘤, 但存在的挑战, 由于免疫抑制剂的分泌因子和下调激活配体在微环境中的实体肿瘤10,11。转化生长因子β (TGF β) 已被认为起抑制作用的致癌作用, 但矛盾的癌细胞也产生 TGF-β1支持肿瘤的发展12,13,14,15. TGF β信号通过下调干扰素反应性和 CD16-mediated 干扰素-γ-γ (干扰素) 产生抑制 NK 细胞的细胞活性体外16,17, 18。
虽然 TGF β信号在肿瘤微环境中的破坏可能是消除癌症的一种可能的方法, 完全阻断 TGF β信号将导致自身免疫性疾病, 由于其抗炎作用, 证明了发展不良副作用包括全身炎症、心血管缺陷和自身免疫在小鼠模型中的19。因此, 了解 TGF β介导的免疫抑制的工作机制将导致确定一个治疗癌症的辅助性治疗靶。
为了阐明 nk 细胞发育所需的分子事件, 威廉斯et al.建立了一个用于将小鼠骨髓造血干细胞分化为 nk 细胞的体外系统20。该系统在很大程度上促进了 nk 细胞发育的机制研究, 包括对 nk 细胞的新祖的鉴定21。然而, 骨髓祖细胞应在系统中培养, 以支持 OP9 基质干细胞为饲养层20,21, 而这种异构的细胞群在很大程度上限制了基因中断工具的进一步应用(例如, siRNA 介导的基因沉默) 专门用于区分 NK 细胞。
在这里, 我们描述一个无馈线系统, 已经开发的进一步修改的在体外系统的威廉姆斯et al20。在我们的系统中, 不需要 OP9 基质饲养细胞, 而使用 OP9 条件培养基, 而不影响 NK 细胞的分化在体外, 这最近导致我们发现, TGF β能够促进癌症进展通过抑制 E4bp4-dependent NK 细胞在肿瘤微环境中的发育22。该系统成功地提供了一种无背景的方法来阐明在特定条件下 NK 细胞发育的分子机制 (例如, 高 TGF β, siRNA 介导的基因沉默, etc。体外。

<table><tbody><tr><td>OP9 cell line</td><td>ATCC</td><td>ATCC&lt;sup&gt;&amp;reg;&lt;/sup&gt; CRL-2749&lt;sup&gt;&amp;trade;&lt;/sup&gt;</td><td></td></tr><tr><td>MEM &amp;alpha;, no nucleosides</td><td>Gibco</td><td>22561021</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10500064</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>PEPROTECH</td><td>217-17</td><td></td></tr><tr><td>Recombinant Murine SCF</td><td>PEPROTECH</td><td>250-03</td><td></td></tr><tr><td>Recombinant Murine Flt3-Ligand</td><td>PEPROTECH</td><td>250-31L</td><td></td></tr><tr><td>Recombinant Murine IL-2</td><td>PEPROTECH</td><td>212-12</td><td></td></tr><tr><td>Lipofectamin RNAiMAX Transfection Reagent</td><td>Invitrogen</td><td>1377815</td><td></td></tr><tr><td>IC Fixation Buffer&amp;nbsp;</td><td>eBioscience</td><td>00-8222-49</td><td></td></tr><tr><td>Flow Cytometry Staining Buffer&amp;nbsp;</td><td>eBioscience</td><td>00-4222-26</td><td></td></tr><tr><td>PE-conjugated anti-mouse CD244</td><td>eBioscience</td><td>12-2441-83</td><td></td></tr><tr><td>Cy3-conjugated anti-mouse NKp46</td><td>Bioss</td><td>bs-2417R-cy3</td><td></td></tr><tr><td>Nonsense control (NC)</td><td>Ribobio</td><td>siN05815122147</td><td></td></tr><tr><td>siRNA against mouse E4BP4 mRNA</td><td>Ribobio</td><td>N/A</td><td>5&amp;prime;-GAUGAGGGUGUA&lt;br/&gt; GUGGGCAAGUCUU-3&amp;prime;</td></tr></tbody></table>

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

自然杀手 (NK) 细胞属于先天免疫系统, 是一线抗癌免疫防御;然而, 它们在肿瘤微环境中被压制, 其基本机制仍然是未知的。nk 细胞缺乏一致、可靠的来源, 限制了 nk 细胞免疫的研究进展。在这里, 我们报告一个体外系统, 可以提供高质量和数量的骨髓来源的小鼠 NK 细胞在无饲养条件下。更重要的是, 我们还证明, siRNA 介导的基因沉默成功地抑制了 E4bp4-dependent NK 细胞成熟, 利用该系统。因此, 这本新的体外NK 细胞分化系统是一种生物材料免疫研究的解决方案。

根据所述协议得到了代表性的结果。在无饲养诱导分化系统下培养的总骨髓悬浮细胞11天;7天, 与0天 (图 1A) 的总细胞数相比, 增殖率显著增加。在系统 (图 1B) 中, 6 天发现了成熟的 NK 细胞, 细胞核胞质比和富含颗粒的细胞质形态。
为了阐明 TGF-β1/Smad3 信号在 ...

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

在这里, 我们提出了一个 mass-produce 基因沉默小鼠 NK 细胞的协议, 使用无馈线分化系统的机械研究体外和在体内。

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

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 ...

a-novel-feeder-free-system-for-mass-production-murine-natural-killer

Research

JoVE Journal

Immunology and Infection

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

7.1K 次观看.  The Chinese University of Hong Kong. 在这里, 我们提出了一个 mass-produce 基因沉默小鼠 NK 细胞的协议, 使用无馈线分化系统的机械研究体外和在体内。

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

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.

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.

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

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

科研

癌症研究

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 inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

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

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

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

免疫与感染

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 are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

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

<meta charset="utf8"></head><body>NK 细胞外囊泡的生物制造和表征

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

<meta charset="utf8"></head><body>可扩展的生物制造工作流程，用于生产和分离基于自然杀伤细胞来源的细胞外囊泡癌症生物治疗药物

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

可扩展的生物制造工作流程，用于生产和分离基于自然杀伤细胞来源的细胞外囊泡癌症生物治疗药物

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

从人类扩增的潜在干细胞中产生自然杀伤细胞

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

发育生物学

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

摘要

探索更多视频

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

可扩展的生物制造工作流程，用于生产和分离基于自然杀伤细胞来源的细胞外囊泡癌症生物治疗药物

可扩展的生物制造工作流程，用于生产和分离基于自然杀伤细胞来源的细胞外囊泡癌症生物治疗药物

可扩展的生物制造工作流程，用于生产和分离基于自然杀伤细胞来源的细胞外囊泡癌症生物治疗药物

鼠单克隆抗体的产生杂交瘤技术

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

从人类扩增的潜在干细胞中产生自然杀伤细胞

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究