Name: fabrication of anisotropic polymeric artificial antigen presenting cells for cd8+ t cell activation
Uploaded: 2018-10-12T13:30:00
Description: Watch this Scientific Journal Video about Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation at JoVE.com

EBA (DGE-1746891) 和 KRR (DGE-1232825) 感谢 NSF 研究生研究奖学金计划的支持。RAM 感谢国家研究服务奖国立卫生研究院 NCI F31 (F31CA214147) 和大学科学家奖学金的成就奖励支持。作者感谢 NIH (R01EB016721 和 R01CA195503), 预防失明詹姆斯和卡罗尔免费催化剂奖的研究, 以及 JHU 彭博-Kimmel 癌症免疫治疗研究所的支持。

这里描述的所有方法都是由约翰霍普金斯大学的机构动物护理和使用委员会 (IACUC) 批准的。
1. 可调谐尺寸球形颗粒的制备
<ol><li>颗粒合成材料的制备<ol>
<li>准备5% 瓦特/w 聚乙烯醇 (PVA) 溶液。<ol>
<li>添加500毫升的去离子 (DI) 水到一个锥形烧瓶与一个磁力搅拌棒和放置在热板搅拌器在 500 rpm 和监测温度与温度计。用锡纸盖上烧瓶, 防止蒸发。</li>
			<li>当水温达到约70摄氏度时, ?...

<ol>
	<li>Eggermont, L. J., Paulis, L. E., Tel, J., Figdor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+efficient+cancer+immunotherapy:+Advances+in+developing+artificial+antigen-presenting+cells.">Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells.</a> Trends in Biotechnology. 32 (9), 456-465 (2014).</li><li>Maus, M. V., Riley, J. L., Kwok, W. W., Nepom, G. T., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+tetramer-based+artificial+antigen-presenting+cells+for+stimulation+of+CD4++T+cells.">HLA tetramer-based artificial antigen-presenting cells for stimulation of CD4+ T cells.</a> Clinical Immunology. 106 (1), 16-22 (2003).</li><li>Oelke, 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=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nature Medicine. 9 (5), 619-624 (2003).</li><li>Rudolf, 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=Potent+costimulation+of+human+CD8+T+cells+by+anti-4-1BB+and+anti-CD28+on+synthetic+artificial+antigen+presenting+cells.">Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells.</a> Cancer Immunology, Immunotherapy. 57 (2), 175-183 (2008).</li><li>Tham, E. L., Jensen, P. L., Mescher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+antigen-specific+T+cells+by+artificial+cell+constructs+having+immobilized+multimeric+peptide-class+I+complexes+and+recombinant+B7-Fc+proteins.">Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins.</a> Journal of Immunological Methods. 249 (1-2), 111-119 (2001).</li><li>Perica, 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=Magnetic+field-induced+T+cell+receptor+clustering+by+nanoparticles+enhances+T+cell+activation+and+stimulates+antitumor+activity.">Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity.</a> ACS Nano. 8 (3), 2252-2260 (2014).</li><li>Steenblock, E. R., Fadel, T., Labowsky, M., Pober, J. S., Fahmy, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+artificial+antigen-presenting+cell+with+paracrine+delivery+of+IL-2+impacts+the+magnitude+and+direction+of+the+T+cell+response.">An artificial antigen-presenting cell with paracrine delivery of IL-2 impacts the magnitude and direction of the T cell response.</a> The Journal of Biological Chemistry. 286 (40), 34883-34892 (2011).</li><li>Zhang, 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=Paracrine+release+of+IL-2+and+anti-CTLA-4+enhances+the+ability+of+artificial+polymer+antigen-presenting+cells+to+expand+antigen-specific+T+cells+and+inhibit+tumor+growth+in+a+mouse+model.">Paracrine release of IL-2 and anti-CTLA-4 enhances the ability of artificial polymer antigen-presenting cells to expand antigen-specific T cells and inhibit tumor growth in a mouse model.</a> Cancer Immunology, Immunotherapy. 66 (9), 1229-1241 (2017).</li><li>Mescher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+contact+requirements+for+activation+of+cytotoxic+T+lymphocytes.">Surface contact requirements for activation of cytotoxic T lymphocytes.</a> The Journal of Immunology. 149 (7), 2402-2405 (1992).</li><li>Steenblock, E. R., Fahmy, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+platform+for+ex+vivo+T-cell+expansion+based+on+biodegradable+polymeric+artificial+antigen-presenting+cells.">A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells.</a> Molecular Therapy. 16 (4), 765-772 (2008).</li><li>Fifis, 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=Size-dependent+immunogenicity:+therapeutic+and+protective+properties+of+nano-vaccines+against+tumors.">Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors.</a> The Journal of Immunology. 173 (5), 3148-3154 (2004).</li><li>Sunshine, J. C., Perica, K., Schneck, J. P., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+shape+dependence+of+CD8++T+cell+activation+by+artificial+antigen+presenting+cells.">Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells.</a> Biomaterials. 35 (1), 269-277 (2014).</li><li>Meyer, R. 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=Biodegradable+nanoellipsoidal+artificial+antigen+presenting+cells+for+antigen+specific+T-cell+activation.">Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation.</a> Small. 11 (13), 1519-1525 (2015).</li><li>Champion, J. A., Katare, Y. K., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+shape:+a+new+design+parameter+for+micro-+and+nanoscale+drug+delivery+carriers.">Particle shape: a new design parameter for micro- and nanoscale drug delivery carriers.</a> Journal of Controlled Release. 121 (1-2), 3-9 (2007).</li><li>Meyer, R. A., Meyer, R. S., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+multidimensional+thin+film+stretching+device+for+the+generation+of+anisotropic+polymeric+micro-+and+nanoparticles.">An automated multidimensional thin film stretching device for the generation of anisotropic polymeric micro- and nanoparticles.</a> Journal of Biomedical Materials Research Part A. 103 (8), 2747-2757 (2015).</li><li>Ho, C. C., Keller, A., Odell, J. A., Ottewill, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monodisperse+ellipsoidal+polystyrene+particles.">Preparation of monodisperse ellipsoidal polystyrene particles.</a> Colloid and Polymer Science. 271 (5), 469-479 (1993).</li><li>Shum, 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=Droplet+microfluidics+for+fabrication+of+non-spherical+particles.">Droplet microfluidics for fabrication of non-spherical particles.</a> Macromolecular Rapid Communications. 31 (2), 108-118 (2010).</li><li>Lan, W., Li, S., Xu, J., Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controllable+preparation+of+nanoparticle-coated+chitosan+microspheres+in+a+co-axial+microfluidic+device.">Controllable preparation of nanoparticle-coated chitosan microspheres in a co-axial microfluidic device.</a> Lab on a Chip. 11 (4), 652-657 (2011).</li><li>Yang, 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=Microfluidic+synthesis+of+multifunctional+Janus+particles+for+biomedical+applications.">Microfluidic synthesis of multifunctional Janus particles for biomedical applications.</a> Lab on a Chip. 12 (12), 2097-2102 (2012).</li><li>Zhou, Z., Anselmo, A. C., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+protein-based,+rod-shaped+particles+from+spherical+templates+using+layer-by-layer+assembly.">Synthesis of protein-based, rod-shaped particles from spherical templates using layer-by-layer assembly.</a> Advanced Materials. 25 (19), 2723-2727 (2013).</li><li>Jang, S. 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=Striped,+ellipsoidal+particles+by+controlled+assembly+of+diblock+copolymers.">Striped, ellipsoidal particles by controlled assembly of diblock copolymers.</a> Journal of the American Chemical Society. 135 (17), 6649-6657 (2013).</li><li>Petzetakis, N., Dove, A. P., O'Reilly, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cylindrical+micelles+from+the+living+crystallization-driven+self-assembly+of+poly(lactide)-containing+block+copolymers.">Cylindrical micelles from the living crystallization-driven self-assembly of poly(lactide)-containing block copolymers.</a> Chemical Science. 2 (5), 955-960 (2011).</li><li>Rolland, J. 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=Direct+fabrication+and+harvesting+of+monodisperse,+shape-specific+nanobiomaterials.">Direct fabrication and harvesting of monodisperse, shape-specific nanobiomaterials.</a> Journal of the American Chemical Society. 127 (28), 10096-10100 (2005).</li><li>Meyer, R. 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=Anisotropic+biodegradable+lipid+coated+particles+for+spatially+dynamic+protein+presentation.">Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.</a> Acta Biomaterialia. 72, 228-238 (2018).</li></ol>

本协议详细介绍了一种用于精确生成各向异性聚合物粒子的通用方法。这里描述的薄膜拉伸技术是可扩展的, 高度重现性和成本低廉。产生各向异性粒子的替代技术受到许多限制, 包括高成本、低吞吐量和有限的粒子大小。薄膜拉伸方法也是有利的, 因为粒子在合成后被修改为各向异性, 因此, 与各种颗粒尺寸和合成技术兼容。图 1详细介绍了自动二维拉伸装置的设置。该装置?...

人工抗原呈现细胞 (aAPC) 已表明承诺作为免疫调节剂, 因为他们可以产生一个强大的抗原特异 T 细胞反应。这些平台的关键是它们能够有效地为 T 细胞活化提供关键信号。脱细胞 aAPC 是一种有吸引力的替代细胞的 aAPC, 因为它们更容易和成本更低的制造, 面临更少的挑战, 在扩大和翻译, 并减轻与细胞疗法相关的风险。脱细胞 aAPC 还允许对信号表示参数和表面物理特性进行高度控制, 这将与 T 细胞1连接。
aAPC 必须重述至少两个信号, 对于 T 细胞激活至关重要。信号1提供抗原识别和发生时 T 细胞受体 (TCR) 识别和参与的 MHC i 类或 II 轴承其同源抗原, 最终通过 TCR 复合信号。为了绕过抗原特异性要求, aAPC 系统经常承受竞赛 CD3 受体的单克隆抗体, 特异刺激 TCR 复合物。重组形式的 mhc, 特别是 mhc 多聚体, 也被用于 aAPC 的表面, 以提供抗原特异性2,3。信号2是指示 T 细胞活动的共刺激信号。为了提供 T 细胞活化所必需的刺激, CD28 受体通常用 aAPC 表面上的竞赛抗体刺激, 尽管共刺激的其他4-1BB 受体已经成功地瞄准了4。信号1和2蛋白通常固定在刚性颗粒表面, 以合成 aAPC。从历史上看, aAPC 是由各种材料制成的, 包括聚苯乙烯4、5和铁葡聚糖6。较新的系统利用生物降解聚合物 (聚乳酸-乙醇酸) (aAPC) 生成可以很容易地耦合到信号蛋白, 适合于体内直接管理, 并可促进持续释放封装细胞因子或可溶性因子, 以增加 T 细胞活化7,8。
除了存在必要的信号蛋白外, 在 aAPC/t 细胞相互作用期间, 受体在足够大的表面积上的参与对于 T 细胞活化是必不可少的。因此, aAPC 的物理参数 (如大小和形状) 会极大地改变其可用接触区域, 并影响其刺激 T 细胞的能力。微米级的 aAPC 在刺激 T 细胞方面比它们的纳米尺度对应9、10更有效。然而, 纳米 aAPC 可以有优越的生物分布和更好的引流到淋巴结, 可能提高其在体内的性能在微 aAPC11。形状是对基于粒子的 aAPC 系统感兴趣的另一个变量。在刺激 T 细胞时, 各向异性 aAPC 最近被证明比各向同性粒子更有效, 主要是由于增强了与靶细胞的相互作用以及减少了非特异细胞吸收。单元格优先绑定到椭球粒子的长轴, 曲率和平坦表面的半径越大, aAPC 和 T 细胞12之间的接触就越多。椭圆形颗粒的长轴也会抑制吞噬, 从而在体内施用12、13的情况下, 与球形颗粒相比, 循环时间增加。由于这些优点, 椭球粒子在体外和体内与球形颗粒相比, 能更好地调节抗原特异 T 细胞的扩张, 这在微 nanoscales12中观察到的效果, 13。制造各向异性粒子有多种策略, 但薄膜拉伸是一种简单、广泛接受的方法, 用于生成一系列不同的粒子形状14。合成后, 粒子被投射到薄膜中, 在一个或两个维度上拉伸, 温度高于粒子材料的玻璃过渡温度。然后将胶片溶解以检索粒子。尽管对各向异性粒子的兴趣越来越大, 但目前制造粒子基 aAPC 的方法大多限于各向同性系统, 改变颗粒形状的方法很难实现, 与某些 aAPC 合成不相容。策略, 缺乏精度和重现性15。我们的薄膜拉伸技术可以手动或以自动方式进行, 以快速生成由各种可生物降解聚合物合成的各向异性粒子, 在一个或两个维度中拉伸到所需的纵横比15。
基于我们以前的工作, 我们开发了一种可生物降解的粒子基方法, 结合可以扩展的薄膜拉伸技术, 快速生成 aAPC, 可调谐尺寸和形状, 以标准化的方式用于 T 细胞扩张, 例如体内或体内。我们的蛋白质共轭策略可用于将任何感兴趣的蛋白质与颗粒表面上的羧基基团进行耦合, 以达到所需的密度, 从而使该 aAPC 系统具有高度的灵活性。我们还描述了 aAPC 的大小、形态和表面蛋白含量的表征方法, 并对其体外功能进行了评价。该协议可以很容易地适应扩大免疫细胞的体内或体内的各种免疫治疗应用。

<table>
	<tbody>
		<tr>
			<td>Poly(vinyl alcohol), MW 25000, 88% hydrolyzed</td>
			<td>Polysciences, Inc.</td>
			<td>02975-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>Fluke</td>
			<td>N/A</td>
			<td>Model name: Fluke 52 II</td>
		</tr>
		<tr>
			<td>Immersion Temperature Probe</td>
			<td>Fluke</td>
			<td>N/A</td>
			<td>Model name: Fluke 80PK 22</td>
		</tr>
		<tr>
			<td>Digital Hotplate &#38; Stirrer</td>
			<td>Benchmark Scientific</td>
			<td>H3760-HS</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipoint stirrer</td>
			<td>Thermo Fisher Scientific</td>
			<td>50093538</td>
			<td></td>
		</tr>
		<tr>
			<td>Resomer RG 504 H, Poly(D,L-lactide-co-glycolide)</td>
			<td>Sigma-Aldrich</td>
			<td>719900</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>IKA</td>
			<td>&#160;0003725001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics &#38; Materials, Inc.</td>
			<td>N/A</td>
			<td>Model number: VC 505</td>
		</tr>
		<tr>
			<td>Sonicator sound abating enclosure</td>
			<td>Sonics &#38; Materials, Inc.</td>
			<td>N/A</td>
			<td>Part number: 630-0427</td>
		</tr>
		<tr>
			<td>Sonicator probe</td>
			<td>Sonics &#38; Materials, Inc.</td>
			<td>N/A</td>
			<td>Part number: 630-0220</td>
		</tr>
		<tr>
			<td>Sonicator microtip</td>
			<td>Sonics &#38; Materials, Inc.</td>
			<td>N/A</td>
			<td>Part number: 630-0423</td>
		</tr>
		<tr>
			<td>High speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Model number: J-20XP (discontinued), alternative model: J-26XP</td>
		</tr>
		<tr>
			<td>High speed centrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>369691</td>
			<td>Model number: JA-17</td>
		</tr>
		<tr>
			<td>High speed polycarbonate centrifuge tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3118-0050</td>
			<td>50 mL, screw cap</td>
		</tr>
		<tr>
			<td>Rectangular disposable petri dish</td>
			<td>VWR International</td>
			<td>25384-322</td>
			<td>75 x 50 x 10 mm</td>
		</tr>
		<tr>
			<td>Square disposable petri dish</td>
			<td>VWR International</td>
			<td>10799-140</td>
			<td>100 mm x 100 mm</td>
		</tr>
		<tr>
			<td>LEAF&#160;Purified anti-mouse CD3&#949; Antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td></td>
		</tr>
		<tr>
			<td>InVivoMab anti-mouse CD28, clone 37.51</td>
			<td>Bio X Cell</td>
			<td>BE0015-1</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E6383</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysulfosuccinimide sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>56485</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse CD3 Antibody</td>
			<td>Biolegend</td>
			<td>100212</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD28 Antibody</td>
			<td>Biolegend</td>
			<td>102109</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;96 Well Solid Polystyrene Microplate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3915</td>
			<td>flat bottom, black polystyrene</td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (+ L-Glutamine)</td>
			<td>ThermoFisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>Heat Inactivated, sterile-filtered</td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Sigma-Aldrich</td>
			<td>17850</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-2 (carrier-free)</td>
			<td>Biolegend</td>
			<td>589102</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Solution (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11120052</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a+&#160;T Cell Isolation Kit, mouse</td>
			<td>Miltenyi Biotech</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Accuri C6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 2 Multi-Detection Microplate Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Running Buffer</td>
			<td>Miltenyi BIotech</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>ThermoFisher Scientific</td>
			<td>22363548</td>
			<td>Sterile, 70 &#181;m nylon mesh</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J (Black 6) Mouse</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>Male, at least 7 weeks old</td>
		</tr>
		<tr>
			<td>U-Bottom Tissue Culture Plates</td>
			<td>VWR</td>
			<td>353227</td>
			<td>Sterile, 96-well tissue culture treated polystyrene plates</td>
		</tr>
		<tr>
			<td>40 V DC Power Supply</td>
			<td>Probotix</td>
			<td>LPSK-4010</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE Coated Wire</td>
			<td>Mouser</td>
			<td>602-5858-100-01</td>
			<td>This is for a 100 ft. spool but an equivalent wire will work</td>
		</tr>
		<tr>
			<td>Stepper Motor Driver</td>
			<td>Probotix</td>
			<td>MondoStep5.6</td>
			<td></td>
		</tr>
		<tr>
			<td>IDC Connector Kit</td>
			<td>Probotix</td>
			<td>IDCM-10-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontroller</td>
			<td>Probotix</td>
			<td>PBX-RF</td>
			<td></td>
		</tr>
		<tr>
			<td>4A Fuses</td>
			<td>Radio Shack</td>
			<td>2701026</td>
			<td>Equivalent fuses will work as well</td>
		</tr>
		<tr>
			<td>DB25 Male to Male Cable</td>
			<td>Probotix</td>
			<td>DB25-6</td>
			<td></td>
		</tr>
		<tr>
			<td>USB-A to USB-B Cable</td>
			<td>Staples</td>
			<td>2094915</td>
			<td>Equivalent cable will work as well</td>
		</tr>
		<tr>
			<td>8-Pin Amphenol Connectors Male and Female</td>
			<td>Mouser</td>
			<td>654-97-3100A-20-7P and 654-97-3106A20-7S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stepper Motor</td>
			<td>Probotix</td>
			<td>HT23-420-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Right Hand Lead Screw</td>
			<td>Roton</td>
			<td>60722</td>
			<td></td>
		</tr>
		<tr>
			<td>Left Hand Lead Screw</td>
			<td>Roton</td>
			<td>60723</td>
			<td></td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>McMaster Carr</td>
			<td>92196A151</td>
			<td></td>
		</tr>
		<tr>
			<td>Neoprene Rubber</td>
			<td>McMaster Carr</td>
			<td>8698K51</td>
			<td></td>
		</tr>
		<tr>
			<td>Right Handed Flanged Lead Nut</td>
			<td>Roton</td>
			<td>91962</td>
			<td></td>
		</tr>
		<tr>
			<td>Left Handed Flanged Lead Nut</td>
			<td>Roton</td>
			<td>91963</td>
			<td></td>
		</tr>
		<tr>
			<td>Linux Control Computer</td>
			<td>Probotix</td>
			<td>LCNC-PC</td>
			<td>Any computer with matching specification and Linux operating system will work</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431097</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4 %</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of anisotropic polymeric artificial antigen presenting cells for cd8+ t cell activation

人工抗原呈现细胞 (aAPC) 是一种有潜力的免疫调节平台, 由于其强大的刺激 T 细胞的能力。脱细胞基质提供了与基于细胞的 aAPC 的关键优势, 包括精确控制信号表示参数和 aAPC 表面的物理特性, 以调节其与 T 细胞的相互作用。由各向异性粒子 (特别是椭球粒子) 构造的 aAPC 在刺激 t 细胞时比球形对应体更有效, 因为可用于 t 细胞接触的增强的结合和更大的表面积, 以及减少非特异性摄取和增强药代动力学特性。尽管增加了对各向异性粒子的兴趣, 但即使是广泛接受的方法来生成各向异性粒子, 如薄膜拉伸, 也可能对实现和使用可重复具有挑战性。
为此, 我们描述了一种快速、标准化的可生物降解各向异性粒子基 aAPC 的协议, 其可调谐大小、形状和信号呈现用于 T 细胞在体内或体内的扩张, 以及方法表征其大小、形态和表面蛋白质含量, 并评估其功能。这种制造各向异性 aAPC 的方法是可扩展的, 可以重现, 因此非常适合为 "现成" 免疫疗法生成 aAPC。

图 1给出了自动2D 薄膜拉伸装置的示意图。给出了一种1D 薄膜拉伸装置的原理图和描述.17担架是由铝零件使用标准铣削和加工技术制造的。与1D 担架类似, 2D 担架由金属夹具和导轨组成。双向引线螺钉用于将线性转换为旋转运动。引线螺钉通过机械丝锥连接到具有足够扭矩的相同步进电机。8步进电机控制导线可焊接到8针耐热的安费?...

Watch this Scientific Journal Video about Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation at JoVE.com

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

在这里, 我们提出了一个协议, 以快速和可重复生成生物启发, 可生物降解的 articifical 抗原呈现细胞 (aAPC) 与可调谐的大小, 形状和表面蛋白呈现的 T 细胞扩张的体内或体内.

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

此方法有助于回答氨基工程领域的关键问题。例如，粒子物理特性对生物材料免疫细胞相互作用和活化的作用是什么？这种技术的主要优点是它能够独立控制仿生粒子的大小和形状。这种技术的影响延伸到癌症的治疗，因为它是一种现成的粒子疗法，可导致体内细胞内调节的免疫反应。虽然这种方法可以提供癌症氨基治疗的见解，它也可以应用于其他系统。如，传染病或1型糖尿病。对于微颗粒合成，首先称出100毫克聚（乳酸共糖酸）或PLGA，放入闪烁小瓶中，然后通过涡流将PLGA溶解在5毫升二氯甲烷中。将均质器放在含有 50 毫升 1%聚乙烯醇或 PVA 溶液的烧杯中，使均质器尽可能靠近烧杯底部，无需接触。以适当的速度打开均质器，并将 PLGA 溶液添加到烧杯中，实现一分钟的均质化。在均质结束时，将聚乙烯醇 PLGA 微颗粒溶液倒入含有 100 毫升 5%PVA 溶液的烧杯中，放在化学罩中的搅拌板上，搅拌溶液至少 4 小时。当溶剂蒸发后，将颗粒溶液倒入50毫升锥形管中，通过离心收集颗粒。用20毫升去离子水更换每个管中的上流水，通过涡流重新悬浮颗粒。然后用新鲜的去离子水将每个管中的最终体积调至50毫升，并像刚才展示的一样，再洗两次颗粒。对于纳米颗粒合成，将200毫克PLGA溶解在5毫升二氯甲烷中，并在含有50毫升1%PVA溶液的烧杯中放置一个声波探针，而不接触烧杯底部。以 12 瓦的速度开始声波化，并立即将 PLGA 溶液添加到烧杯中，进行两分钟的声波化，以生成直径约为 200 纳米的纳米粒子。声波化后，将1%聚乙烯醇PLGA纳米颗粒溶液倒入含有100毫升5PVA溶液的烧杯中，在搅拌板上进行4小时的化学烟气罩溶剂蒸发。当所有溶剂蒸发时，将颗粒溶液倒入 50 毫升锥形管中进行离心，以去除任何微颗粒并去除中超剂。然后将纳米颗粒转移到高速离心管中，在去离子水中进行三次洗涤，如所证明的。对于聚合物颗粒制造，在上次洗涤后，将颗粒重新悬浮在大约一毫升的新鲜去离子水中，并将薄膜铸造溶液添加到颗粒中，最终浓度为每毫升颗粒 2.5 毫克。将 10 毫升等分中的颗粒悬浮液转移到 75 乘 50 毫米矩形培养皿中，进行一维拉伸。或在15毫升等分成100由100毫米方培养皿的二维拉伸。在化学罩中隔夜干燥后，使用钳子去除薄膜，用剪刀修剪边缘。对于 1D 拉伸，将一个薄膜的两个短边放在两块氯丁橡胶之间，用于安装在自动薄膜拉伸装置的一轴的铝块上。然后使用 Allen 扳手拧紧橡胶顶部的金属手柄，将薄膜固定到位。对于 2D 拉伸，将所有四个边安装到四个铝块上，以在两个轴上拉伸薄膜，并在一个轴上记录铝块之间的薄膜长度，用于一个 D 拉伸或两个轴进行 2D 拉伸，并基于所需的折数拉伸计算在一个或两个方向上拉伸薄膜所需的距离。然后将装有薄膜的拉伸装置放在90摄氏度的烤箱中，放在少量水的更大烧杯旁边，使薄膜温度超过10分钟。拉伸完成后，让薄膜冷却到室温20分钟。对于 1D 拉伸，将薄膜从边缘的拉伸设备中剪切出来。对于 2D 拉伸，剪切并保存两个轴上均匀拉伸的胶片中心方形。将薄膜放在 50 毫升锥形管中，每个管不超过两个薄膜，并将大约 25 毫升的去离子水添加到每个管中进行涡旋。当薄膜溶解后，如所证明，在去离子水中清洗颗粒三次。上次洗涤后，将颗粒重新悬浮在大约一毫升的新鲜脱离子水中。然后在零下80摄氏度下冷冻颗粒一小时或过夜冻干。第二天早上，在新鲜准备好的 MES 缓冲液中，以每毫升 20 毫克重新悬浮冻干的微颗粒或纳米颗粒，并进行涡旋。将 100 微升颗粒溶液添加到含有 900 微升 MES 缓冲液的聚丙烯微离心管中，并加入 100 微升新鲜准备的 EDC NHS 溶液。通过涡旋混合颗粒，并在室温下在逆变器上孵育颗粒30分钟。在孵化结束时，通过离心收集颗粒，通过旋转或重新悬浮PBS一毫升的纳米颗粒，在2至3瓦的声波下再悬浮在PBS的一毫升中。接下来，向微粒添加8微克合适的信号一个蛋白质和10微克抗小鼠CD28抗体，或向纳米粒子添加16微克的信号一个蛋白质和20微克的抗小鼠CD28。使用 PBS 将每个管中的最终体积达到 1.1 毫升，并在 4 摄氏度的逆变器上孵育颗粒。第二天，在去离子水中洗三次颗粒，如证明。这些PLGA纳米和微粒是使用单一乳液技术合成的，使用传输和扫描电子显微镜进行演示和成像。均质化后，微粒的平均直径约为3微米。球面微粒子的纵横比例约为1，而1D拉伸前向椭圆粒子的纵横比较大约3个半，2D拉伸椭圆粒子的纵横比约为1.2，大致维持1的纵横比。结合效率结果揭示了球面和椭圆形微人造抗原呈现细胞（AAPC和纳米-AAPC）表面的蛋白质含量相似，表明AAPC合成过程中蛋白质耦合以浓度依赖的方式发生。发现蛋白酸椭圆形AAPLC在亚饱和剂量下诱导比球形AAP高浓度T细胞增殖，在01毫克剂量下达到最佳分离。七天后，对T细胞的人工计数显示，与微和纳米尺度的球形细胞相比，蛋白酸酯AAPC以剂量依赖的方式更有效地刺激T细胞。按照这个程序，AAPC可以在体内进行管理，以测量其疗效和药代动力学。该技术开发后，为免疫工程领域的研究人员探索各向异性对AAPC疗效的影响铺平了道路。

Research

JoVE Journal

Environment

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

巴西田间放生转基因 伊 蚊的大规模生产

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

科研

环境科学

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

从好氧颗粒污泥结构胞外聚合物的提取

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) ...

Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process

碳纳米材料的功能化和分散使用环境友好的超声波臭氧分解过程

Functionalization of carbon nanomaterials is often a critical step that facilitates their integration into larger material systems and devices. In the ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

基于实验室诱导的昼夜循环缺氧和 pH 值的双壳软体动物连续气门目瞪口呆测量用应变计监测仪 (通用)

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

用原位曝光法测定淡水珍珠贻贝 Bioindication 的流环境适宜性

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

气泡柱光生物反应器中绿色微藻的培养及中性脂质的测定

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...