外显体应用是药物开发和再生医学的新兴工具。我们建立了一种高纯度的外体分离方案,将外体与称为CB-SC的新鲜干细胞分离出来,用于机械研究。我们还与人类单核细胞共同培养CB-SC衍生的外体,导致它们分化成表型的分形巨噬细胞。
干细胞教育者(SCE)疗法是治疗1型糖尿病和其他自身免疫性疾病的一种新型临床方法。SCE 治疗通过密码机循环分离患者的血液单核细胞(如淋巴细胞和单核细胞),在 SCE 设备中用粘附脐带血源干细胞 (CB-SC) 共同培养患者的血液单核细胞,然后将这些”受过教育的”免疫细胞返回到患者的血液中。外显子是纳米大小的细胞外囊泡,在30\u2012150 nm之间,存在所有生物流体和细胞培养基。为了进一步探索SCE治疗的分子机制,并确定从CB-SC释放的外体的行为,我们调查哪些细胞在用CB-SC治疗期间对这些外体进行噬菌体。通过与人类外周血单核细胞(PBMC)共同培养具有Dio标签的CB-SC衍生外体,我们发现CB-SC衍生的外显体主要由人类CD14阳性单细胞组成,导致单细胞分化为2型巨噬细胞(M2),具有主轴样形和M2相关表面分子标记的表达。在这里,我们提出了一个协议,用于分离和表征CB-SC衍生外体,以及CB-SC衍生外体与人类单核细胞共同培养和M2分化监测的协议。
脐带血干细胞(CB-SC)是从人脐带血中鉴定的独特干细胞类型,与其他已知类型的干细胞(如中生血缘干细胞)和造血干细胞(HSC)1等不同。基于其独特的免疫调节特性和紧贴培养皿表面的能力,我们在临床试验2、3中开发出一种被指定为干细胞教育者(SCE)疗法的新技术。在SCE治疗期间,患者的外周血单核细胞(PBMC)通过细胞分离器收集和循环,并在体外与粘附的CB-SC共同培养。这些”受教育”细胞(CB-SC治疗的PBMC)然后返回到患者的循环在闭环系统。临床试验已经证明了SCE疗法治疗自身免疫性疾病的临床安全性和有效性,包括1型糖尿病(T1D)2、4和脱发(AA)5。
外显体是一个纳米粒子系列,直径范围为30\u2012150 nm,存在于所有生物流体和细胞培养介质6中。外显子富含许多生物活性分子,包括脂质、mRNA、蛋白质和微RNA(miRNA),并在细胞到细胞之间的通信中发挥重要作用。最近,外显体已经变得更加有吸引力的研究人员和制药公司,因为他们的治疗潜力在诊所7,8,9。最近,我们的机械研究表明,CB-SC释放的外体有助于SCE治疗10的免疫调节。
在这里,我们描述了通过CB-SC释放的外细胞来探索针对单细胞的SCE治疗机制的协议。首先,使用超离心方法从CB-SC衍生的调节介质中分离出CB-SC释放的外体,并经流式细胞学、西印(WB)和动态光散射(DLS)验证。其次,CB-SC衍生的外体被标记为绿色荧光嗜白染料:迪奥。第三,他们与PBMC共同培养,通过流细胞学检查Dio标记的CB-SC衍生外显子体在PBMC的不同亚细胞的阳性百分比。该协议为研究干细胞免疫调节的外体作用提供了指导。
外显体的应用是临床诊断、药物开发、再生医学的新兴领域。在这里,我们提出了一个详细的协议,关于CB-SC衍生的外体的准备和外体的功能研究,人类单细胞的分化。目前的方案表明,功能性CB-SC衍生的外显体通过顺序离心和高纯度的超离心分离,并表现出对单核细胞的免疫调节。
与其他常规协议相比,超滤是一种基于不同于其他细胞外囊泡(EV)的分子量和排除尺寸的分?…
The authors have nothing to disclose.
我们感谢波达尔先生和路德维希先生通过哈肯萨克UMC基金会的慷慨资助。我们感谢赵老师的英文编辑。
1.5 ml Microcentrifuge tube | Fisher Scientific | 05-408-129 | |
15 ml conical tube | Falcon | 352196 | |
24-well plate | Falcon | 351147 | Non-tissue culture plate |
3,3'-Diostadecyloxacarbocyanine perchlorate(Dio) | Millipore sigma | D4292-20MG | Store at 4 °C |
300 Mesh Grids | Ted Pella | 1GC300 | |
50 mL conical tube | Falcon | 352070 | |
6-well plate | Falcon | 353046 | Tissue culture plate |
96-well plate | Falcon | 353072 | Tissue culture plate |
ACK Lysis Buffer | Lonza | 10-548E | 100 ml |
Amicon-15 10kDa Centrifuge Fliter Unit | Millipore sigma | UFC901024 | |
Anti-Human Alix | Biolegend | 634501 | store at 4 °C, RRID: AB_2268110 |
Anti-Human Calnexin | Biolegend | 699401 | store at 4 °C, RRID: AB_2728519 |
Anti-Human CD11c antibody, Pe-Cy7 | BD Bioscience | 561356 | store at 4 °C, RRID: AB_10611859 |
Anti-Human CD14 antibody, Karma Orange | Beckman Coulter | B36294 | store at 4 °C, RRID: AB_2728099 |
Anti-Human CD163 antibody, PE | BD Bioscience | 556018 | store at 4 °C, RRID: AB_396296 |
Anti-Human CD19 antibody, PC5 | Beckman Coulter | IM2643U | store at 4 °C, RRID: AB_131160 |
Anti-Human CD206 antibody, FITC | BD Bioscience | 551135 | store at 4 °C, RRID: AB_394065 |
Anti-Human CD209 antibody, Brilliant Violet 421 | BD Bioscience | 564127 | store at 4 °C, RRID: AB_2738610 |
Anti-Human CD270 antibody, PE | Biolegend | 318806 | store at 4 °C, RRID: AB_2203703 |
Anti-Human CD3 antibody, Pacfic Blue | Biolegend | 300431 | store at 4 °C, RRID: AB_1595437 |
Anti-Human CD34 antibody, APC | Beckman Coulter | IM2427U | store at 4 °C, RRID: N/A |
Anti-Human CD4 antibody, APC | BD Bioscience | 555349 | store at 4 °C, RRID: AB_398593 |
Anti-Human CD45 antibody, Pe-Cy7 | Beckman Coulter | IM3548U | store at 4 °C, RRID: AB_1575969 |
Anti-Human CD56 antibody, PE | Beckman Coulter | IM2073U | store at 4 °C, RRID: AB_131195 |
Anti-Human CD63 antibody,PE | BD Bioscience | 561925 | store at 4 °C, RRID: AB_10896821 |
Anti-Human CD8 antibody, APC-Alexa Fluor 750 | Beckman Coulter | A94686 | store at 4 °C, RRID: N/A |
Anti-Human CD80 antibody, APC | Beckman Coulter | B30642 | store at 4 °C, RRID: N/A |
Anti-Human CD81 antibody, FITC | BD Bioscience | 561956 | store at 4 °C, RRID: AB_394049 |
Anti-Human CD86 antibody, APC-Alexa Fluor 750 | Beckman Coulter | B30646 | store at 4 °C, RRID: N/A |
Anti-Human CD9 antibody, FITC | ThermoScientic | MA5-16860 | store at 4 °C, RRID: AB_2538339 |
Anti-Human Galectin 9 antibody, Brilliant Violet 421 | Biolegend | 348919 | store at 4 °C, RRID: AB_2716134 |
Anti-Human OCT3/4 antibody, eFluor660 | ThermoScientic | 50-5841-82 | store at 4 °C, RRID: AB_11218882 |
Anti-Human SOX2 antibody, Alexa Fluor 488 | ThermoScientic | 53-9811-82 | store at 4 °C, RRID: AB_2574479 |
BCA Protein Assay Kit | ThermoFisher Scientific | 23227 | |
Bovine Serum Albumin | Millipore Sigma | A1933 | |
Buffy coat | New York Blood Center | 40-60 ml/unit | |
Cell scraper | Falcon | 353085 | |
Disposable semi-micro cuvette | VWR | 97000590 | |
Dissociation buffer | Gibco | 131510014 | 100 ml |
Dual-Chanber cell counting slides | Bio-Rad | 1450015 | |
Exosome-Human CD63 Detection reagent | ThermoFisher Scientific | 10606D | store at 4 °C |
Ficoll-Paque PLUS density grandient media | GE Health | 17-1440-03 | 500 ml |
Fixed-Angle Rotor(25°) | Thermo Scientifc | 75003698 | Maxium 24,700 x g |
Gallios flow cytometer | Beckman Coulter | 3 lasers 10 Color Max |
|
Human cord blood | Cryo-Cell International | 40-100 ml/unit | |
Human Fc Block | BD Bioscience | 564220 | store at 4 °C |
Immun-Blot PVDF membrane | Bio-Rad | 1620177 | |
Millex-GP Syringe Filter Unit, 0.22 µm | Millipore Sigma | SLGP033RS | |
Optima XE-90 Ultracentriguge | Beckman Coulter | ||
Orbital Shaker MP4 | BioExpress | S-3500-1 | |
PBS | ThermoFisher Scientific | 10010049 | 500 ml |
Propidium Iodide | BD Bioscience | 56-66211E | store at 4 °C |
Nikon Eclipse Ti2 microscope | Nikon instruments Inc | Eclipse Ti2 | NIS-Elements software version 5.11.02 |
Hochest 33342 | Thermo Scientifc | 62249 | 5 ml |
Revert microsopy | Fisher scientific | 12563518 | |
Rotor 41 Ti | Beckman Coulter | 331362 | Maxium 41,000 rpm |
Sorvall St 16R Centrifuge | Thermo Scientifc | 75004381 | |
Swinging Bucket Rotor | Thermo Scientifc | 75003655 | |
TC-20 cell counter | Bio-Rad | ||
ThermoScientific Forma 380 Steri Cycle CO2 Incubator | ThermoFisher Scientific | ||
Transmission electron microscopy | JEOL | JEM-2100 PLUS | |
Ultracentrifuge tube | Beckman Coulter | 331372 | |
X'VIVO 15 Serum-free medium | Lonza | BEBP04-744Q | 1000 ml Culture medium, store at 4 °C |