促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶
Summary
This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.
Abstract
Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.
Introduction
胸腺是负责病原体 – 反应性，自我耐受T细胞的不同群体的产生即获得性免疫系统的功能所必需的主要淋巴器官。它是一个动态的器官，其中显影胸腺细胞，从骨髓如淋巴细胞祖细胞移居，通过胸腺基质的海绵状三维（3-D）矩阵迁移，经受谱系说明书和分化，最终移民如成熟T细胞。此井编程过程的成功在很大程度上取决于迁移胸腺细胞和住宅胸腺上皮细胞（TECs的）中，胸腺基质的主要群体是建立和维护的胸腺微环境的完整性必不可少之间的串扰。
根据他们的解剖位置和独特的功能，的TECs可以分为两个子集：所述的TECs在皮层（cTECs）是responsible选择自身MHC（主要组织相容性复合物）限制性T细胞（阳性选择），并且在髓质是用于消除自身反应性T细胞（阴性选择）1,2-必需的的TECs（mTECs）。许多因素（ 如老化，感染，辐射，药物治疗）可能会导致不可逆的损伤胸腺上皮细胞，导致受损适应性免疫。尽管多次努力，恢复胸腺功能一直具有挑战性，由于重现胸腺微环境的难度。值得注意的是，胸腺三维（3-D）的配置是的TECs的生存和功能是至关重要的，而在2维环境中培养的TECs迅速降低基因thymopoiesis 3,4-临界的表达。
EAK16-II（AEAEKAKAEAEAKAK）及其C末端histidinylated类似物EAKIIH6（AEAEKAKAEAEAKAKHHHHHH）是低分子量，两亲性寡肽可溶于去离子水：R，但是当暴露在离子强度超过20毫摩尔NaCl（在人体液正常盐浓度为154毫摩尔）更高发生凝胶化，形成β-纤维。此环境响应特性使得他们多才多艺的积木，形成三维结构。上EAKII-H6 His标签提供一个对接机构，通过该抗His的IgG / Fc结合的重组蛋白A / G（αH6：PA / G）的配合物可以作为一个适配器锚蛋白药物和其它生物分子（ 例如 ，在水凝胶复合5-8抗体）。
我们以前表明，锚定在水凝胶的荧光标记IgG分子可以在注射部位长达13天9被保留。此外，当αH6：用抗CD4的IgG锚pA的/ G适配器加到EAK16-II / EAKIIH6（EAK）水凝胶，CD4 + T细胞是专门捕获10。使用类似的技术，我们最近证明的TECs的c的3-D聚合乌尔德在EAK水凝胶促进携带特定的TEC-抗-EpCAM抗体复合适配器（αH6：PA / G：αEpCAM）。当裸鼠的肾囊下移植，嵌在EAK水凝胶将TEC集群可以有效地支持体内11,12官能性T细胞的发展。
在这里，我们示出了我们的方法来快速并有效地净化用荧光激活细胞分选（FACS）的TECs和生成与EAK水凝胶系统的3-D TEC聚集体。
Protocol
Representative Results
Discussion
同时的TECs是胸腺基质的主要人口和播放的胸腺的结构和功能重要作用，它们仅占大约0.1％-0.5％的总胸腺细胞构成的。它们也可用作细胞死亡的高百分数以下胶原酶消化偶尔观察到的， 即 ，治疗以解离来自细胞外基质（ECM）的TECs脆弱的细胞。其稀有性（〜20余万小鼠胸腺）和脆弱使其成为具有挑战性的任务来隔离的TEC。高度纯化的胶原酶了TEC隔离最近利用已显著增加活细胞的以下酶消化<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院的一部分授予R21 AI113000（WSM）和R01 AI123392（YF）的支持。
Materials
| 1. TEC Isolation | |||
| 70% Ethanol | Decon Laboratories | 2701(1 Gallon) | Ethanol 200 Proof, deionized water |
| Dissecting scissors, straight | Fine Science Tools, Inc. | 91460-11 | |
| Graefe forceps, straight | Fine Science Tools, Inc. | 11053-10 | |
| Graefe forceps, curved | Fine Science Tools, Inc. | 11052-10 | |
| Washing solution | 1X PBS, 0.1% BSA, 2mM EDTA | ||
| 1x PBS (Phosphate buffered saline) | Gibco | 10010-023 | |
| BSA (Bovine serum albumin) | Sigma | A1470-100G | |
| EDTA (ethylenediaminetetraacetic acid) | Invitrogen | 15575-038 | |
| Digestion solution | 9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI | ||
| RPMI-1640 | Gibco | 11879-020 | |
| Liberase TM Research Grade | Roche | 05 401 127 001 | referred as "purified collagenase" |
| 1M HEPES | Lonza | 17-737E | |
| Dnase I | Roche | 10 104 159 001 | |
| 50mL Centrifuge tube | Corning | 430290 | |
| 60mm tissue culture dish | Falcon | 353002 | |
| 1/2cc U-100 Insulin syringe 28G1/2 | Becton Dickinson | 329461 | |
| 5mL Polystyrene round-bottom tube | Falcon | 352058 | |
| 5ml glass pipet | Fisher Healthcare | 13-678-27E | Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets. |
| MACSmix tube rotator | Miltenyi | 130-090-753 | |
| 100um Cell strainer | Falcon | 352360 | |
| Density gradient medium: OptiPrep | Axis-Shield | ||
| Name | Company | Catalog Number | Comments |
| 2. Cell sorting | |||
| 5mL Polypropylene round-bottom tube | Falcon | 352063 | |
| Anti-mouse CD16/CD32 (Fc Block) | BD Biosciences | 553142 | Use as undiluted, 2uL per sample |
| Anti-mouse CD45-PercpCy5.5 | eBioscience | 45-0451-80 | Use at 1:150, 10uL per sample |
| Anti-mouse CD326 (EpCAM)-PE | eBioscience | 12-5791-82 | Use at 1:100, 10uL per sample |
| BD Influx | BD Biosciences | ||
| Single cell analysis software | FlowJo | ||
| Name | Company | Catalog Number | Comments |
| 2. EAK gel assembly | |||
| Anti-His-Tag | AnaSpec | 29673 | "anti-His-Tag IgG" |
| Purified anti-mouse CD326 (EpCAM) | BioLegend | 118202 | "anti-EpCAM IgG" |
| Recombinant protein A/G | Pierce Biotechnology | ||
| 1.5mL Safe-Lock Tubes, Biopur, Sterile | Fisher Healthcare | 05-402-24B | referred as "1.5mL microcentrifuge tube" |
| 96-well, Tissue culture plate, Round-bottom with low evaporation lid | BD Falcon | 353917 | |
| Rocking platform: Nutator Mixer no.1105 | BD Clay Adams | ||
| 10% sucrose | Sigma | S0389 | Prepare with sterile distilled water |
| EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2) | American Peptide Company | custom synthesized, 10mg/mL | |
| EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2) | American Peptide Company | custom synthesized, 7.5mg/mL | |
| Complete medium | RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol | ||
| RPMI-1640 | Gibco | 11879-020 | |
| FBS (Fetal Bovine Serum) | Atlanta Biologicals | S11150 | Heat inactivated before use |
| Pen/Strep | Gibco | 15140-122 | |
| L-glutamine 200mM (100x) | Gibco | 25030-081 | |
| NEAA (non-essential amino acid) 100x | Gibco | 11140-050 | |
| 1M HEPES | BioWhittaker | 17-737E | |
| 2-Mercaptoethanol (100X) | Millipore | ES-007-E | |
| Platform shaker: The Belly Dancer | Stovall Life Sciences Inc. | model: USBDbo |
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