从表型分析和人体皮肤淋巴细胞分离<em>离体</em>细胞培养
Summary
We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.
Abstract
人体皮肤具有重要的屏障功能，并包含有助于从病原体组织稳态和保护各种免疫细胞。由于皮肤相对容易获得，它提供了一个理想的平台，研究外周免疫调节机制。免疫固有细胞在健康的皮肤进行免疫监视，而且还起到炎性皮肤疾病，如牛皮癣的发展中起重要作用。尽管新兴的见解，我们对生物学基本各种炎症皮肤病的了解仍然有限。有必要从皮肤活检样本中分离出质量好（单）的细胞群。到目前为止，隔离程序已经严重缺乏获得足够数量的活菌的阻碍。隔离和随后的分析也已受到的免疫细胞谱系标志物的损失，由于引起的电流离解过程来获得机械和化学应力单细胞悬浮液。在这里，我们描述了一种改进方法，通过使用一种自动组织离解器和胶原酶处理组合机械皮肤分离以分离从健康的和涉及银屑病人类皮肤T细胞。这种方法在单细胞悬浮液的制备保持最免疫谱系标记，例如CD4，CD8，Foxp3的和CD11c的表达。成功的CD4 + T细胞的分离和随后的表型和功能的分析的例子被示出。
Introduction
皮肤，因为身体和环境之间的主要接口，提供了针对外部的物理，化学和生物损伤如伤人，紫外线辐射和微生物防御的第一道防线。皮肤由两个主要隔间，表皮和真皮层，并含有多种免疫细胞包括Langerhans细胞，巨噬细胞，树突细胞（DC），以及约20十亿记忆T细胞，本近两倍的数量在整个血容量1 ，2。数据越来越多支持皮肤有重要的免疫功能，无论是在组织的动态平衡和各种病理状态的概念。驻留在正常皮肤的免疫细胞被认为以进行免疫监视3，并已显示出在炎性疾病如牛皮癣4的发展中的作用。在银屑病皮损部位，CD4 +和CD8 +浸润T细胞OBServed并且它表明CD4和CD8的比例的变化取决于疾病状态5。但是，细胞的这些群体是很难研究，因为现有技术仅允许少数细胞的分离。
从人体皮肤T细胞分离当前广泛使用的技术相结合的机械皮肤解离酶处理。人体皮肤活检被广泛切碎，并与像胰蛋白酶，胶原酶和/或EDTA 6-8酶的培养。考虑到皮肤的阻挡薄纸这对拉力和机械解聚高度抗T细胞分离的确立方法产生非常少的细胞，并且甚至活细胞数目降低，这使得这些细胞群的体外细胞培养困难和具有挑战性的。
在这里，我们报告的改进方法结合mechan隔离来自健康和参与的银屑病人的皮肤淋巴细胞使用自动组织离解，而不是广泛的切碎建立的方法，一起用胶原酶酶消化皮肤的iCal分离。制备单细胞悬浮液后观察到的各种可行的免疫细胞亚群，包括DC和T细胞。重要的是，表面标记CD3，CD4和CD8的表达保存完好。由此制备的细胞，是准备用于体外细胞培养物或流式细胞分析使用。此协议已被成功地用于从牛皮癣患者的皮损衍生的单一皮肤活检（直径4 mm）的分析。结果表明，皮肤驻患者的T细胞产生更多的炎性细胞因子如IL-17和IFNγ在比较健康志愿者9。
Protocol
Representative Results
Discussion
在这里，我们提出了一个协议，以有效地隔离人体皮肤活检的皮肤驻地T细胞。这个协议的优点是相对高的数字可行淋巴细胞的隔离，并表示相关的表面标记。确定的细胞亚群为：的CD11c +树突，CD4 +和CD8 + T细胞和Foxp3 + CD25 +细胞。重要的是，孤立的皮肤驻地T细胞的体外培养是很好的可行性，并允许进行后续功能分析。
人体皮肤?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
从牛皮癣患者的皮肤活检好心由Andreas Koerber博士经过科学使用口头或书面的知情同意书（在德国埃森大学皮肤科）提供。
XH也由国家自然科学基金委员会61263039和国家自然科学基金委员会11101321的支持。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Disposable Biopsy Punch | Kai Europe | BP-40F | 4 mm |
| Disposable sterile scalpels | Dalhausen | 1100000510 | |
| gentleMACS C tube | Miltenyi Biotech | 130-093-237 | Blue-capped, used as the dissociation tube. |
| gentleMACS Dissociator | Miltenyi Biotech | 130-093-235 | Automated tissue dissociator. Using the "program spleen_01" for dissociation of the skin biopsy. |
| Cell strainer | BD | 352350 | 70 µm nylon |
| 96-well U-bottom plate | Greiner Bio-One | 650180 | |
| RPMI 1640 | Life Technologies | 22409-015 | |
| Sodium pyruvate | Life Technologies | 11360-039 | |
| GlutaMAX | Life Technologies | 35050-061 | |
| Penicillin/Streptomycin | Life Technologies | 15140-122 | |
| Human Pooled Serum (HPS) | in house prepared | ||
| Collagenase I | Sigma-Aldrich | C2674 | Type 1-A, suitable for cell culture |
| DNase I | Calbiochem | 260913 | |
| PBS | B Braun | 3623140 | |
| BSA | Sigma-Aldrich | A4503-500G | |
| Fixable Viability Dye (FVD) APC-eFluo780 | eBioscience | 65-0865-18 | Stain dead cells prior to cell fixation; dilute with PBS at 1:1000 |
| Fixation/Permeabilization Concentrate | eBioscience | 00-5123-43 | |
| Fixation/Permeabilization Diluent | eBioscience | 00-5223-56 | |
| Permeabilization Buffer (10x) | eBioscience | 00-8333-56 | |
| BV421 Mouse anti-human CD45 | BD | 563879 | Clone: HI30; dilution factor 1:50 |
| FITC Mouse anti-human CD14 | Dako | T0844 | Clone: TUK4; dilution factor 1:50 |
| PE Mouse anti-human CD56 | Dako | R7127 | Clone: MOC-1; dilution factor 1:50 |
| ECD Mouse anti-human CD3 | Beckman – Coulter | A07748 | Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining. |
| PC5.5 Mouse anti-human CD4 | Beckman – Coulter | B16491 | Clone: 13B8.2; dilution factor 1:200 |
| PeCy7 Mouse anti-human CD11c | Beckman – Coulter | A80249 | Clone: BU15; dilution factor 1:50 |
| APC Mouse anti-human CD1c | Miltenyi Biotech | 130-090-903 | Clone: AD5-8E7; dilution factor 1:10 |
| APC-AlexFluo700 Mouse anti-human CD8 | Beckman – Coulter | A66332 | Clone: B9.11; dilution factor 1:400 |
| APC-AlexFluo750 Mouse anti-human CD19 | Beckman – Coulter | A94681 | Clone: J3-119; dilution factor 1:50 |
| PeCy7 Mouse anti-human CD25 | eBioscience | AD5-8E7 | Clone: BC96; dilution factor 1:50 |
| PE Rat anti-human CLA | Miltenyi Biotech | 130-091-635 | clone: HECA-452; dilution factor 1: |
| eFluo450 Rat anti-human Foxp3 | eBIoscience | 48-4776-42 | Clone: PCH101; intracellular staining; dilution factor 1:50 |
| AlexFluo488 Mouse anti-human IL-17A | eBioscience | 53-7179-42 | Clone: eBio64DEC17; intracellular staining; dilution factor 1:50 |
| PeCy7 Mouse anti-human IFNg | eBioscience | 25-7319-41 | Clone: 4S.B3; intracellular staining; dilution factor 1:400 |
| Flow-Count Fluorospheres | Beckman – Coulter | 7547053 | Counting beads, for flow cytometry |
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