Summary

从表型分析和人体皮肤淋巴细胞分离<em>离体</em>细胞培养

Published: April 08, 2016
doi:

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

注:从发生后科学运用口头或书面的知情同意择期整形手术个人腹部皮肤剩获得来自健康人的皮肤活检。利用人体皮肤的批准,并按照医学伦理委员会的Radboud大学医疗中心,奈梅亨,荷兰和德国埃森大学人类研究机构的规定。 1.准备从人体皮肤的单细胞悬浮液(工作在无菌流柜如果后续细胞培养是必需的) 制备细胞培养基:RPMI 1640 +青霉素/链霉素(最终浓度100单位/ …

Representative Results

这里介绍的协议将产生2200±178000 760之间的615(平均值±SEM,健康志愿者的皮肤)至±用一个4 mm皮肤活检时(平均值±SEM,牛皮癣患者的皮损)从人体皮肤可行的淋巴细胞。 不同类型的 CD45 +细胞在来自健康个体包括CD4 + T细胞(〜45%),CD8 + T细胞(〜30%)的皮肤衍生的单细胞悬液进行鉴定,和的CD11c +树突状(〜5% ),而少数B细胞(…

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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Cite This Article
He, X., de Oliveira, V. L., Keijsers, R., Joosten, I., Koenen, H. J. Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture. J. Vis. Exp. (110), e52564, doi:10.3791/52564 (2016).

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