鼠淋巴结基质细胞的分离
Summary
Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.
Abstract
Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host.
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.
Introduction
淋巴结有专门的隔间，其中对外国和自身抗原的适应性免疫应答发起和协调。这里介绍的过程描述很短的酶消化结合机械式自动移液获得淋巴结单细胞悬液，并获得可行的淋巴结基质细胞的维持几个分子的表面表达。
淋巴结基质细胞形成淋巴结的支架，履行三大职能：第一，他们筛选体液样品抗原，病原体及其病原相关分子模式（的PAMP），以及细胞因子与危险相关分子模式（D-AMPS）目前在体内。第二，它们吸引和指示的抗原呈递细胞（APC）和淋巴细胞相互作用，并启动适应性免疫应答;第三，他们为淋巴细胞1的稳态和分化的结构性环境-3。在炎症过程中淋巴结基质细胞产生的生长因子，细胞因子和趋化因子，适应肿胀从而组织树突状细胞（DC）之间的相互作用，T，和B细胞。免疫反应的编排是唯一可能的，因为由不同的基质细胞群体形成的复合结构的体系结构。
淋巴结基质细胞是CD45阴性细胞中，并且可以通过CD31的表达或GP38在成纤维细胞和内皮细胞1-6相区别。 GP38 + CD31 –定义T区网状细胞（TRC，也被称为FRC：成纤维网状细胞），GP38 + CD31 +限定淋巴管内皮细胞（LEC），GP38 – CD31 +限定血液内皮细胞（BEC）。此外，该亚群的特征显示其他淋巴结基质细胞的存在。事实上，小周细胞样细胞群进行了表征内GP38 – CD31 –人口7。因此，在分离过程中的适应是有利的，用于鉴定不同淋巴结基质细胞的功能特性和特征。
的淋巴结基质细胞消化之前开发协议淋巴结基质细胞的研究是利用组织切片和显微镜不限于原位观测。然而，结构和功能研究表明，淋巴结基质细胞的重要特征。淋巴结基质细胞与平足，胶原和细胞外基质（ECM）蛋白结合以形成所谓的管道系统中的复杂的三维结构，其输送淋巴和相关联的低分子量蛋白质的淋巴结的囊下窦的高内皮微静脉中的T细胞区8。 DC是在与基质细胞紧密接触，并且可以观察到突出到管状CONduit结构来样液和检测抗原8。淋巴结基质细胞（储税券和淋巴管内皮细胞）与DC的相互作用是由趋化因子CCL21和CCL19 9,10释放和呈现介导的。 CCL19和CCL21通过CCR7受体促进树突状细胞和T细胞迁移至淋巴结的T细胞区4,11确认。尽管使用类似的趋化因子，树突状细胞和T细胞有不同的迁徙路线进入淋巴结12。后来，使用淋巴结和纯淋巴结基质细胞分离的酶消化，功能研究，对不同淋巴结基质细胞的作用和其与DC和T / B细胞6,13相互作用的能力进行的。首先，IFN-γ的产生的效应T细胞和淋巴结基质细胞之间的交调失真引起的生产所示以抑制T细胞应答和增殖中的次级淋巴器官14-16的代谢物的一氧化氮。二，淋巴Ñ赋基质细胞已报告，以支持监管DC亚群的分化通过产生IL-10的17的，并通过产生IL-7 6,18的调节幼稚T细胞稳态。三，淋巴结基质细胞Toll样受体的表达表明，基质细胞很容易受到信号从组织损伤时释放的感染或自身分子而得。的确，淋巴结基质细胞与TLR3聚配体的治疗（I：C）诱导主要组织相容性复合体I类表达和共同抑制分子PD-L1的表达上调的适度上调，但不是共刺激分子，从而导致外周组织的急剧变化抗原表达19。几个小组已经表明淋巴结基质细胞表达外周组织中的抗原并诱导自身反应性T细胞19,21-27的耐受性。因此，了解淋巴结基质细胞和其它迁移和再之间的相互作用目前主席淋巴结细胞，将有助于找到新的目标分子，以允许在炎症期间激活或抑制免疫应答。因此，淋巴结的出版酶的分离的实施是必要的。
先前公布的协议使用胶原酶类酶消化具有低机械应力6,19,20的不同组合。然而，长期的孵化与消化酶或消化酶的不同组合，可能会降低分析的激活状态，并确定新的淋巴结基质细胞所需的各种表面分子。根据不同的基质细胞的分析的类型，链路协议或弗莱彻协议可能是更适合的。在所描述的过程中，稍短酶消化结合了自动机械解聚作用，以减少可行淋巴结基质细胞的表面标记物的降解。此过程使高度可重复的分离和区分淋巴结基质细胞群与低变异性和超过95％的存活率。将新鲜分离的淋巴结基质细胞可直接用于表面标志物表达，蛋白质分析和转录的研究，以及建立基质细胞系在体外进行功能测定。
Protocol
Representative Results
Discussion
淋巴结基质细胞的研究最近成为研究的热点，由于两个出版消化协议6,13的发展。这两种协议是足够获得单个淋巴结基质细胞，但不同之处在于使用消化酶和消化的时间。自基质细胞和其表面标志物是酶消化和机械应力敏感，一个优化的协议是必须的。
可行的淋巴结基质细胞从新鲜解剖淋巴结的隔离是为了进行表型和功能的分析的第一步。因此，在限定的酶浓度要仔?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.
Materials
| DMEM | LifeTechnologies-Gibco | 41965-039 | |
| FCS | Final concentration 2% | ||
| CaCl2 | Sigma | 499609 | Final concentration 1.2 mM |
| Collagenase IV | Worthington Biochemical Corporation | >160 units per mg dry weight, use at final concentration of 1 mg/ml | |
| Collagenase D | Roche | 11088882001 | use at 3.5 mg/ml |
| DNAse I | Roche | 11284932001 | use at 40 µg/ml |
| stiring magnets | FAUST | 5 mm long-2 mm ø | |
| Polystyren Round-Bottom Tubes 5ml | Falcon-BD Bioscience | ||
| Magnetic stirrer with heating funktion | IKA-RCT-standard | 9720250 | |
| Petridishes 100 mm, sterile | TPP | 6223201 | |
| 25G needles | Terumo | ||
| anti mouse CD45 Ab | Biolegend | Clone 30-F11 | |
| anti mouse CD11c Ab | Biolegend | Clone N418 | |
| anti mouse Podoplanin Ab | Biolegend | Clone 8.1.1 | |
| anti mouse CD31 Ab | Biolegend | Clone MEC13.3 | |
Eppendorf Xplorer plus, Multichannel |
Eppendorf | 4861 000.821/830 | 1.250 µl max. volume |
| anti mouse CD140a | Biolegend | Clone APA5 | |
| anti mouse CD80 | Biolegend | Clone 16-10A1 | |
| anti mouse CD40 | Biolegend | Clone 1C10 | |
| anti mouse I-Ab | Biolegend | Clone AF6-120.1 | |
| anti mouse CD274 (PD-L1) | Biolegend | Clone 10F.9G2 | |
| LIVE/DEAD Fixable Near-IR Dead Cell stain kit | Invitrogen | L10119 |
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