Summary

从人类母胎界面分离白细胞

Published: May 21, 2015
doi:

Summary

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis – the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Abstract

怀孕的特点是在生殖组织,并在母胎界面白细胞(底蜕膜及蜕膜顶叶)的渗透。此接口是母体和胎儿组织之间的接触解剖位置;因此,它是作用在怀孕期间免疫位点。白细胞浸润在母胎界面起到交付的植入了核心作用,维护妊娠和时序。因此,这些白细胞的表型和功能性的刻画将提供洞察导致怀孕紊乱的机制。有几个协议,以隔离从蜕膜和蜕膜顶叶白细胞浸润了说明;然而,由于缺乏在试剂,酶和孵化的时间一致性使得难以比较这些结果。本文描述的是一种新的方法,结合使用温和的机械和酶迪斯引产技术,以保护细胞外和细胞内标志物的可行性和完整性白细胞从在母胎界面的人体组织中分离。除了免疫,细胞培养和细胞分选,这个协议的未来的应用是多种多样的。按照此协议,分离的白细胞可用于确定DNA甲基化,靶基因的表达, 在体外白细胞功能( ,吞噬作用,细胞毒性,T细胞增殖,和可塑性, ),以及生产的活性氧在母胎界面。此外,使用所描述的协议,该实验室已经能够描述新和罕见的白细胞在母胎界面。

Introduction

怀孕的特征在于三种不同的免疫学阶段:1)植入并用促炎症反应相关的早期胎盘( ,植入类似于一个“开放性伤口”); 2)孕中期和孕大部分的孕晚期时免疫稳态通过主要是消炎的状态在母胎界面实现; 3)分娩,促炎症状态1-7。免疫细胞在母胎界面发挥在炎症反应的调节中起重要作用,他们的丰度和在整个孕期6-9本土化的改变。

在人类中,母胎界面代表了母性(蜕膜)和胎儿(绒毛膜滋养层或)组织之间的直接接触面积。该接口包括:1)蜕膜顶叶的行宫腔未包括的胎盘和是并列以绒毛膜绒毛的;和2)的底蜕膜,位于它是由间质滋养层10( 图1)侵入胎盘的基板。接触的这些区域的亲密创建胎儿抗原暴露于母体免疫系统11-13的条件。不足为奇的是,白细胞包括多达蜕膜细胞8,9,14,15 30-40%除了典型的基质型细胞和腺细胞8,14,16。白细胞在母胎界面的作用涵盖了包括滋养细胞浸润17的限制,螺旋动脉18,19的重塑,维护产妇宽容12,20和劳动力21-26启动多个进程。两者的适应性和免疫系统, ,T细胞,巨噬细胞,中性粒细胞,B细胞,树突细胞,和NK细胞,已在蜕膜组织确定了先天四肢,以及它们的白细胞比例和激活状态已示出,以改变空间和时间整个妊娠期6-10,12,14,24,27-30。在白细胞种群和/或功能的扰动与自然流产31,先兆子痫32,宫内生长受限32,33,和早产7,24相关联。因此,在人类母胎界面的表型特征和白细胞功能的研究将有助于失调在怀孕紊乱的免疫途径的阐明。

一种用于确定表型和白细胞的功能特性被流式细胞术的最有力的工具,技术,允许多个参数同时34-36的定量分析。分析白细胞通过流式细胞术,在单细胞悬浮液中的白细胞的隔离是必需的。因此,一个方法来分离浸润亮氨酸从母胎界面kocytes是需要研究其表型和功能特性。

几种方法已经被描述,从人类母胎界面10,14,25,27,37-39隔离白细胞。虽然一些应用机械分解10,25,27,38,别人用酶消化37,40进行组织分离。因为机械解聚产生较低产量和降低的生存力41,和酶解可影响生存力和细胞表面标记物保留42,本文所描述的方法结合温和机械解离与酶预处理,以增加分离的白细胞的产量而不影响细胞生存力。方法类似的组合已经被证明是有效的,从在母胎界面39蜕膜组织白细胞的隔离。因此,本文描述的协议涉及机甲nical分解与自动组织离解,增加一致性,同时节省时间和劳动时与反对手术刀,刀片,或手术剪10,28比传统的切碎。选择用于组织解离酶是的Accutase。不同于常用的胶原酶43,分散酶44,和胰蛋白酶45的Accutase(细胞分离液)结合了一般的蛋白和有助于高效而温和的解离46,47胶原溶解活动。解离后,将白细胞从蜕膜细胞通过密度梯度离心的总人口富集。各种密度梯度介质先前已经利用,其中最常见的是珀(胶体二氧化硅粒子的悬浮液)48和聚蔗糖(蔗糖的具有高合成分子量的聚合物)49。隔离的蔗糖聚合物的卓越效率一直previously所示50,和本文中进一步描述的方案证明该密度梯度介质能产生单核白细胞的足够高的纯度。

因此,本文描述的协议相结合的机械组织分解用自动组织离解,酶消化用细胞分离溶液,并用密度梯度介质(1.077 + 0.001g /每毫升)以分离从人蜕膜白细胞白细胞的分离。该协议已经被证明维持细胞表面抗原连同细胞活力。分离的白细胞可用于多种应用,包括免疫流式细胞仪和体外功能研究。

Protocol

该协议适用于由底蜕膜及蜕膜在顶叶准备免疫流式细胞仪检测白细胞隔离。此外,可用于细胞分选,细胞培养,RNA分离和细胞学分离的细胞。与之前在此协议中提到的样品的工作,人类伦理委员会批准,必须从本地研究伦理委员会和伦理审查委员会获得的。收集和利用人力样本用于研究目的的批准了儿童健康和人类发展(NICHD)的尤尼斯·肯尼迪·施莱佛国立研究所的机构审查委员会,健康?…

Representative Results

。在母胎界面人类组织(底蜕膜及蜕膜顶叶)的解剖见图1此过程包括基板,其中包括底蜕膜( 图1A – D)的清扫。通过除去从基板(图1C)的胎盘绒毛(胎侧)得到的蜕膜。蜕膜是顶叶轻轻刮绒毛膜( 图1E – F)收集。 图2显示了孤立的巨噬细胞(CD14 +),从使用磁性细胞分选在足月妊娠蜕膜收集顶叶的形态。…

Discussion

在人类母胎界面白细胞浸润的功能和表型特征的表征是必不可少的,导致妊娠疾病的免疫机制的理解。若干技术以分离从人母胎界面白细胞整个怀孕10,14,25,28,37,42,43了描述。然而,每一个这些技术是截然不同的,使用不同的酶或酶组合,需要不同的解离时间,不指定组织的数量时,以及最重要的是,并不总是指定分离的细胞的生存力。本文所描述的协议允许浸润在人类母胎界面白细胞(蜕…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由儿童健康和人类发展的尤尼斯·肯尼迪·施莱佛国立研究所,美国国立卫生研究院/ DHHS支持。这项工作也得到了支持,在某种程度上,在孕产妇,围产期和儿童健康的韦恩州立大学围产期倡议。 我们非常感谢莫林McGerty(韦恩州立大学)为她的手稿的读数。

Materials

Dissection
Sterile dissection tools: surgical scissors, forceps, and fine-tip tweezers  Any vendor 20012-027
1X phosphate buffered saline (PBS) Life Technologies (1X PBS)
Large and small Petri dishes Any vendor
Dissociation
Accutase Life Technologies A11105-01 (cell detachment solution)
Sterile 2 mL safe-lock conical tubes Any vendor
50 mL conical centrifuge tubes Any vendor
100 µm cell strainers FALCON/Corning 352360
5 mL round bottom polystyrene test tubes Any vendor
Transfer pipettes Any vendor
C tubes Miltenyi Biotec 130-093-237
Cell Culture
RPMI culture medium 1640  Life Technologies 22400-089 (1X) (10% FBS and 1% P/S)
Plastic chamber slides Thermo Scientific 177437
Incubator Thermo Scientific Corporation HEPA Class 100
Water bath Fisher Scientific ISOTEMP 110
Cell counter Nexelcom Cellometer Auto2000
Microscope Olympus Olympus CKX41
Cell Separation
MS columns Miltenyi Biotec 130-042-201
Cell separator Miltenyi Biotec 130-042-109
30μm pre-separation filters Miltenyi Biotec 130-041-40
Multistand Miltenyi Biotec 130-042-303
15mL safe-lock conical tubes Any Vendor
MACS buffer  (0.5% bovine serum albumin, 2mM EDTA and 1X PBS)
Reagents
FcR Blocking Miltenyi Biotec 130-059-901 (Fc Block)
Anti-human cell surface antigen antibodies  BD Biosciences (Table 1)
Bovine serum albumin  Sigma A7906
LIVE/DEAD viability dye BD Biosciences 564406
Lyse/Fix buffer  BD Biosciences 346202
FACS buffer  (1% BSA, 0.5% Sodium Azide, and 1X PBS)
Staining buffer  BD Biosciences 554656
Trypan Blue solution 0.4% Life Technologies 15250-011
Ficoll GE Healthcare 17-1440-02 20% density gradient media
Additional Instruments
Incubator with shaker Thermo Scientific MAXQ 4450
Flow cytometer BD Biosciences LSR-Fortessa
Centrifuge  Beckman Coulter SpinChron DLX
Vacuum system Any vendor
Automatic tissue dissociator  Miltenyi Biotec gentleMACS Dissociator

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Cite This Article
Xu, Y., Plazyo, O., Romero, R., Hassan, S. S., Gomez-Lopez, N. Isolation of Leukocytes from the Human Maternal-fetal Interface. J. Vis. Exp. (99), e52863, doi:10.3791/52863 (2015).

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