Isolation of Leukocytes from the Human Maternal-fetal Interface

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

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Introduction

Pregnancy  is characterized by three distinct immunological phases: 1) implantation and early placentation associated with a pro-inflammatory response (i.e., implantation resembles an ‘open wound’); 2) the second trimester and most of the third trimester of pregnancy when immune homeostasis is achieved through a predominantly anti-inflammatory state at the maternal-fetal interface; and 3) parturition, a pro-inflammatory state^1-7. Immune cells play an important role in the regulation of the inflammatory response at the maternal-fetal interface where their abundance and localization change throughout pregnancy^6-9.

Protocol

This protocol is appropriate for leukocyte isolation from the decidua basalis and decidua parietalis in preparation for immunophenotyping by flow cytometry. Furthermore, the isolated cells can be used for cell sorting, cell culture, RNA isolation, and cytology. Before working with the samples mentioned in this protocol, human ethical approval must be obtained from the Local Research Ethics Committee and Institutional Review Boards. The collection and utilization of human samples for research purposes were approved by the Institutional Review Boards of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institu....

Results

The dissection of human tissues at the maternal-fetal interface (decidua basalis and decidua parietalis) is shown in Figure 1. This procedure includes the dissection of the basal plate, which includes the decidua basalis (Figure 1A-D). The decidua basalis is obtained by removing the placental villi (fetal side) from the basal plate (Figure 1C). The decidua parietalis is collected by gently scraping the chorionic membrane (Figure 1E-

Discussion

Characterization of the functional and phenotypic properties of infiltrating leukocytes at the human maternal-fetal interface is essential to the understanding of the immune mechanisms that lead to pregnancy disorders. Several techniques have been described in order to isolate leukocytes from the human maternal-fetal interface throughout pregnancy ^{10,14,25,28,37,42,43}. However, each of these techniques is distinct, uses different enzymes or enzyme combinations, requires different dissociation times, does .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
30 μm pre-separation filters	Miltenyi Biotec	130-041-40
Multistand	Miltenyi Biotec	130-042-303
15 ml safe-lock conical tubes	Any Vendor
MACS buffer			(0.5% bovine serum albumin, 2 mM 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			(0.1% BSA, 0.05% Sodium Azide, and 1X PBS, pH=7.4)
Staining buffer	BD Biosciences	554656
Trypan Blue solution 0.4%	Life Technologies	15250-011
Ficoll	GE Healthcare	17-1440-02	20% density gradient media (1.077 + 0.001 g/ml)
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