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.

In humans, the maternal-fetal interface represents an area of direct contact between maternal (decidua) and fetal (chorion or trophoblast) tissues. This interface includes: 1) the decidua parietalis that lines the uterine cavity not covered by the placenta and is in juxtaposition to the chorion laeve; and 2) the decidua basalis, located in the basal plate of the placenta where it is invaded by interstitial trophoblasts¹⁰ (Figure 1). The intimacy of these areas of contact creates conditions for fetal antigenic exposure to the maternal immune system^11-13. Not surprisingly, leukocytes comprise up to 30-40% of the decidual cells^8,9,14,15 in addition to typical stromal-type cells and glandular cells^8,14,16. The role of leukocytes at the maternal-fetal interface encompasses multiple processes that include the limitation of trophoblast invasion¹⁷, remodeling of spiral arteries^18,19, maintenance of maternal tolerance^12,20, and initiation of labor^21-26. Leukocytes of both the adaptive and innate limbs of the immune system, i.e., T cells, macrophages, neutrophils, B cells, dendritic cells, and NK cells, have been identified in the decidual tissues, and their proportions and activation status have been shown to vary spatially and temporally throughout gestation^{6-10,12,14,24,27-30}. Perturbations in the leukocyte population and/or function are associated with spontaneous abortion³¹, preeclampsia³², intrauterine growth restriction^32,33, and preterm labor^7,24. Therefore, the study of the phenotypic characteristics and functionality of leukocytes at the human maternal-fetal interface will facilitate the elucidation of the immunological pathways dysregulated in pregnancy disorders.

One of the most powerful tools used to determine the phenotype and functional properties of leukocytes is flow cytometry, technology that allows the quantitative analysis of multiple parameters simultaneously^34-36. To analyze leukocytes by flow cytometry, isolation of the leukocytes in a single-cell suspension is required. Therefore, a method to separate infiltrating leukocytes from the maternal-fetal interface is needed to study their phenotypic and functional properties.

Several methods have been described to isolate leukocytes from the human maternal-fetal interface^{10,14,25,27,37-39}. While some apply mechanical disaggregation^10,25,27,38, others use enzymatic digestion^37,40 for tissue dissociation. Because mechanical disaggregation produces a lower yield and reduced viability⁴¹, and enzymatic dissociation can affect viability and cell surface marker retention⁴², the method described herein combines gentle mechanical dissociation with enzymatic pre-treatment to increase the yield of isolated leukocytes without compromising cell viability. A similar combination of methods has been demonstrated to be effective in the isolation of leukocytes from the decidual tissues at the maternal-fetal interface³⁹. Therefore, the protocol described herein involves mechanical disaggregation with an automatic tissue dissociator that increases consistency while saving time and labor when compared to traditional mincing with opposing scalpels, razor blades, or surgical scissors^10,28. The enzyme chosen for tissue dissociation was Accutase. Unlike commonly used collagenase⁴³, dispase⁴⁴, and trypsin⁴⁵, Accutase (a cell detachment solution) combines both general proteolytic and collagenolytic activities that contribute to efficient yet gentle dissociation^46,47. After dissociation, the leukocytes are enriched from the total population of the decidual cells by density gradient centrifugation. Various density gradient media have been previously utilized, the most common of which are Percoll (a suspension of colloidal silica particles)⁴⁸ and Ficoll (a polymer of sucrose with a high synthetic molecular weight)⁴⁹. The superior efficiency of isolation by the sucrose polymer has been previously shown⁵⁰, and the protocol described herein further proves that this density gradient media produces a sufficiently high purity of mononuclear leukocytes.

Hence, the protocol described herein combines the mechanical tissue disaggregation with an automatic tissue dissociator, enzymatic digestion with a cell detachment solution, and leukocyte separation with a density gradient media (1.077 + 0.001 g/ml) to isolate leukocytes from human decidual tissues. This protocol has been proven to preserve cell surface antigens along with cell viability. The isolated leukocytes can be used for multiple applications that include immunophenotyping with flow cytometry and functional studies in vitro.

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 Institutes of Health (NIH), Department of Health and Human Services (DHHS, Bethesda, MD, USA) and Wayne State University (Detroit, MI, USA). Written informed consent was obtained from all pregnant women prior to the collection of tissue samples.
NOTE: While working with animal blood, cells, or hazardous agents as mentioned in this protocol, it is essential that proper biosafety and laboratory safety actions be followed.

1. Dissection of the Human Decidual Tissues

NOTE: The basal plate is the base beneath and attached to the placenta and represents the maternal surface. The chorioamniotic membranes include the amnion and the chorion. The basal plate includes the decidua basalis and the chorion includes the decidua parietalis (Figure 1).

1. Decidua Basalis
   1. Dissect a piece of the basal plate from one cotyledon of the placenta (Figures 1A and 1B).
   2. Place it on a sterile cutting board with the placental villi facing upward. The side showing the placental villi is usually bloody red with hairy tissue in appearance. The basal plate is smooth and pale red in color.
   3. Use sharp, fine-point scissors and forceps to remove the villous tissue and blood vessels. Keep the tissue soaked in 1x PBS (Ca²⁺- and Mg²⁺- free) during the process (Figure 1C). Collect 2 to 3 of these pieces and rinse them thoroughly with 1x PBS to remove the blood (Figure 1D).
2. Decidua Parietalis
   1. Dissect a piece of the chorioamniotic membranes (approximately 10 cm x 10 cm, Figure 1E). Place it on the board with the chorion facing upward. The chorionic side contains bloody clots and is usually light yellowish in color.
   2. Use fine-point forceps to remove as many of the blood clots as possible (Figure 1E). Rinse the membrane in sterile 1x PBS to clean the excess blood from the membrane until the 1x PBS runs clear.
   3. Use a disposable sterile cell scraper to gently scrape the decidual layer from the membrane. Apply sterile 1x PBS on the membrane while completing this process (Figure 1F). Collect the decidual tissues and place them in a 50 ml plastic tube with sterile 1x PBS (Figure 1G).

2. Mechanical Disaggregation and Enzymatic Digestion

1. Wash the tissue dissected from the decidua basalis (from Step 1.1.3) or the decidua parietalis (from Step 1.2.3) with sterile 1x PBS in a 50 ml plastic tube. Collect tissue pellets by centrifugation at 300 x g for 5 min at RT.
2. Aspirate the supernatant located above the tissue pellet carefully without disturbing the pellet.
   NOTE: At this point, the pellets are very loose because they contain red blood cells. If the volume of the pellet is about or less than 3 ml, re-suspend the pellet in 6 ml of cell detachment solution pre-warmed to 37 °C. If the pellet is larger than 3 ml of volume, add more cell detachment solution (add about 2 times the volume of the tissue samples).
3. Transfer the homogenized tissues (decidua basalis + cell detachment solution or decidua parietalis + cell detachment solution) to a C tube.
4. Place the C tube in the automatic dissociator and run the corresponding program.
   NOTE: The program for the decidua basalis runs for 17 sec with 668 total rounds per run. The program for the decidua parietalis runs for 37 sec with 235 total rounds per run. These programs have been customized to isolate leukocytes from the decidua basalis or the decidua parietalis with good yield and cell viability.
5. Following the mechanical disaggregation of the tissues, digest the tissues with a commercially available cell detachment solution such as accutase; (a cocktail that contains proteolytic and collagenolytic enzymes) for 45 min at 37 °C with gentle agitation.

3. Isolation of Leukocytes

1. Following incubation, add 10 ml of 1x PBS to the digestion mixture and pass it through a 100 µm cell strainer into a 50 ml plastic tube. Depending on the stickiness of the digestion mixture, one may need to use several cell strainers for one mixture.
2. Fill the tube with 1x PBS and centrifuge at 300 x g for 5 min at RT. Remove the 1x PBS above the cell pellets carefully and re-suspend the pellets with 5 ml of ice-cold FACS buffer.
   NOTE:To study the polymorphonuclear and mononuclear leukocytes together, go to Step 6.1; to study the mononuclear leukocytes, continue to Step 3.3.
3. Add 5 ml of 20% density gradient media (1.077 + 0.001 g/ml) to a 15 ml plastic tube and slowly overlay the cell suspension on top of the density gradient media. While layering the sample, it is important not to mix the density gradient media with the cell suspension.
4. Centrifuge with a swing-out rotor at 500 x g for 30 min at 4 ˚C without the brake. It is very important to turn off the brake to avoid mixing the cells and losing the gradient. Leukocytes will be found in the interface between the density gradient media and the FACS buffer.
5. Remove the upper layer that contains the FACS buffer and cell debris very carefully using a pipette. The band at the interface contains the decidual mononuclear cells and a portion of the polymorphonuclear leukocytes.
6. Transfer the cells at the interface completely to a new 15 ml plastic tube. Add at least 3 times more volume of FACS buffer.
7. Centrifuge at 300 x g for 5 min to pelletize the cells. Aspirate the supernatant and wash the cells with FACS buffer. Pelletize the cells with another centrifugation at 300 x g for 5 min at RT.
   NOTE: To perform the cell culture, see Step 4. To perform cell sorting, see Step 5. To perform immunophenotyping, see Step 6.

4. Applications – Cell culture

1. Re-suspend the cells from Step 3.7 in 1 ml of RPMI culture medium 1640. Count the viable cells using an automatic cell counter or a hemocytometer and a trypan blue solution 0.4%.
2. Add up the amount of RPMI culture medium to make a cell concentration of 1 x 10⁶ cells/ml. Cells are now ready for culture.

5. Applications – Isolation of Macrophages for Primary Cell Culture

1. To isolate CD14+ cells, magnetically label the cells from Step 3.7 with CD14 microbeads (20 µl beads per 10⁷ cells), incubate for 15 min at 4 ˚C, and separate CD14+ cells from other types of cells by applying a magnetic field. After 2 washes with MACS buffer and removal of the magnetic field, elute the magnetically retained CD14+ cells with MACS buffer.
2. Following centrifugation, re-suspend the cell pellets in RPMI culture medium 1640. The cells are ready for plating (Figure 2).

6. Applications – Immunophenotyping

1. To use the cells for immunophenotyping, re-suspend the cells from Step 3.7 in 1 ml of 1x PBS. Count the viable cells using an automatic cell counter or a hemocytometer and a trypan blue solution 0.4%.
2. Label the cells with a fixable viability dye (Figure 3). Wash the cells twice with FACS buffer and re-suspend the cells in stain buffer. Incubate with an FcR blocker for 15 min at 4 °C.
3. Add extracellular antibodies conjugated to fluorochromes. For example, in this protocol, use anti-CD3, anti-CD19, anti-CD14, anti-CD15, and anti-CD56 antibodies (Table 1). Incubate for 30 min at 4 °C in darkness.
4. After 2 washes with 1x PBS, the cells will be analyzed in 500 µl of stain buffer using a flow cytometer. A representation of the gating strategy used to analyze the leukocyte sub-populations found in the decidual tissues is shown in Figure 4.

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

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