This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH/DHHS. This work was also supported, in part, by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. &#160;We gratefully acknowledge Maureen McGerty (Wayne State University) for her critical readings of the manuscript.

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...

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&#160;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 ...

Pregnancy &#160;is characterized by three distinct immunological phases: 1) implantation and early placentation associated with a pro-inflammatory response (i.e., implantation resembles an &#8216;open wound&#8217;); 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 state1-7. 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 pregnancy6-9.
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 system11-13. Not surprisingly, leukocytes comprise up to 30-40% of the decidual cells8,9,14,15 in addition to typical stromal-type cells and glandular cells8,14,16. The role of leukocytes at the maternal-fetal interface encompasses multiple processes that include the limitation of trophoblast invasion17, remodeling of spiral arteries18,19, maintenance of maternal tolerance12,20, and initiation of labor21-26. 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 gestation6-10,12,14,24,27-30. Perturbations in the leukocyte population and/or function are associated with&#160;spontaneous abortion31, preeclampsia32, intrauterine growth restriction32,33, and preterm labor7,24. 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 simultaneously34-36. 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 interface10,14,25,27,37-39. While some apply mechanical disaggregation10,25,27,38, others use enzymatic digestion37,40 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&#160;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 scissors10,28. 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 dissociation46,47. 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Sterile dissection tools: surgical scissors, forceps, and fine-tip tweezers&#160;</td>
			<td>Any vendor</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td></td>
			<td>(1X PBS)</td>
		</tr>
		<tr>
			<td>Large and small Petri dishes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissociation</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td>(cell detachment solution)</td>
		</tr>
		<tr>
			<td>Sterile 2 mL safe-lock conical tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainers</td>
			<td>FALCON/Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom polystyrene test tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>RPMI culture medium 1640&#160;</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td>(1X) (10% FBS and 1% P/S)</td>
		</tr>
		<tr>
			<td>Plastic chamber slides</td>
			<td>Thermo Scientific</td>
			<td>177437</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific Corporation</td>
			<td>HEPA Class 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>ISOTEMP 110</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Nexelcom</td>
			<td>Cellometer Auto2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Olympus CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Separation</td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#956;m pre-separation filters</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL safe-lock conical tubes</td>
			<td>Any Vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS buffer&#160;</td>
			<td></td>
			<td></td>
			<td>(0.5% bovine serum albumin, 2mM EDTA and 1X PBS)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>FcR Blocking</td>
			<td>Miltenyi Biotec</td>
			<td>130-059-901</td>
			<td>(Fc Block)</td>
		</tr>
		<tr>
			<td>Anti-human cell surface antigen antibodies&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Bovine serum albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD viability dye</td>
			<td>BD Biosciences</td>
			<td>564406</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyse/Fix buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>346202</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td></td>
			<td></td>
			<td>(1% BSA, 0.5% Sodium Azide, and 1X PBS)</td>
		</tr>
		<tr>
			<td>Staining buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>20% density gradient media</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</td>
		</tr>
		<tr>
			<td>Incubator with shaker</td>
			<td>Thermo Scientific</td>
			<td>MAXQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSR-Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>SpinChron DLX</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum system</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic tissue dissociator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>gentleMACS Dissociator</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from the human maternal-fetal interface

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&#160;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&#160;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.

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-<stro...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Human Maternal-fetal Interface at JoVE.com

Isolation of Leukocytes from the Human Maternal-fetal Interface

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.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

isolation-of-leukocytes-from-the-human-maternal-fetal-interface

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JoVE Journal

Immunology and Infection

15.8K 조회수.  NICHD/NIH/DHHS. 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. 