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Oct 2021

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Isolation of First-Trimester and Full-term Human Placental Hofbauer Cells
妊娠早期和足月人胎盘霍夫鲍尔细胞的分离   

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Abstract

The placenta is the crucial organ that regulates the health of both mother and fetus during pregnancy. The human placenta is composed of villous tree-like structures that embed into the maternal decidua. Within the stroma of the villi resides a population of fetally-derived macrophages, the Hofbauer cells (HBC). HBC are the only fetal immune cells found within the placenta in the steady-state and are thought to play a crucial role in placental function. From the 10th week of gestation, maternal blood flow into the intervillous space begins, resulting in the placental villi becoming bathed in maternal blood. To study HBC it is necessary to develop techniques that allow for their specific isolation and distinction from maternal blood monocytes and decidual macrophages. Here, we describe a protocol that explains step-by-step the strategy we have developed that allows the specific isolation of HBC.

Keywords: Placenta (胎座), Tissue processing (组织处理), Hofbauer cells (霍夫鲍尔细胞), Macrophages (巨噬细胞)

Background

The human placenta is a highly specialized organ that is crucial for both maternal and fetal health during pregnancy. It is made up of highly branched villous tree-like structures that are bathed in maternal blood from approximately the 10th week of gestation (Jauniaux et al., 2000). Anchoring villi attach the placenta to the endometrium, which is transformed during placentation into the decidua. A population of fetal tissue-resident macrophages, the Hofbauer cells (HBC), are abundant within the stroma of placental villi. Early studies determined that these cells are of fetal origin based on Y-chromosome detection in pregnancies with male fetuses (Kim et al., 2008). HBC are the only immune cell population on the fetal side of the placental barrier in the steady-state (Thomas et al., 2021). Accordingly, they are thought to have important roles in the regulation of placental growth and homeostasis and to act as immune sentinels, protecting the fetus from infection in utero. However, compared to other adult tissue-resident macrophages, HBC remain poorly understood. A range of protocols has been used to isolate HBC, as excellently reviewed elsewhere (Tang et al., 2011). However, a standard technique has not been established across the field. Many of the protocols used are suboptimal for the isolation of tissue macrophages. For example, unnecessarily long digestion times (sometimes up to 3 h) and the failure to use DNase I greatly affect HBC viability. Furthermore, HBC are typically isolated as total CD14+ cells from placental tissue for phenotypic and functional analysis. However, we have previously shown that maternal cells can comprise up to 30% of CD14+ cells isolated from first-trimester placental digests (Thomas et al., 2021). By adding common HLA allotype antibodies to our flow cytometry panel, we can distinguish maternal from fetal cells and ensure the isolation of a pure population of HBC. Therefore, in all future research of HBC using in vitro assays, additional steps that are described here should be performed to ensure that true fetally-derived HBC are studied with optimal viability.


In this article, we provide step-by-step protocols to isolate HBC from first-trimester and full-term human placental tissue with high purity. Procedures B and C describe how to obtain single-cell suspensions from first-trimester and full-term placental tissue, respectively. Procedure D describes how to obtain HBC from placental digests with high purity by fluorescence-activated cell sorting (FACS). Following these protocols allows investigating HBC properties with downstream in vitro assays to understand their functional properties further. A scheme of the overall procedure of HBC isolation from first-trimester and full-term placenta is shown in Figure 1.



Figure 1. Isolation of HBC from human placenta. A schematic outlining the procedure for isolating HBC from first-trimester and full-term placenta.


Materials and Reagents

  1. Disposable scalpels, No. 22 (Swann Morton, catalog number: 508)

  2. Pasteur pipette (ELKay, catalog number: 127-P503-STR)

  3. Muslin gauze (Winware food grade, catalog number: E948)

  4. Cell strainers 30 µm (Miltenyi Biotec, catalog number: 130-041-407)

  5. Cell strainers 70 µm (BD Falcon, catalog number: 352350)

  6. Cell strainers 100 µm (BD Falcon, catalog number: 352360)

  7. Cell strainers 400 µm (Pluriselect, catalog number: 43-50400-50)

  8. Petri dishes 140 mm (Sterilin, catalog number: SC269)

  9. Falcon 50 ml round bottom polypropylene test tubes (Corning, catalog number: 352070)

  10. Polypropylene round-bottom FACS tubes 5 ml (Falcon, catalog number: 352063)

  11. Dissection forceps (Scientific Laboratory Supplies, catalog number: SR04010)

  12. Surgical straight scissors (SAMCO, catalog number: E101/01)

  13. 250-ml glass Duran bottles (Scientific Laboratory Supplies, catalog number: F151164 )

  14. Funnel (Scientific Laboratory Supplies, catalog number: FUN1260)

  15. Collagenase type V (Sigma-Aldrich, catalog number: C9263)

  16. DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, catalog number: D8417)

  17. DNase I (Roche, catalog number: 10104159001)

  18. Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E9884)

  19. Fetal calf serum (heat inactivated) (Biosera, catalog number: FB -1001)

  20. Flow cytometry antibodies (see Table 1)


    Table 1. Flow cytometric antibodies

    Antibody Company Dilution
    Antibody CD3 FITC, clone: UCHT1 Biolegend, catalog number: 300406 1:100
    Antibody CD14 PE-Dazzle 594, clone: HCD14 Biolegend, catalog number: 325634 1:100
    Antibody CD19 FITC, clone: SJ25C1 Biolegend, catalog number: 363008 1:100
    Antibody CD20 FITC, clone: 2H7 Biolegend, catalog number: 302304 1:100
    Antibody CD45 PerCp-Cy5.5, clone: 2D1 Biolegend, catalog number: 368503 1:40
    Antibody CD66b FITC, clone: G10F5 Biolegend, catalog number: 305103 1:100
    Antibody CD335 FITC, clone: 9E2 Biolegend, catalog number: 331921 1:100
    Antibody FOLR2 APC, clone: 94b/FOLR2 Biolegend, catalog number: 391705 1:1,000
    Antibody HLA-A2 APC-Cy7, clone: BB7.2 Biolegend, catalog number: 343310 1:50
    Antibody HLA-A3 BV650, clone: GAP.A3 BD Biosciences, catalog number: 747774 1:100
    Antibody HLA-B7 PE, clone: BB7.1 Biolegend, catalog number: 372404 1:100


  21. Pancoll (density 1.077 mg/ml) (Pan Biotech, catalog number: P04-601000)

  22. Penicillin/streptomycin solution (Sigma-Aldrich, catalog number: P0781)

  23. RPMI-1640 Medium (Sigma-Aldrich, catalog number: R7388)

  24. Trypsin-250 (Pan Biotech, catalog number: P10-025100P)

  25. Glucose (Sigma-Aldrich, catalog number: D9434)

  26. NaCl (MP Biomedicals, catalog number: 151944)

  27. KCl (MP Biomedicals, catalog number: 194738)

  28. Disodium hydrogen orthophosphate (Fisher Scientific, catalog number: S/4520/53)

  29. Potassium dihydrogen orthophosphate (Fisher Scientific, catalog number: P/4800/53)

  30. Phosphate-buffered saline (PBS) 1× concentration

  31. Human serum (Sigma-Aldrich, catalog number: H4522)

  32. Mouse serum (Sigma-Aldrich, catalog number: M5905)

  33. Rat serum (Sigma-Aldrich, catalog number: R9759)

  34. Brilliant stain buffer (BD Horizon, catalog number: 563794)

  35. Trypsin/EDTA, 0.2% solution (see Recipes)

  36. The RF10 medium (see Recipes)

  37. The FACS buffer (see Recipes)

  38. Blocking buffer (see Recipes)

Equipment

  1. BD FACSAriaTM III Cell Sorter (or similar), 4-laser cell sorter for containment level 2 samples fitted with a 100 µm nozzle (BD Biosciences, model: FACSAriaTM III)

  2. Temperature controlled centrifuge with lids for buckets (Eppendorf Centrifuge 5810R, catalog number: F151380)

  3. Hot plate magnetic stirrer (DLAB, catalog number: DL2255-380H)

  4. Waterbath (VWR, catalog number: 462-0557)

  5. Haemocytometer (VWR, catalog number: BR718605)

  6. Light microscope (Leica, catalog number: 11964479)

Software

  1. BD FACSDivaTM software

    A collection of tools for flow cytometer and application setup, data acquisition, and analysis. It runs on the Microsoft® Windows 10 64-bit operating system.

  2. FlowJoTM (Becton, Dickinson, and Company; 2019) software

    A package for analyzing flow cytometry data. Files produced by modern flow cytometers are written in the Flow Cytometry Standard format with a .fcs file extension. FlowJo will import and analyze cytometry data regardless of which flow cytometer is used to collect the data. It is available for both Mac and PC.

Procedure

  1. Biological samples

    It is essential that all work using human tissue receives prior approval from the appropriate institutional and/or national review boards; donors must always provide informed consent. For this work, tissue samples were obtained with written informed consent from all participants under ethical approval from the East of England–Cambridge Central Research Ethics Committee (17/EE/0151).

    All work with unscreened human placental tissue is regarded as a potential hazard for group 3 blood-borne pathogens and should be carried out in containment level 2 facilities.


  2. Obtaining a single-cell suspension from first-trimester placental tissue

    HBC are fetal macrophages found within the stroma of the villi, which make up the placenta. A series of enzymatic digestions ensures the breakdown of the tissue to obtain a single-cell suspension with high HBC yield and viability (see Note 2). This single-cell suspension will also contain other cell types that make up the placenta, including trophoblast cells and fibroblasts, as well as placenta-associated maternal monocytes/macrophages (PAMM) (Thomas et al., 2021). A purified population of HBC can be obtained from this digest by FACS, a method described in further detail in Procedure D.

    1. Prepare reagents and equipment

      The same materials and equipment are required for Procedures B. Media, reagents, and tools must be sterile (see Note 1).

      1. Prepare the Trypsin/EDTA solution (see Recipes)

      2. Prepare the RF10 medium (see Recipes)

      3. Prepare the collagenase V stock solution (10 mg/ml) by dissolving it in RF10. Distribute the solution to 2.5 ml aliquots and store at -20 °C for up to 6 months.

      4. Prepare the DNase I stock solution (10 mg/ml) by dissolving it in sterile water. Distribute the solution to 100 µl aliquots at -20 °C for up to 6 months.

      5. Prepare PBS with DNase I (20 µg/ml) by diluting an aliquot of DNase I (from Procedure B1d) in 50 ml of 1× PBS. Keep sterile at 4 °C and make fresh each time (see Note 3).

      6. Before beginning placental tissue processing, warm Trypsin/EDTA (0.2% solution), collagenase V, 1× PBS, and Pancoll to 37 °C. Set a heated shaker to 37 °C, with 500 rotations per min (RPM), and set a magnetic stirrer hot plate to 37 °C.

    2. Processing first-trimester placental tissue to obtain a single-cell suspension

      1. Process the placenta immediately upon receipt.

      2. Rinse the placenta in 1× PBS gently with a magnetic stirrer for 10 min at room temperature (RT).

      3. Transfer placenta to a Petri dish.

      4. Remove any blood clots with forceps.

      5. Hold down one end of the placental unit and gently scrape the villi from the chorionic membrane with a scalpel (see Figure 2A).

      6. Discard the fetal membrane.

      7. Using a Pasteur pipette (with the tip cut off), transfer scraped villi into 75 ml of pre-warmed 0.2% trypsin-EDTA in a sterile 250-ml glass Duran bottle. Place on the hot plate magnetic stirrer heated to 37 °C and stir gently for 7 min.

      8. Add 2 ml of FCS to stop the trypsinisation of the tissue.

      9. Filter the trypsined tissue through a sterile muslin gauze in a funnel.

      10. Rinse with PBS at RT.

      11. Keep both the digested tissue that ran through the gauze and the remaining tissue on the gauze.

      12. Retrieve the undigested tissue from the gauze using a scalpel and place it in a 50 ml Falcon tube. Add 2.5 ml of collagenase V and 100 µl of DNase I and add PBS to a final volume of 25 ml (final concentration of 1 mg/ml Collagenase V and 40 µg/ml DNase I).

        Note: The addition of DNase I is crucial; macrophage yield and viability are greatly reduced in the absence of DNase I.

      13. Digest for 20-30 min at 37 °C on the heated shaker at 500 RPM.

      14. Filter the resultant cell suspension through the sterile muslin gauze in a funnel. Discard any undigested tissue and centrifuge the filtrate for 5 min at 120 × g, RT, to pellet the cells. Discard the supernatant.

      15. Pool the cells from steps i and n and wash in PBS with DNase I (see Note 3).

        Note: The cells from Step B2i will mainly contain maternal and trophoblast cells. We have found that HBC are sometimes also found in this digest, and pooling cells from Steps B2i and B2n will help to maximize HBC yield. However, it not essential to pool the cells from Steps B2i and B2n, as few HBC are found in the cellular suspension from Step B2i.

      16. Gently resuspend the cell pellet in 20 ml of PBS with DNase I.

      17. Filter the cell suspension through a 400 µm strainer.

      18. Layer the cell suspension onto 10 ml of Pancoll in a 50 ml Falcon tube.

      19. Centrifuge for 20 min at 600 × g, RT, acceleration 0, break 1.

      20. Collect the cells at the interface using a Pasteur pipette.

      21. Wash with PBS with DNase I and centrifuge for 5 min at 120 × g, RT. Discard the supernatant.

      22. Repeat the previous step.

      23. Resuspend cells in PBS with DNase I and pass through a 70 µm strainer, count the cells with a haemocytometer, and place them on ice (see Notes 2 and 5).



      Figure 2. Scraping of placental villi. Images showing scraping of placental villi from (A) first-trimester and (B) full-term placenta.


  3. Obtaining a single-cell suspension from full-term placental tissue

    Human placenta collected from full-term pregnancies differs dramatically from those in the first trimester. As pregnancy progresses, placental villi continue sprouting, and the vascular network matures to increase the surface area and vascularisation of the organ and meet the needs of the growing fetus. Full-term placental villi have a more compact fibrous stroma with muscular arteries, which are absent in the first trimester (Castellucci and Kaufmann, 1982). For this reason, the digestion of full-term placental tissue for the isolation of HBC differs from that described in Procedure B for first-trimester placenta and is outlined in detail below.

    1. Prepare reagents and equipment

      Prepare reagents and equipment as described in Step B1.

    2. Process full-term placental tissue to obtain a single-cell suspension

      1. Process the placenta immediately upon receipt.

      2. Cut out a lobe from the whole placenta using surgical scissors.

        Note: For consistency between donors, always isolate the lobe from the same region.

      3. Rinse the placenta in PBS gently with a magnetic stirrer for 10 min at RT.

      4. Transfer to a Petri dish.

      5. Remove any blood clots with forceps.

      6. Remove and discard a thin layer (~1 mm) of tissue from the basal side (side which faces the mother) using surgical scissors.

        Note: This step ensures the removal of any decidual tissue from the placenta.

      7. Hold down one end of the placental unit and gently scrape the villi from the chorionic membrane with a scalpel (see Figure 2B).

      8. Discard the fetal membrane and any thick connective tissue fibers and visible blood vessels.

      9. Using a Pasteur pipette (with the tip cut off), transfer scraped villi into 75 ml of pre-warmed 0.2% trypsin-EDTA in a sterile glass Duran bottle (250 ml). Place on the hot plate magnetic stirrer heated to 37 °C and stir gently for 15 min (see Note 4).

      10. Add 2 ml of FCS to stop the trypsinisation of the tissue.

      11. Filter the trypsined tissue through a sterile muslin gauze in a funnel.

      12. Rinse the gauze with PBS at RT.

      13. Keep both the digested tissue that ran through the gauze and the remaining tissue on the gauze.

      14. Retrieve the undigested tissue from the gauze and place in a 50 ml Falcon tube. Add 2.5 ml of collagenase V and 200 µl of DNase I and add PBS to a final volume of 25 ml (final concentration of 1 mg/ml collagenase V and 80 µg/ml DNase I).

        Note: The addition of DNase is crucial; macrophage yield and viability are greatly reduced in the absence of DNase I.

      15. Digest for 60 min at 37 °C on the heated shaker at 500 RPM (see Note 4).

      16. Filter the digested tissue through a sterile muslin gauze in a funnel. Discard any undigested tissue and centrifuge the filtrate for 5 min at 120 × g, RT, to pellet the cells. Discard the supernatant.

      17. Repeat Steps B2o-B2w.


  4. Isolating Hofbauer cells from first-trimester and full-term placental digests

    In this protocol, single-cell digests obtained from Procedures B and C are stained with fluorescent-conjugated antibodies for the isolation of HBC by FACS. The protocol includes an optimized panel and gating strategy that allows isolating a highly purified population of HBC without contamination of maternal monocytes/macrophages that are found in placental digests.

    1. Prepare reagents and equipment (see Note 1)

      1. Prepare the FACS buffer (see Recipes). Keep sterile and store at 4 °C for up to 1 month.

      2. Prepare blocking buffer (see Recipes). Filter through a 0.2 µm sterile filter and store at 4 °C for up to 1 month.

        Note: All serum is heat inactivated by placing in a water bath at 56 °C for 30 min.

      3. Prepare 0.9 mM DAPI in sterile water. Store at -20 °C indefinitely.

      4. Prepare RF10 as described in Step B1b.


    2. Isolate HBC by FACS

      1. Pass cells through a 30 µm filter into a polypropylene FACS tube.

      2. Centrifuge for 5 min at 120 × g, 4 °C. Discard supernatant.

      3. Resuspend cells in 150 µl of human blocking buffer per 10 × 106 cells.

      4. Make antibody cocktail in 50 µl of Brilliant stain buffer per tube (follow dilutions in Table 1, for a final volume of 200 µl). The same antibody cocktail is used for first-trimester and full-term placental cells.

      5. Add the antibody cocktail to the cells and mix by gently vortexing.

      6. Incubate on ice for 20 min.

      7. Add 2 ml of cold FACS buffer and centrifuge for 5 min at 120 × g, 4 °C. Discard supernatant.

      8. Repeat previous step.

      9. Resuspend the cells in 1 ml of cold FACS buffer.

      10. Stain cells with DAPI (1:10,000) for 10 min prior to starting the sorting.

      11. See representative images of the gating strategy used to identify HBC within first-trimester and full-term placental digests in Figure 3 (see Note 6).

      12. Cells are sorted at 4 °C into appropriate collection tubes and buffers.



    Figure 3. Fluorescent activated cell sorting gating strategy to isolate HBC. (A, B) Representative images of the gating strategy used to distinguish fetal from maternal cells using antibodies to common HLA allotypes and identity CD45+CD14+FOLR2+ HBC (red gate) within placental digests using BD FACSDiva and Flowjo software. (A) First-trimester placental samples; fetal cells are HLA-A3+, and maternal cells are HLA-A3-. (B) Full-term placental cells; fetal cells are HLA-B7-, and maternal cells are HLA-B7+. Lineage markers (Lin) include CD3, CD19, CD20, CD66b, and CD335 in FITC.

Data analysis

Isolation of viable cells from placental tissues

Our protocol for generating single-cell suspensions from placental digests yields cells with ~90% viability from first-trimester placental tissue and ~80% viability from full-term placental tissue. After enriching viable HBC by cell sorting, we obtain HBC with ~95% viability as determined by cell counting with a hematocytometer and microscope (see Note 5).


Flow cytometric gating strategy to distinguish HBC from maternal macrophages

To separate contaminating maternal CD14+ monocytes and macrophages from fetal CD14+ HBC, we developed a flow cytometric gating strategy that can be easily adapted to the configuration of different cell sorters (Table 1). We demonstrate that anti-HLA typing antibodies can be used to distinguish the more abundant fetal CD14+ HBC from the less abundant maternal CD14+ monocytes and macrophages from both first-trimester and full-term placental digests (Figure 3) (see Note 6).

Notes

There are several parameters critical to the success of the protocols described here.

  1. It is essential to use sterile reagents and equipment throughout the protocols as HBC are responsive to bacterial products, which can alter the properties of the cells (Thomas et al., 2021). Once a single-cell suspension has been obtained, it is essential to keep it at 4 °C to avoid macrophage loss through adherence to plastic and minimize cell activation.

  2. The addition of DNase I during the digestion and subsequent PBS washing steps is crucial to ensure high HBC viability.

  3. The longer digest with both trypsin and collagenase V for full-term placental tissue is necessary to ensure complete digestion of the tissue and successful isolation of HBC. However, over-digestion and prolonged exposure to digestion enzymes reduce HBC viability.

  4. The total cell counts from first-trimester and full-term placental tissue digests vary between samples. Generally, a total of 20 × 106-50 × 106 cells can be expected from placental tissue that has been digested successfully. The yield of HBC sorted by FACS also varies between samples but is ~1-3% of all cells.

  5. To ensure the isolation of HBC by FACS with high purity, we include three HLA allotyping antibodies in our flow cytometric panel. Because HBC are fetal cells containing genetic material from the mother and father, one can expect an HLA allotype mismatch between the fetus and the mother in some cases. In these cases, we can ensure that sorted HBC are of fetal origin without the contamination of maternal cells, which are often present in placental digests (Figure 3). The specificity of the anti-HLA antibodies used in our staining panel has been validated by quantitative PCR (qPCR) on DNA from blood samples of HLA-typed donors. However, a shortcoming of the protocol described here is that if there is no HLA allotype mismatch between the fetus and mother for HLA-B7, HLA-A3, or HLA-A2, the protocol will fail to separate maternal from fetal cells. In this scenario, it is necessary to include additional markers in the flow cytometric panel that are expressed specifically by HBC. For example, we have previously shown that first trimester HBC do not express HLA-DR, whereas maternal macrophages do. Therefore, the addition of HLA-DR to the flow cytometric panel will allow the separation of first-trimester HBC from maternal macrophages in the absence of an HLA allotype mismatch (Thomas et al., 2021). HLA-DR cannot be used to distinguish maternal cells from full-term placental HBC because HBC express HLA-DR by full-term (Sutton et al., 1983).

Recipes

  1. Trypsin/EDTA [(0.2% solution); 0.2% (w/v) Trypsin - 250/0.02% (w/v) EDTA/PBS solution)]

    0.3 g Glucose

    12 g NaCl

    0.3 g KCl

    1.725 g disodium hydrogen orthophosphate

    0.3 g potassium dihydrogen orthophosphate

    2 g trypsin

    0.2 g EDTA

    1. Dissolve chemicals in 1 L of water

    2. Sterile-filter the solution using a 0.22 µm filter unit

    3. Distribute the stock solution to 75 ml aliquots and store at -20 °C for up to 6 months

  2. The RF10 medium

    Roswell Park Memorial Institute (RPMI) 1640 Medium

    10% heat-inactivated fetal calf serum

    1× penicillin/streptomycin solution

    100 units of penicillin, and 0.1 mg/ml streptomycin

  3. The FACS buffer

    2% heat-inactivated fetal calf serum and 2 mM EDTA in 1× PBS. Keep sterile and store at 4 °C for up to 1 month.

  4. Blocking buffer

    5% human serum, 1% rat serum, 1% mouse serum, 5% FCS, and 2 mM EDTA in 1× PBS

Acknowledgments

We thank the following for assistance: 1. The Flow Cytometry Core Facility at the Department of Pathology; 2. Lucy Gardner, Imogen Duncan, and Ritu Rani for their help collecting and processing placental samples; 3. Donors who participated in this study and the hospital staff; 4. Professor Stephen Charnock-Jones and Dr. Irving Aye Department of Obstetrics & Gynaecology, University of Cambridge, for help collecting full-term placental samples. This work was supported by the Wellcome Trust, Royal Society, Centre for Trophoblast Research, and Department of Pathology, University of Cambridge, UK. N.McG, is funded by a Wellcome Sir Henry Dale and Royal Society Fellowship (grant number 204464/Z/16/Z). J.T is funded by a Wellcome Trust PhD Studentship (grant number 215226/Z/19/Z).

This protocol was developed and adapted based on the previous work of others (Tang et al., 2011) and our own findings (Thomas et al., 2021).

Competing interests

The authors have no conflicts of interest to declare.

References

  1. Castellucci, M. and Kaufmann, P. (1982). A three-dimensional study of the normal human placental villous core: II. Stromal architecture. Placenta 3(3): 269-285.
  2. Jauniaux, E., Watson, A. L., Hempstock, J., Bao, Y. P., Skepper, J. N. and Burton, G. J. (2000). Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol 157(6): 2111-2122.
  3. Kim, J. S., Romero, R., Kim, M. R., Kim, Y. M., Friel, L., Espinoza, J. and Kim, C. J. (2008). Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology.Histopathology 52(4): 457-464.
  4. Sutton, L., Mason, D. Y. and Redman, C. W. (1983). HLA-DR positive cells in the human placenta. Immunology 49(1): 103-112.
  5. Tang, Z., Abrahams, V. M., Mor, G. and Guller, S. (2011). Placental Hofbauer cells and complications of pregnancy. Ann N Y Acad Sci 1221: 103-108.
  6. Thomas, J. R., Appios, A., Zhao, X., Dutkiewicz, R., Donde, M., Lee, C. Y. C., Naidu, P., Lee, C., Cerveira, J., Liu, B., Ginhoux, F., Burton, G., Hamilton, R. S., Moffett, A., Sharkey, A. and McGovern, N. (2021). Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells. J Exp Med 218(1).

简介

[摘要]胎盘是调节孕期母亲和胎儿健康的重要器官。人类的胎盘由绒毛状的树状结构组成,这些结构嵌入母体的蜕膜中。内的绒毛基质驻留群体fetally衍生巨噬细胞中,霍夫鲍尔细胞(HBC)。HBC是在胎盘内处于稳态的唯一胎儿免疫细胞,被认为在胎盘功能中起着至关重要的作用。从10个妊娠周,母体血液流入空间intervillous的开始,导致母体血液的胎盘绒毛变得沐浴。学习HBC 有必要开发一种技术,使其能够与母体血液单核细胞和蜕膜巨噬细胞进行特异性分离和区分。在这里,我们描述了一种协议,该协议逐步解释了我们开发的允许HBC特定隔离的策略。


[背景]人类胎盘是一个高度专业化的器官,在怀孕期间是母亲和胎儿的健康是至关重要的。它由高度分支的绒毛树状结构组成,大约在妊娠第10周时就浸入母体血液中(Jauniaux et al 。,2000)。固定的绒毛将胎盘附着在子宫内膜上,子宫内膜在胎盘形成过程中转化为蜕膜。胎盘绒毛基质内有大量胎儿组织驻留巨噬细胞,即霍夫鲍尔细胞(HBC)。早期研究确定该这些细胞是基于在与男性胎儿妊娠的Y染色体检测胎儿来源的(金等人,2008) 。在稳态下,HBC是胎盘屏障胎儿侧的唯一免疫细胞群(Thomas等,2021)。因此,它们被认为在胎盘生长和体内平衡的调节中起着重要的作用,并起着免疫哨兵的作用,保护胎儿免受子宫内感染。然而,与其他成人组织驻留巨噬细胞相比,HBC仍然知之甚少。协议的范围已被使用于isolat é HBC,如极好地在别处审查(唐等人。,2011) 。但是,尚未在整个领域建立标准技术。对于组织巨噬细胞的分离,使用的许多方案都不理想。例如,不必要的较长的消化时间(有时长达3小时)以及未能使用DNase I都会严重影响HBC的生存能力。此外,通常从胎盘组织中分离出作为总CD14 +细胞的HBC,以进行表型和功能分析。然而,我们以前曾表明,母体细胞可以包含至多CD14的30%+从第一分离的细胞-三个月胎盘消化(托马斯等人,2021。) 。通过将常见的HLA同种异型抗体添加到我们的流式细胞仪中,我们可以区分母体和胎儿细胞,并确保分离出纯HBC群体。因此,在使用HBC的所有未来的研究在体外试验中,此处所描述的额外步骤应该被执行,以确保真正fetally衍生HBC进行了研究与最佳可行性。

在本文中,我们提供了一步一步协议来从第一分离HBC -三个月和足月人胎盘组织具有高纯度。程序小号乙和Ç描述如何获得从第一单细胞悬液-分别三个月和足月胎盘组织。程序D描述了如何通过荧光激活细胞分选(FACS)从胎盘消化物中获得高纯度的HBC。以下这些协议允许investigati纳克下游HBC性质体外测定,以了解其功能特性进一步。甲舍姆ë从第一HBC隔离的整体过程的-三个月和足月胎盘示于图1 。


图1.从人胎盘中分离出HBC。的示意性概述用于从第一分离HBC的程序-三个月和足月胎盘。

关键字:胎座, 组织处理, 霍夫鲍尔细胞, 巨噬细胞



材料和试剂


一次性手术刀,第22号(Swann Morton,目录号:508)
巴斯德吸管(ELKay ,目录号127-P503-STR )
薄纱布(Winware食品级,目录号:E948)
30 µm细胞过滤器(Miltenyi biotec ,目录号:130-041-407)
70 µm细胞过滤器(BD Falcon,目录号:352350)
细胞过滤器100 µm(BD Falcon,目录号:352360)
细胞过滤器400 µm(Pluriselect ,目录号:43-50400-50)
140 mm培养皿(Sterilin ,目录号:SC269)
鹘50ml圆底聚丙烯试管小号(康宁,目录号:352070)
聚丙烯圆底FACS管5 ml(Falcon,目录号:352063)
解剖钳(科学实验室用品,目录号:SR04010)
手术直剪刀(SAMCO,目录号:E101 / 01)
250毫升杜兰玻璃瓶(科学实验室用品,目录号:F151164 )
漏斗(科学实验室用品,目录号:FUN1260 )
V型胶原酶(Sigma-Aldrich,目录号:C9263)
DAPI(4',6-二mid基-2-苯基吲哚)(Sigma-Aldrich,目录号:D8417)
DNase I(罗氏(Roche),目录号:10104159001)
乙二胺四乙酸EDTA(Sigma-Aldrich,目录号:E9884)
胎牛血清(热灭活的)(Biosera ,目录号:FB -1001)
流式细胞术抗体(见表1 )


表1.流式细胞仪抗体


Pancoll (密度1.077 mg / ml)(Pan Biotech ,目录号:P04-601000)
青霉素/链霉素溶液(Sigma-Aldrich,目录号:P0781)
RPMI-1640培养基(Sigma-Aldrich,目录号:R7388)
胰蛋白酶250(Pan Biotech,目录号:P10-025100P)
葡萄糖(Sigma-Aldrich,目录号:D9434)
NaCl(MP Biomedicals,目录号:151944)
氯化钾(MP Biomedicals ,目录号:194738)
正磷酸氢二钠(Fisher Scientific,目录号:S / 4520/53)
正磷酸二氢钾(Fisher Scientific,目录号:P / 4800/53)
pH值osphate缓冲盐水(PBS)1×浓度
人血清(Sigma-Aldrich,目录号:H4522)
小鼠血清(Sigma-Aldrich,目录号:M5905)
大鼠血清(Sigma-Aldrich,目录号:R9759)
出色的污渍缓冲液(BD Horizon,目录号:563794)
胰蛋白酶/EDTA,0.2%溶液(请参阅食谱)
              RF10介质(请参阅食谱)
FACS缓冲区(请参阅食谱)
阻塞缓冲区(请参见配方)


设备


BD FACSAria TM III细胞分选仪(或类似产品),4激光细胞分选仪,用于装有100 µm喷嘴的2级安全壳样品(BD Biosciences ,型号:FACSAria TM III )
带桶盖的温控离心机(Eppendorf离心机5810R,目录号:F151380)
热板磁力搅拌器(DLAB,目录号:DL2255-380H)
水浴(VWR,目录号:462-0557)
血细胞计数器(VWR,目录号:BR718605)
光学显微镜(Leica,目录号:11964479)


软件


BD FACSDiva TM软件
一对流式细胞仪和应用程序的安装,数据采集工具,采集,分析。它运行微软®的Windows 10的64位操作系统。


的FlowJo TM (碧迪,和公司; 2019)软件
甲包用于分析流式细胞术数据。现代流式细胞仪产生的文件以流式细胞术标准格式写入,扩展名为.fcs。无论使用哪种流式细胞仪收集数据,FlowJo都会导入并分析细胞计数数据。Mac和PC均可使用。


程序


生物样本
至关重要的是,所有的工作使用人体组织接收来自事先批准的适当的机构和/或国家审查委员会; 捐助者必须始终提供知情同意。对于这项工作,在英格兰东部-剑桥中央研究伦理委员会(17 / EE / 0151)的伦理许可下,在所有参与者的书面知情同意下获得组织样本。


与未公开的人胎盘组织所有的工作都视为组3种血液潜在危险-源性致病菌,并应在开展遏制级别2的设施。


获得单-从第一细胞悬液-三个月胎盘组织
HBC是在绒毛基质内发现的胎儿巨噬细胞,它构成胎盘。一系列酶消化的确保了组织的击穿,以获得一个单一的-以高收率HBC和活力细胞悬浮液(见注2)。这种单-细胞悬浮液也含有其他细胞类型即弥补胎盘,包括滋养层细胞和成纤维细胞,以及胎盘相关的母体单核细胞/巨噬细胞(PAMM) (托马斯等人,2021。) 。可以通过FACS(程序D中进一步详细描述的方法)从该消化物中获得HBC的纯化种群。


准备试剂和设备
同样的材料和设备所需要的程序小号B.媒体,试剂,和工具必须是无菌的(见注1)。


制备的胰蛋白酶/ EDTA溶液(见配方)
准备的RF10中(见食谱)
制备的Ç通过溶解ollagenase V原液(10毫克/毫升)它在RF10。将溶液分装成2.5毫升等分试样,并在-20°C下保存长达6个月。
制备的DNA酶I原液(10毫克/毫升)通过溶解它在无菌水中。在-20°C下将溶液以100 µl等分试样分配最多6个月。
通过将DNase I的等分试样(来自过程B1d)稀释在50 ml的1 × PBS中来制备含DNase I的PBS(20 µg / ml)。保持无菌于4°C并每次新鲜(见注3)。
之前开始胎盘组织处理,瓦特臂Ť rypsin / EDTA(0.2%溶液),胶原酶V,1 × PBS ,并Pancoll至37℃。设置一个加热摇床至37℃,用每分钟500转(RPM) ,并设置一个磁力搅拌器加热板,以37℃。
过程荷兰国际集团第一-三个月胎盘组织以获得单-细胞悬浮液
收到后立即处理胎盘。
冲洗的胎盘1×与在室温下(RT)10分钟磁力搅拌器轻轻PBS。
转移胎盘到P ETRI菜。
用镊子清除任何血块。
按住胎盘单元的一端,并且轻轻刮去从绒毛膜绒毛用手术刀(S EE图2A )。
丢弃胎膜。
使用巴斯德吸管(与顶端切断),转移刮下绒毛到75mL的预热0.2%胰蛋白酶-EDTA在无菌250米升玻璃Duran瓶中。在地方的热板磁力搅拌器加热至37℃并轻轻搅拌7分钟。
加入2mL的FCS以停止胰蛋白酶消化的组织的。
过滤trypsined通过ST组织erile漏斗薄纱纱布。
在室温下用PBS冲洗。
将消化过的穿过纱布的组织和剩余的组织都保留在纱布上。
使用手术刀从纱布中取出未消化的组织,并将其放入50 ml F alcon管中。添加2.5毫升的胶原酶V和100微升DNA酶I的并添加PBS至25毫升的最终体积(终浓度为1mg / ml胶原酶V和40微克/毫升DNA酶I的)。
注意:DNase I的添加至关重要。米acrophage产量和活力都在无DNA酶I的大大降低


在500 RPM的加热振荡器上于37°C消化20-30分钟。
通过漏斗中的无菌细纱布过滤所得的细胞悬液。丢弃任何未消化的组织,并离心滤液5分钟,在120 ×克,RT ,以沉淀细胞。丢弃上清液。
合并来自步骤s i和n的细胞,并用DNase I在PBS中洗涤(请参见注释3)。
注意:来自S tep B2 i的细胞将主要包含母体和滋养细胞。我们发现,HBC有时也在此消化中发现,从汇集细胞的步骤B2 i和B2 ñ将有助于最大限度地提高产量HBC 。然而,由于来自S tep B2 i的细胞悬浮液中发现的HBC很少,因此合并来自步骤B2 i和B2 n的细胞不是必需的。


轻轻重悬细胞沉淀在20ml的PBS中的DNA酶I
通过400 µm过滤器过滤细胞悬液。
层中的细胞悬浮液到10毫升的Pancoll在50ml Falcon管中。
以600 × g离心20分钟,RT,加速度0,断裂1。
使用巴斯德移液器在界面处收集细胞。
用DNase I用PBS洗涤,并在120 × g室温下离心5分钟。丢弃上清液。
重复了前面的步骤。
用DNase I将细胞重悬于PBS中,并通过70 µm过滤器,用血球计数器计数细胞,然后将其置于冰上(参见注释2和5)。




图2.刮除胎盘绒毛。从胎盘绒毛(的图像,显示刮甲)第一-三个月和(乙)足月胎盘。


获得单-细胞悬浮液从足月胎盘组织
从足月妊娠收集胎盘的显着不同的在第一三个月。随着怀孕的进行,胎盘绒毛继续发芽,血管网络成熟,以增加器官的表面积和血管形成,并满足胎儿成长的需要。足月胎盘绒毛的纤维间质较致密,带有肌肉动脉,在孕早期不存在(Castellucci和Kaufmann,1982)。出于这个原因,足月胎盘组织的用于从在步骤B对首先描述的不同HBC的隔离消化-三个月胎盘和在下面详细所述。


准备试剂和设备
按照步骤B1所述准备试剂和设备。


处理足月胎盘组织以获得单-细胞悬浮液
收到后立即处理胎盘。
使用手术剪刀从整个胎盘切出一个小叶。
注意:为确保供体之间的一致性,请始终将叶与同一区域隔离开。


冲洗的在PBS中胎盘轻轻地用在10分钟磁力搅拌器RT 。
转移到一个P ETRI菜。
用镊子清除任何血块。
使用外科剪刀从基底侧(面对母亲的一侧)上去除并丢弃一薄层组织(约1毫米)。
注意:此步骤可确保从胎盘中清除所有蜕膜组织。


按住胎盘单元的一端,并且轻轻刮去从绒毛膜绒毛用手术刀(S EE图2B )。
丢弃胎膜,结缔组织纤维和可见血管。
使用巴斯德吸管(与顶端切断),转移刮下绒毛到75mL的预热0.2%胰蛋白酶-EDTA在无菌玻璃Duran瓶中(250 ml的)。在地方的热板磁力搅拌器加热到37℃并轻轻搅拌15分钟(见注4)。
加入2mL的FCS以停止胰蛋白酶消化的组织的。
通过漏斗中的无菌薄纱布过滤经胰蛋白酶处理的组织。
在室温下用PBS冲洗纱布。
将消化过的穿过纱布的组织和剩余的组织都保留在纱布上。
从纱布中取出未消化的组织,放入50 ml F alcon管中。添加2.5毫升的胶原酶V和200μl的DNA酶I的并添加PBS至终体积25毫升(1毫克/毫升胶原酶V和80微克/毫升脱氧核糖核酸酶I的终浓度)。
注意:DNase的添加至关重要。米acrophage产量和活力都在无DNA酶I的大大降低


在37°C的加热的振荡器上以500 RPM的速度消化60分钟(请参见注释4)。
通过在漏斗中的无菌平纹细布纱布过滤消化的组织。丢弃任何未消化的组织,并离心滤液5分钟,在120 ×克,RT ,以沉淀细胞。丢弃上清液。
重复小号TEPS B2o-B2W。


Isolati NG霍夫鲍尔从第一单元-孕早期和足月胎盘摘要
在这个协议中,单-从过程获得的细胞消化小号乙和C被染色与HBC的通过FACS分离荧光缀合的抗体。该协议包括一个优化的面板和门控策略的是允许isolati纳克HBC的高度纯化的人口没有了在胎盘消化母体发现的单核细胞/巨噬细胞的污染。


准备试剂和设备(请参阅注1)
制备的FACS缓冲液(见ř ecipes)。保持无菌并在4°C下保存最多1个月。
制备封闭缓冲液(见ř ecipes) 。通过过滤器一个在4℃下为至多0.2微米的无菌过滤器和存储1月份。
注意:将所有血清在56°C的水浴中加热30分钟即可使其热失活。


在无菌水中准备0.9 mM DAPI。无限期储存在-20°C下。
如步骤B1b中所述准备RF10 。


Isolat è通过FACS HBC
使细胞通过30 µm过滤器进入聚丙烯FACS管。
在120 × g,4°C下离心5分钟。丢弃上清液。
于150μl重悬细胞的人类阻挡每10个缓冲× 10 6细胞。
在每管50 µl的Brilliant染色缓冲液中制成抗体混合物(按照表1中的稀释比例,最终体积为200 µl)。同一抗体混合物用于孕中期和足月胎盘细胞。
添加的抗体鸡尾酒的细胞,并通过轻轻混合涡旋。
在冰上孵育20分钟。
加入2mL的冷FACS缓冲液,并离心5分钟,在120 ×克,4℃。丢弃上清液。
重复上一步。
重悬细胞于1ml的冷FACS缓冲液。
染色的细胞用DAPI(1:10,000)用于启动排序之前的10分钟ING 。
参见图3中用于鉴定早孕和足月胎盘消化物中HBC的门控策略的代表性图像(请参见注释6)。
将细胞在4°C下分选到合适的收集管和缓冲液中。




图3.荧光激活细胞分选门控策略以分离HBC。(A,B)使用BD FACSDiva和Flowjo软件使用常见HLA同种异型抗体和胎盘消化物中CD45 + CD14 + FOLR2 + HBC(红门)身份的抗体用于区分胎儿与母体细胞的门控策略的代表性图像。(A)首先-三个月胎盘样品; 胎儿细胞是HLA-A3 + ,和母体细胞是HLA-A3 - 。(B)全-术语的胎盘细胞; 胎儿细胞是HLA-B7 - ,和母体细胞是HLA-B7 + 。谱系毫安- [R KERS(LIN)包括CD3,CD19,CD20,CD66b ,和CD335在FITC。


数据分析


从胎盘组织中分离活细胞


我们的协议生成来自胎盘消化收率细胞单细胞悬浮液与〜从第一90%存活率-三个月胎盘组织和〜80%从足月胎盘组织生存力。通过细胞分选富集可行的HBC后,我们获得了〜95%的HBC,如通过血细胞计数器和显微镜进行细胞计数所确定的(参见注释5)。


流式细胞术门控策略以区分HBC和母体巨噬细胞


为了从胎儿CD14 + HBC中分离出污染性的母体CD14 +单核细胞和巨噬细胞,我们开发了一种流式细胞术门控策略,可以很容易地适应不同细胞分选仪的配置(表1 )。我们证明,抗HLA分型抗体可用于区分更丰富的胎儿CD14 +从较不丰富的母体CD14 HBC +来自第一单核细胞和巨噬细胞-三个月和足月胎盘消化(图3 )(见注6) 。


笔记


有几个参数,关键在了成功的协议小号描述。


在整个方案中必须使用无菌试剂和设备,因为HBC对细菌产物有反应,这会改变细胞的特性(Thomas等,2021)。一旦已经获得单细胞悬浮液,它是必不可少的,以保持它在4℃下,以避免通过遵守塑料巨噬细胞损失和最小化细胞活化。
DNA酶I的过程中加入的消化和随后的PBS洗涤荷兰国际集团的步骤是,以确保高HBC生存能力的关键。
对于足月胎盘组织,用胰蛋白酶和胶原酶V进行更长的消化是确保完整消化组织和成功分离HBC所必需的。但是,过度消化和长时间暴露于消化酶会降低HBC的生存能力。
总细胞数小号从第一-三个月和足月胎盘组织消化变种ý样本之间。通常,从已经成功消化的胎盘组织中可以预期总共有20 × 10 6 -50 × 10 6个细胞。通过FACS分选的HBC的产量在样品之间也有所不同,但约占所有细胞的1-3%。
为确保通过高纯度FACS分离HBC ,我们在流式细胞仪中提供了三种HLA异型分型抗体。由于HBC是包含母体和母体遗传物质的胎儿细胞,因此在某些情况下,可以预期胎儿和母体之间的HLA同种异型不匹配。在这些情况下,我们可以确保排序HBC是胎儿来源的无母体细胞的污染,其通常存在于胎盘消化(图3 )。在我们的染色面板中使用的抗HLA抗体的特异性已被证实从HLA分型献血者的血样通过DNA上的定量PCR(qPCR) 。然而,这里描述的方案的一个缺点是,如果有没有对胎儿和母亲为HLA-B7,HLA-A3之间HLA同种异型错配,或HLA-A2,协议将无法从胎儿细胞分离母体。在这种情况下,有必要在流式细胞仪中包括由HBC特异性表达的其他标记。例如,我们先前已经表明,孕早期的HBC不表达HLA-DR,而母亲的巨噬细胞则表达HLA-DR 。因此,加入HLA-DR的,以流式细胞术面板将允许第一分离-三个月HBC从母体的巨噬细胞在不存在HLA同种异型的失配(托马斯等人,2021。) 。HLA-DR不能用于区分足月胎盘HBC中的母体细胞,因为HBC通过足月表达HLA-DR (Sutton等人,1983)。


菜谱


胰蛋白酶/ EDTA [(0.2%溶液);0.2%(w / v )胰蛋白酶-250 / 0.02%(w / v )EDTA / PBS溶液)]
0.3克葡萄糖


12克氯化钠


0.3克氯化钾


1.725 g正磷酸氢二钠


0.3克正磷酸二氢钾


2克胰蛋白酶


0.2克EDTA


将化学药品溶于1升水中
使用0.22 µm过滤器单元对溶液进行无菌过滤
分发储备溶液至75个在毫升分装并储存- 20℃下进行长达6个月
Ť他RF10媒体
罗斯威尔公园纪念学院(RPMI)1640中


10%热灭活胎牛血清


1×青霉素/链霉素溶液


100单位的青霉素和0.1 mg / ml链霉素


Ť他流式细胞仪缓冲
1%PBS中2%热灭活的胎儿血清和2 mM EDTA 。保持无菌并在4°C下保存最多1个月。


B锁定缓冲区
1×PBS中的5%人血清,1%大鼠血清,1%小鼠血清,5 %FCS和2 mM EDTA


致谢


感谢您的协助:1 .病理学系的流式细胞仪核心设施;2.露西·加德纳(Lucy Gardner),伊莫金·邓肯(Imogen Duncan )和里图·拉尼(Ritu Rani)帮助收集和处理胎盘样品;3 d onors谁参加了这项研究,并在医院的工作人员; 4.教授斯蒂芬·查诺克-琼斯和妇产科的欧文埃系博士妇科,剑桥大学,用于帮助收集足月胎盘样本。这项工作得到了英国剑桥大学皇家学会,滋养细胞研究中心和惠康基金会的支持。N.McG由惠康爵士亨利·戴尔爵士和皇家学会奖学金资助(授权号204464 / Z / 16 / Z)。JT由Wellcome Trust博士生资助(资助号215226 / Z / 19 / Z)。


该协议是根据其他人的先前工作(Tang等人,2011)和我们自己的发现(Thomas等人,2021)开发和修改的。


利益争夺


作者没有利益冲突要声明。


参考


Castellucci ,M。和Kaufmann,P。(1982)。正常人胎盘绒毛核心的三维研究:II。基质结构。胎盘3(3):269-285。              
Jauniaux ,E.,Watson,AL,Hempstock ,J.,Bao,YP,Skepper ,JN和Burton,GJ(2000)。孕妇动脉血流和胎盘氧化应激的发作。人类早期妊娠失败的可能因素。美国病理学杂志157(6):2111-2122。
Kim,JS,Romero,R.,Kim,MR,Kim,YM,Friel,L.,Espinoza,J.和Kim,CJ(2008)。Hofbauer细胞和母体T细胞参与病因不明的绒毛炎。组织病理学52(4):457-464。
Sutton,L.,Mason,DY和Redman,CW(1983)。人胎盘中的HLA-DR阳性细胞。免疫学49(1):103-112。
Tang,Z.,Abrahams,VM,Mor ,G. and Guller ,S.(2011年)。胎盘霍夫鲍尔细胞与妊娠合并症。Ann NY Acad Sci 1221:103-108。
Thomas,JR,Appios ,A.,Zhao,X.,Dutkiewicz ,R.,Donde ,M.,Lee,CYC,Naidu,P.,Lee,C.,Cerveira ,J.,Liu,B.,Ginhoux , F.,Burton,G.,Hamilton,RS,Moffett,A.,Sharkey,A. and McGovern,N.(2021)。孕早期人胎盘巨噬细胞霍夫鲍尔细胞的表型和功能表征。J Exp Med 218(1)。              
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Appios, A., Thomas, J. R. and McGovern, N. (2021). Isolation of First-Trimester and Full-term Human Placental Hofbauer Cells. Bio-protocol 11(11): e4044. DOI: 10.21769/BioProtoc.4044.
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