Co-culture of placental explants with isolated CD4 and CD8 T cells: a functional model to define the consequences of placental inflammation
Materials and Methods
- RPMI-1640 medium (Cat. # 21875, Gibco, UK) supplemented with:
- 5% Fetal bovine serum (FBS) (Gibco, UK)
- Insulin solution 1 µg/ml
- Streptomycin sulphate 100 µg/ml
- Penicillin G 100 IU/ml
- Retinol acetate 0.1 µg/ml
- Calcium chloride
- L-alanine 25 µg/ml
- L-cysteine 200 µg/ml
- Ascorbic acid 50 µg/ml
- Sterile PBS with Ca2+ and Mg2+
- Sterile PBS without Ca2+ and Mg2+
- 70% ethanol
- 0.01% trypsin in sterile PBS
- LymphoprepTM (Cat. # 07801, Stem Cell Technologies, UK)
- EasySepTM buffer (Stem Cell Technologies, UK)
- EasySepTM CD8 T cell isolation kit, positive selection (Cat. # 18013, Stem Cell Technologies, UK)
- EasySepTM CD4 T cell enrichment kit, negative selection (Cat. # 19012, Stem Cell Technologies, UK)
- Recombinant human (rh) interleukin (IL)-2 10 ng/ml (Life Technologies, UK)
- RhIL-15 10 ng/ml (Life Technologies, UK)
- Anti-CD3 antibody 5 µg/ml (BD Pharmingen, UK)
- Anti-CD28 antibody 1 µg/ml (BD Pharmingen, UK)
- CellTrackerTM CMPTX red dye (Life Technologies, UK)
- CellTrackerTM CMFDA green dye (Life Technologies, UK)
- Sterile Petri dishes
- 24-well culture plates
- 12-well culture plates
- Sterile Pasteur pipettes
- SepMate centrifugation tubes (Stem Cell Technologies, UK)
- 15 ml and 50 ml centrifuge tubes
- 5 ml Eppendorf tubes
- 5 ml, 10 ml and 25 ml sterile stripettes
- 5 ml round-bottomed polystyrene falcon tubes
- Sterile dissecting scissors and forceps
- Centrifuge (Beckman Coulter)
- EasySepTM magnet (Stem Cell Technologies, UK)
- Humidified incubator 37°C, 21% O2, 5% CO2
- Laminar air flow
- Inverted microscope (Olympus CK2, Olympus, Southend-on-Sea, UK)
- Fluorescent microscope (Zeiss Observer Z1 AX10, Zeiss, Welwyn Garden City, UK)
- Flow cytometer (BD Accuri C6, BD Biosciences, Oxford, UK)
Placentas and blood
Trypsinization and syncytiotrophoblast morphology
Trypsinization and placental function
Trypsinization, cytokines and placental function
EasySep cell isolation
T cell labeling
System A activity
1.Prepare 4 ml sterile PBS supplemented with 5 µg/ml anti-CD3 antibody.
2.Add 1 ml antibody solution to 4 wells of a 12-well plate.
3.Incubate for 2 h at 37°C.
4.Wash twice with 1 ml sterile PBS.
5.Prepare 1 L RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), 1 µg/ml insulin, 100 µg/ml streptomycin sulphate, 100 IU/ml penicillin G, 0.1 µg/ml retinol acetate, 25 µg/ml L-alanine, 200 µg/ml L-cysteine and 50 µg/ml ascorbic acid. Filter prepared medium through 0.2 µm bottle top vacuum filter (Corning, Germany).
6.Receive placenta within 30 min of delivery. Take four biopsies of approximately 1 cm3 from random areas of parenchyma, place in pre-warmed sterile PBS. All subsequent steps take place in the laminar flow hood.
7.Cut approximately 30 smaller fragments (~4–5 mm3) of villous tissue from each of the large biopsies, taking care to avoid fetal chorionic plate, decidua and large blood vessels. Transfer to sterile petri dishes with 10 ml pre-warmed sterile PBS.
8.Wash twice in sterile PBS.
9.Transfer tissue fragments to an empty petri dish. Add 10 ml 0.01% trypsin, incubate at 37°C for 15 min.
10.Remove trypsin solution then neutralize with two changes of RPMI medium.
11.Add 1 ml RPMI medium to 16 wells of a 24 well culture dish and place three fragments in each
12.Culture for 24 h in a humidified incubator at 37°C, 21% O2.
13.Add 15 ml Lymphoprep to a SepMate tube.
14.In a separate centrifuge tube, mix blood with an equal volume of EasySep buffer. Carefully transfer the blood/buffer mix to the SepMate tube ensuring the tube is kept vertical.
15.Centrifuge at 1200 g for 10 min at room temperature with the brake on.
16.Pour off the supernatant (which contains the peripheral blood mononuclear cells) into a new centrifuge tube; do not invert the SepMate tube for longer than two seconds. NOTE the tube that the supernatant is poured into does not have to be a SepMate tube.
17.Centrifuge for 8 min at 300 g at RT.
18.Remove supernatant with a stripette taking care not to disturb the pellet.
19.Add ~10 ml EasySep buffer and re-suspend the cells. Centrifuge for 8 min at 300 g at RT.
20.Repeat steps 6 and 7.
21.Remove supernatant and re-suspend cells in 4 ml EasySep buffer. Pour into a 5 ml round-bottomed falcon tube.
22.Centrifuge for 8 min at 300 g at RT, remove supernatant.
23.Re-suspend pellet with 100 µl EasySep buffer. Add 10 µl EasySep CD8 positive selection cocktail. Mix well and incubate for 15 min at RT.
24.Mix the magnetic nanoparticles by vigorous pipetting (do not vortex). Add 10 µl of magnetic nanoparticles. Mix well, incubate for 10 min at RT.
25.Bring the cell suspension to a total of 2500 µl using EasySep buffer. Mix by gentle pipetting. Place the tube in the magnet for 5 min ensuring that tube does not have the cap on.
26.Whilst the tube is still in the magnet invert it, pouring the supernatant into a new 5 ml falcon tube. Leave inverted for 2–3 seconds but do not shake or blot any drops. Save the pour off for the isolation of CD4 T cells.
27.Take the tube out of the magnet, add ~2500 µl EasySep buffer. Mix by gentle pipetting, place the tube back in the magnet and set aside for 5 min.
28.Whilst the tube is still in the magnet invert it, pouring off the supernatant. (There is no need to save this supernatant.)
29.Repeat steps 15 and 16.
30.Re-suspend in 2500 µl EasySep buffer, centrifuge for 8 min at 300 g at RT.
31.Remove supernatant, re-suspend in 2 ml RPMI medium with 1 µg/ml anti-CD28, 10 ng/ml rhIL-2 and 10 ng/ml rhIL-12.
32.Use the supernatant/first pour off from the CD8 selection, centrifuge for 8 min at 300 g at RT.
33.Remove the supernatant, re-suspend cells in 250 µl EasySep buffer.
34.Add 12.5 µl EasySep Human CD4 T cell enrichment cocktail. Mix well and incubate for 10 min at RT.
35.Vortex the D magnetic particles for 30 seconds. Add 25 µl magnetic particles, mix well and incubate for 5 min at RT.
36.Bring the cell suspension to 2500 µl by adding EasySep buffer (~2200 µl) and mix gently.
37.Place the tube, without cap, into the magnet for 5 min.
38.With the tube in the magnet, invert, pouring the supernatant into a new 5 ml tube. The cells are in the pour off.
39.Centrifuge for 8 min at 300 g at RT.
40.Remove supernatant, re-suspend in 2 ml RPMI medium with 1 µg/ml anti-CD28 to activate T cells, 10 ng/ml rhIL-2 and 10 ng/ml rhIL-15 to stimulate proliferation of T cells.
41.Plate cells into the coated wells of a 12-well plate.
42.Incubate overnight in a humidified incubator at 37°C, 21% O2.
43.Remove medium from wells, transferring into two 5 ml Eppendorfs (one for CD4 cells, one for CD8 cells).
44.Add 500 µl ice-cold sterile PBS (without Ca2+ and Mg2+), gently scrape the bottom of the wells to release the cells. Transfer to the Eppendorfs containing the medium.
45.Examine the plates on an inverted microscope to check how many cells remain attached.
46.Repeat step 2.
47.Centrifuge at 1300 rpm for 5 min at RT.
48.Dilute 1 µl CellTracker red in 1 ml pre-warmed RPMI medium and 1 µl CellTracker green in 1 ml pre-warmed RPMI medium.
49.After centrifugation, remove supernatant, re-suspend cells in pre-warmed medium with CellTracker (we used red for CD4 cells and green for CD8 cells). Incubate at 37°C for 45 min.
50.Centrifuge at 1300 rpm for 5 min at RT.
51.Re-suspend in 1 ml pre-warmed medium. Incubate at 37°C for 30 min.
52.Centrifuge at 1300 rpm for 5 min at RT.
53.Re-suspend in 1 ml pre-warmed sterile PBS (with Ca2+ and Mg2+). Centrifuge at 1300 rpm for 5 min at RT.
54.Re-suspend in 480 µl medium supplemented with rhIL-2 and rhIL-15.
55.Collect medium from 2 wells for analysis.
56.Aspirate medium from other wells.
57.Add 10 µl cell suspension (~16,000 cells (range 4,000-42,000) to each explant.
58.Incubate for 24 h in a humidified incubator at 37°C, 21% O2.
59.Add 300 µl RPMI medium with rhIL-2 and rhIL-15 to each well.
60.Incubate for 24 h in a humidified incubator at 37°C, 21% O2.
61.Collect medium from 2 wells for analysis.
62.Aspirate medium from other wells.
63.Add 1 ml RPMI medium with rhIL-2 and rhIL-15 to each well.
64.Incubate for 24 h at 37°C, 21% O2, 95% humidity.
65.Collect medium from two wells for analysis.
Trypsinization and syncytiotrophoblast morphology
T cell labeling
System A activity
Proliferation and apoptosis
Isolated T cells
Isolated T cells in explants
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