Source: Rizzuto, G. A., et al. Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface. J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation of Organ Culture Plates
NOTE: The following steps should be performed using sterile technique in a tissue culture hood. It is ideal for the dissecting microscope to be located in a sterile field. However, this is not absolutely necessary as long as micro-dissection is performed expeditiously, and instruments are dipped frequently in 70% ethanol.
2. Establishment of Organ Cultures
NOTE: This step describes how to place decidual organ cultures and villous organ cultures in separate transwells. The tissues are not in contact. Researchers interested in a co-culture technique where decidua and villi are in direct contact with each other can refer to prior literature.
3. Preparation of L. monocytogenes Cultures
NOTE: For a detailed protocol on the long-term storage of L. monocytogenes, and the culture and growth of this organism in brain heart infusion (BHI) broth and agar, please refer to Wang et al.
4. Infection of Organ Cultures with L. monocytogenes
Table 1: Media recipes. Components and concentrations for preparation of Wash buffer, Collection medium, Villous medium, and Decidual medium
Wash buffer | Collection medium | Villous medium | Decidual medium |
PBS | DMEM/F-12 with GlutaMAX | DMEM/F-12 with GlutaMAX | DMEM/F-12 with GlutaMAX |
Penicillin 100 IU/ml | Fetal Bovine Serum 2.5% | Fetal Bovine Serum 20% | Fetal Bovine Serum 2.5% |
Streptomycin 100 μg/ml | Penicillin 100 IU/ml | Penicillin 100 IU/ml | 17β-estradiol 300 pg/ml |
Gentamicin 50 μg/ml | Streptomycin 100 μg/ml | Streptomycin 100 μg/ml | Progesterone 20 ng/ml |
Amphotericin B 1.25 μg/ml | Gentamicin 50 μg/ml | ||
Amphotericin B 1.25 μg/ml |
Figure 1: Villous organ cultures – Representative gross and microscopic images.
(A) Two terminal villous trees with a gestational age of 6 weeks, as viewed under a dissecting microscope. Note the "fluffy" ends (arrowheads) and prominent fetal vasculature coursing through the branches of the tree on the left that make this piece suitable for organ culture. (B) Immunofluorescence microscopy of organ culture (gestational age 8.3 weeks) 72 hr post-infection with Listeria monocytogenes, highlighting heavy bacterial burden [green] in extravillous trophoblasts. DAPI is shown in blue, and βHCG+ syncytiotrophoblasts in red. Scale bars = 1 mm (A), 250 µm (B).
The authors have nothing to disclose.
Sterilization pouches | Fisher Scientific | 01-812-54 | For autoclaving individual dissecting tools |
Fine mesh strainer | Cuisinart (Amazon.com) | NA | Wrap completely in aluminum foil and autoclave prior to tissue collection. |
Carboy with spigot | Fisher Scientific | 03-007-647 | For large volume preparation of Wash buffer. |
Ice packs | Nortech labs | GB8818 | These do not have to be purchased, rather they can be recycled/reused from any routine laboratory shipment that includes them in the packaging. |
70% Ethanol | VWR | V1001 | 70% solution made by adding dH20 to 190 or 200 proof research grade alcohol. |
10% Bleach | Waxie Sanitary Supply | CLO 30966 | 10% solution made by adding dH20. |
Light Box | Litebox Lumina (dickblick.com) | 55305-2009 | Note replacement bulbs also purchased on Blick (55305.0100) |
Micro dissecting forceps | Stoelting | 52102-43 | 4in, 1×2 0.5mm, Slight Curve |
Micro dissecting forceps | Stoelting | 52102-06 | 4in, Straight Fine, Sharp |
Micro dissecting vannas spring scissors | Stoelting | 52130-01P | McPherson-Vannas Spring Scissors, 8.5cm, 0.33 Tip, Slight Curve |
Dissecting microscope | Leica Microsystems | MZ16 or M60 | We have had success with the listed models. External gooseneck flexible light sources are helpful but not necessary. |
50 mL conical tubes | Sigma-Aldrich (Corning) | CLS4558 | |
Phosphate Buffered Saline | Gibco (ThermoFisher Scientific) | 10010023 | We purchase from our university Tissue Culture Core facility, alternate options such as this are available. |
10X Phosphate Buffered Saline | Teknova | P0195 | For preparation of Wash buffer we use 10X PBS |
DMEM/F-12 nutrient mixture (Ham's) with GlutaMAX | Gibco (Life Technologies) | 10565-018 | We purchase this specific media formulation, containing 2.438 g/L sodium bicarbonate, 55 mg/L sodium pyruvate, and 4.5 g/L glucose |
Gentamicin | Thermofisher Scientific | 15710072 | 1000X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw. |
Penicillin/Streptomycin | Gibco (ThermoFisher Scientific) | 15140-122 | 100X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw. |
Amphotericin B | Gibco (ThermoFisher Scientific) | 15290-018 | 500X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw. |
progesterone | Sigma-Aldrich | P8783 | A 1 mM stock solution is made by reconstituting 15.7 mg progesterone powder in 15.7 mL Ethanol and adding 34.3 mL PBS. Solution is sterile filtered, aliquoted, and stored at -20 °C. |
17β-estradiol | Sigma-Aldrich | E2758 | A 10 μM stock solution is made by reconstituting 13.5 mg estradiol powder in 10 mL ethanol and adding 40 mL PBS. Solution is sterile filtered, aliquoted, and stored at -20 °C. |
Bottle-top vaccum filter (0.22 μm) | Sigma-Aldrich (Corning) | CLS430769 | For sterile filtration of Collection media after preparation |
6-well tissue culture plate | BD Falcon | 353224 | Polystyrene, Tissue culture treated |
6-well transwells | Millipore | PICM03050 | Insert – 30mm diameter, 0.4μm pore size hydrophilic PTFE membrane |
Extracellular Matrix (for example, Matrigel Matrix) | BD Biosciences | 354234 | We have utilized Matrigel Matrix in our studies. It is a solid at room temperature and at -20 °C. Avoid repeat freeze/thawing. Thaw bottle to viscous solution at 4 °C, and prepare ~300μL aliquotsin the cold room with chilled pipette tips. Store aliquots at -20 °C. |
Paraformaldehyde, 16% w/v aqueous solution | Alfa Aesar | 30525-89-4 | For tissue fixation, a fresh preparation of 4% paraformaldehyde is made by diluting this stock in PBS. |
Tissue culture incubator, maintained at 37 °C, 5% CO2, 3% oxygen (optional for villous organ cultures) | For some experiments, hypoxia may be preferred. This can be established multiple ways, including addition of exogenous nitrogen via gas cylinder, Tygon tubing, and a regulator. | ||
Bench top centrifuge |