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 the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.
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, Prior to Collection Day
2. Collection of Fresh Tissue
CAUTION: When working with fresh human tissue, researchers must follow Universal Precautions (OSHA) for preventing transmission of Bloodborne Pathogens, and must have received proper institutional training. Proper personal protective equipment, including gloves, eye protection, and lab coats are necessary.
3. 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.
4. 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.
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).
Figure 2: Decidual organ culture – Representative gross and microscopic images. (A) Decidua parietalis [left] and decidua capsularis [right] at gestational age 6 weeks, as viewed under a dissecting microscope. (B) H&E-stained section of 14.3 weeks gestational age decidua parietalis organ culture shows glands and vasculature organized heterogeneously throughout the decidual stroma. The (diamond, ◆) and the (asterisk, *) highlight representative vessels and glands, respectively. The brown line at the lower edge is the transwell membrane, on the edge. (C) H&E-stained section of 14.3 weeks gestational age decidua parietalis organ culture at higher magnification demonstrates muscular-walled spiral arterioles embedded in the decidual stroma. Maternal immune cells are small with dark round nuclei and are irregularly distributed throughout the stroma. (D) Immunofluorescence microscopy of organ culture 48 hr post-infection with L. monocytogenes, highlighting large zones of bacteria [red] in the decidual stroma, and maternal CD14+ macrophages [green] lining a tortuous vascular space and scattered in the stroma. Scale bars = 1 mm (A), 250 µm (B), 50 µm (C and D).
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 |
Micro dissecting forceps | Stoelting | 52102-43 | 4 inches, 1 x 2 x 0.5 mm3 , Slight Curve |
Micro dissecting forceps | Stoelting | 52102-06 | 4 inches, Straight Fine, Sharp |
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 |
6-well tissue culture plate | BD Falcon | 353224 | Polystyrene, Tissue culture treated |
6-well transwells | Millipore | PICM03050 | Insert – 30 mm diameter, 0.4 μm 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 ~ 300 μL aliquots in 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. |