Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of 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

1. Autoclave strainers/forceps for collection, and scissors/forceps for micro-dissection.
2. Prepare an adequate volume (500 ml per specimen) of Wash buffer (refer to Table 1 for the recipe).
3. Prepare an adequate volume (100 ml per specimen) of 0.22 µM sterile-filtered Collection media (refer to Table 1 for the recipe).
4. Aliquot Extracellular Matrix (ECM, refer to Table of Reagents for details) for long-term storage.
5. Store ECM in solid form at -20 °C. For best performance, avoid repeat freeze-thaw. Thaw bottle to a viscous solution at 4 °C, and prepare 300 µl aliquots in the cold room with chilled pipette tips. Store aliquots at -20 °C.

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.

1. Transport autoclaved strainers (one per specimen), autoclaved forceps, Wash buffer (carboy), Collection media (25 ml per specimen in a 50 ml conical tube), spray bottle, 10% bleach, 70% ethanol, tissue collection tray, sharpie, and ice bucket/cooler to hospital/clinic.
   NOTE: Specimens are received in a research-designated space located in a room close to the operating suite. A sterile tissue culture hood is not required, but the collection should occur expeditiously and the researcher should appropriately sanitize surfaces, as described below.
2. Place carboy of Wash buffer adjacent to the sink, and spray the spigot with 10% bleach and 70% ethanol. Place a glass tissue collection tray on the light source (light pad or lightbox), and sanitize with 10% bleach and 70% ethanol. Allow to air dry.
3. Have the clinician carry specimens from the operating suite to the specimen preparation room. At the sink, pour the specimen atop a sterile hand-held strainer, rinse the tissue several times in Wash buffer, and transfer the entire specimen in the buffer to a sanitized glass tray atop the light source.
   NOTE: Assessment of tissue by the clinician is of the highest priority, thus the following steps are only performed after the clinician has verbally indicated that he/she has finished the evaluation.
4. Use sterile forceps to select placental villi and decidua (Figures 2A and 3A) for collection, and transfer to separate 50 ml conical tubes. Label the tubes with gestational age.
   NOTE: Preferred gestational ages are less than 9 weeks and less than 16 weeks for placenta and decidua, respectively. At the institution, only a fraction of each specimen is procured for research purposes, as adequate tissue must remain for clinical pathologic diagnosis.
5. Store and transport the specimen on ice to the research lab.
6. To collect more than one specimen, sanitize the glass tray as described above with 10% bleach and 70% ethanol between the collection of each specimen.

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.

1. Thaw ECM vial(s) on ice. Dilute the ECM 1:1 with ice-cold Collection media, and mix by pipetting. Pipette slowly to avoid introducing bubbles. Each well of a 6-well tissue culture plate requires 100 µl of this suspension and will accommodate 3-4 organ cultures.
2. Place transwell inserts (pore size 0.4 µM) into each well of the 6-well tissue culture plate.
3. Coat each transwell insert with a thin layer (100 µl) of the ECM/media suspension.
4. Place the plate on ice and immediately proceed to the establishment of organ cultures. Alternatively, prepare the plate prior to tissue collection, in which case wrap it in para-film and store it at 4 °C for later use. Storage longer than 6 hr is not recommended as the ECM will dry out.

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.

1. Rinse specimens twice in Collection media, and centrifuge at 1,000 x g between each rinse. Transfer tissue to a sterile petri dish and place it on the stage of a dissecting microscope. Keep a 6-well plate on ice adjacent to the microscope.
2. Micro-dissect tissue with sterile springer dissecting scissors and forceps, to establish 2-3 mm villous organ cultures (Figure 1A).
   NOTE: Villous organ cultures are small, well-vascularized "trees" with 2 or 3 branches, and with prominent extravillous trophoblast (EVT) cell columns11. It is important to utilize only villous tissue from < 9-week first trimester specimens, as the invasion of extravillous trophoblasts into ECM is most pronounced at these young ages.
3. Select well-vascularized villi with prominent EVT cell columns. EVT cell columns are visualized as bulbous, "fuzzy", "fluffy" ends (refer to arrowheads in Figure 1A).
4. Use sterile forceps to transfer 3 or 4 villous trees into each transwell. Adjust branches so the tree is positioned flat atop ECM and separate clumped branches.
5. Micro-dissect decidual tissue with sterile springer dissecting scissors and forceps, to establish 3 mm3 decidual organ cultures (Figure 2A).
   NOTE: Specimens contain two distinct types of decidual tissues, decidua parietalis and decidua capsularis (left and right tissue fragments, respectively, Figure 2A).
6. Trim with scissors to create 3 mm3 pieces of decidua parietalis. Decidua capsularis is not used for these cultures. Use forceps to tease away any clotted maternal blood.
7. Use sterile forceps to transfer 3-4 pieces of decidua into each transwell.
8. Add 1 ml of Collection media to the bottom of the well. Incubate plate overnight at 37 °C in 5% CO₂
9. For experiments where it is important to mimic normal first-trimester placental oxygen tension, maintain villous organ cultures in relative hypoxia (3% O₂).
   NOTE: Laboratory options include small hypoxia incubator chambers that fit inside existing incubators or a tri-gas incubator that introduces external N₂ gas to lower oxygen concentration.
10. Add 1 ml of Villous or Decidual medium (Table 1) to the top of the transwell after 12-16 hr of culture.
    NOTE: The absence of media during the first overnight in culture allows the extravillous trophoblast cell columns to invade (villous culture), and for the tissue (both villous and decidual) to become securely embedded in ECM. In our experience, decidual organ cultures remain viable for 72 hr and villous organ cultures for 96 hr. We recommend experimental endpoints that fall within this timeframe.  See Table 1 for the composition of Villous and Decidual medium.  The villous medium has a high percentage of FBS, while the Decidual medium is supplemented with pregnancy hormones (progesterone, 17β-estradiol) that maintain the decidualized state (Table 1).

Table 1: Media recipes. Components and concentrations for preparation of Wash buffer, Collection medium, Villous medium, and Decidual medium.