Protocol for culturing the endocrine junctional zone of the mouse placenta in serum-free medium

Summary The lack of a suitable explant culture system restricts the study of factors secreted by the mouse placenta into maternal circulation. Here, we present a protocol for culturing the endocrine junctional zone of the mouse placenta, free from the decidua and labyrinthine layers in serum-free medium. We describe steps for dissecting and separating layers, dicing tissue, and culture setup. We then detail medium processing for downstream analysis. This model allows the investigation of placental signals that may regulate maternal physiology. For complete details on the use and execution of this protocol, please refer to Yung et al. (2023).1


SUMMARY
The lack of a suitable explant culture system restricts the study of factors secreted by the mouse placenta into maternal circulation. Here, we present a protocol for culturing the endocrine junctional zone of the mouse placenta, free from the decidua and labyrinthine layers in serum-free medium. We describe steps for dissecting and separating layers, dicing tissue, and culture setup. We then detail medium processing for downstream analysis. This model allows the investigation of placental signals that may regulate maternal physiology. For complete details on the use and execution of this protocol, please refer to Yung et al. (2023). 1

BEFORE YOU BEGIN
Timing: 13-16 days (for step 1) Timing: 0.5-3 h (for step 2) 1. Set up a timed mating: generate a pregnant female for collection of placentas at embryonic day 12.5 (E12.5). a. Put male and virgin female mice (10-16 weeks old) together, check for vaginal plug the following and subsequent mornings. b. A pregnant female is identified on the basis of presence of a vaginal plug. Noon of the day that the vaginal plug is identified, is defined as E0.5. c. Separate the pregnant female from the male and house for an additional 12 days to reach a gestational age of E12.5. 2. Preparation of reagents and defined Junctional zone (Jz) explant medium (Jz-medium).
a. Prepare and autoclave phosphate buffered saline (PBS Store at 4 C for a maximum period of 6 months. Dissection of the uterine horns from the animal can be carried on a clean bench in a clean dissecting room. Ideally the separation of the placenta from amniotic sac and dissection of the junctional zone and dicing the junctional zone should be carried out in a Class I microbiological safety cabinet (MSC). It is possible to use a Class 2 MSC but adaptations are needed to house the dissecting microscope. Alternatively, these steps can be carried out on a disinfected (70% ethanol) bench in tissue/cell culture room with minimal air movement (i.e., using a bench away from doors and windows with any air conditioning switched off and avoiding opening and closing the door).
A humidified 37 C incubator capable of maintaining an oxygen concentration of 10%, and carbon dioxide concentration of 5% is necessary.
CRITICAL: As the Jz will be cultured, steps must be taken to avoid bacterial and fungal contamination. Good laboratory practice and antiseptic technique are essential to minimize contamination. Use of the anti-mitotic agent and/or additional antibiotics are the last resort for suppressing microbial growth as some of these agents may induce lowgrade cellular stress.
Culture under ambient atmospheric oxygen causes cell stress, hence the more physiological level of 10% is used.
Optional: Conditioned medium is harvested and can be concentrated using a Vivaspin (5000 MWCO) centrifugal concentrator (2 mL, 6 mL or 20 mL, Vivaproducts Inc., USA) depending on the volume of the medium. A refrigerated centrifuge for 15 mL and/or 50 mL tubes (Cat# 5810R, Eppendorf, UK) is required.

STEP-BY-STEP METHOD DETAILS
Timing: approximately 3 h The section describes the step-by-step procedures to setup the Jz explants culture. This includes taking out the uterine horns from pregnant mouse, dissecting out placenta, separating placenta into different regions and setting up the explant culture.

Dissecting out junctional zone from placenta
Note: All dissecting instruments used below must be sterile or autoclaved a. Removal of uterine horns from the pregnant animal at E12.5. We have not tested the procedure at other gestational ages. However, the junction zone decreases in size after E16. i. Cull the pregnant animal by cervical dislocation.
ii. Sterilize the abdomen of the animal with 70% ethanol, blot dry with clean paper towel to remove excess ethanol. iii. Make a skin incision in the abdomen with blunt scissors and the skin peeled back.
Note: This can be done by hand or using blunt scissors.
Note: Take care to prevent the outer skin surface touching the abdominal muscle and so avoid possible cross-contamination. Note: Keep the container on ice during transport.
Note: Take the uteri to the Class I MSC (or the bench as described above) for the following steps.
b. Carefully separate Individual implantations containing the embryo and placenta from the uterine horns and keep under ice-cold PBS in a sterile dish ( Figure 1A).
Note: Use sufficient PBS to submerge the entire implantation.
i. Prepare a 35 mm dish containing ice-cold PBS for each implantation and leave it on the top of the ice. ii. Using blunt forceps take the uterine horns out of the container and place in a 100 mm dish containing ice-cold PBS and rinse briefly to remove material loosely attached to the tissue. iii. Using blunt forceps, grasp the junction between the two uterine horns and remove from the PBS. iv. Using fine scissors separate the implantations from the uterine horns by cutting between each one.
Note: The myometrium may contract and expel the amniotic sac.
v. Place each implantation in a separate 35 mm dish with cold PBS prepared in step b.i. above. Place the lid on to minimize contamination. vi. Keep the dish with the implantation, amniotic sac and placenta on ice until the next step. c. Separating the maternal decidua and dissecting out the labyrinth region are performed under a stereo-microscope ( Figure 1A(b-e). For a better view, a schematic diagram of the images in Figure 1A is presented in Figure 1B. i. Process one implantation at a time in the 35 mm dish in ice-cold PBS.
ii. Using fine forceps, first carefully remove the remaining uterine tissue still enclosing amniotic sac and placenta. If necessary, uses forceps to gently tear the uterine tissue apart.
Note: Occasionally, the decidua separates from the placenta and adheres to the myometrium. If this happens, jump to step d below.
iii. Using the same fine forceps, peel off the decidua layer from the basal (outer convex) surface of the placenta. Use the amniotic sac as an anchor to hold the placenta in position. iv. Access the decidual layer from the edge of the placenta. ll OPEN ACCESS  v. Peel off from the edge of the placenta gently and gradually extend towards the center of the disc to remove entire decidua layer ( Figures 1C and 1D). vi. Once the decidual layer is removed, separate the amniotic sac from the placenta.
Note: At the position where the umbilical cord connects to the placenta and the embryo, use the fine forceps to cut the umbilical cord as close as possible to the chorionic plate of the placenta in order to minimise chorion/amnion contamination. d. Carefully dissect the labyrinth from the junctional zone with ultrafine forceps.
i. Lay the placenta flat with chorionic surface with umbilical cord attached facing upwards.
Note: Identify the labyrinth as the central red area as it contains maternal and fetal blood vessels. The junctional zone is the paler, almost colourless rim.
ii. Use one forceps to gently hold the placenta and use another forceps to separate along the edge of the labyrinth. Push closed forceps into the boundary between the two and allow them to open slowly to tease the tissue apart. Continue around the disc. iii. After completing the round, flip the placenta over. Use one set of forceps to lift up the edge of junctional zone layer and the other forceps to slowly tease apart the two layers towards the center of the disc until the two regions are completely separated.
Note: If necessary, clean up the dissected junctional zone by removing any remaining labyrinth tissue which is pink in colour ( Figure 1F).
iv. Transfer the junctional zone to a new dish containing pre-cooled DMEM/F12 medium and keep on top of the ice until step 2). 2. Dicing junctional zone tissue.
a. Dice the dissected junctional zone tissue into cubes approximately 1-1.5 mm 3 in size using fine forceps inside an insert in dish containing the 2 mL Jz-medium under the stereo-microscope. b. Place the junctional zone tissue from one or two placenta in a 15 mm or 24 mm diameter Netwellä respectively.
Note: If the placentas are from animals with the same genotype the same dish containing Jzmedium may be re-used. Otherwise clean forceps and new inserts are required for each placenta.
Note: Clean the forceps by dipping in 70% ethanol following by serial rinsing in 3 pots PBS to remove residual ethanol.
3. Set up ex vivo junctional zone explant cultures a. Transfer the 15 mm or 24 mm diameter inserts containing junctional zone explants into either a 12-well plate or a 6 well plate with well containing 1 mL or 3 mL Jz-medium respectively.
Note: Make sure all tissue explants are submerged under the medium.
4. Culture incubation a. Incubate the plate in a humidified 37 C incubator containing 5% CO 2 and 10% O 2 balance N 2 for 48 h. b. Check viability of the tissue by adding MTT (50 mg/mL) to the culture medium at a final concentration of 500 mg/mL. After incubation for 1 h in the incubator dark blue/purple staining indicates viable tissues (Figure 2). In the higher magnification image, a few explants are not stained after MTT incubation ( Figure 2B, arrows).

OPEN ACCESS
Collecting conditioned medium for downstream analysis -Day 3 Timing: Approximately 3 h Timing: 1-3 h (for step 5) This section describes step by step procedures for collecting and processing conditioned medium and harvesting explant tissues ready for downstream analysis.
5. Collecting and concentrating conditioned medium a. At the end of incubation, collect the junctional zone explant tissue from the inserts and place in a 1.5 mL tube and snap freeze in liquid nitrogen for subsequent analysis (Western blotting, PCR etc). b. For the conditioned medium (CM) from each well, it can either collect separately or pool together in 15 mL or 50 mL centrifuge tubes. Centrifuge the CM at 4000 rpm (1771 g) for 30 min at 4 C to remove tissue debris. c. Transfer the supernatant (free of cell debris) to a new tube. d. If the clarified CM is to be concentrated, transfer it to a Vivaspin centrifugal concentrator and centrifuge according to the manufacturer's instructions until the desired reduction in volume is achieved. Transfer the concentrated CM to a 1.5 mL tube.
Note: The CM is generally concentrated by 20 to 30-fold. Optimization is required to identify correct extent of concentration for different downstream processes.
e. Store both junctional zone explant tissue fragments and concentrated conditioned medium at -80 C until further analysis.
CRITICAL: To avoid any contamination during explant culture, it is crucial to keep the entire procedure as clean as possible. All plasticware, glassware and instruments must be sterilized prior to use. Equipment and bench surfaces need to be disinfected with 70% ethanol prior to use.

LIMITATIONS
We also have not tested extending the incubation beyond 48 h so do not know whether it is suitable for prolonged culture. We did not test the effect of changing the culture medium during culture.
This junctional zone explant model can only be used for placenta containing distinct junctional and labyrinth zones. Therefore, it is likely to be suitable for studying the rat placenta, but it will not be suitable for other species.
We have not tested this method using placentas at gestations later than E12.5. It is known that the labyrinth volume increases as gestation advances, while junctional zone and decidua increase their ll OPEN ACCESS volume and peak at E16.5 before declining at E18.5. 2 Nonetheless, we anticipate it may be useful for studies at other gestational ages.

TROUBLESHOOTING Problem
Contamination of cultures.

Potential solution
Good general laboratory practice is essential. Ensure the room used for dissecting the animal is clean and does not have excessive air movement (such as from open windows or directly under ventilation openings). Dissection of the junctional zone with the stereo-microscope can be carried out in a Class 1 MSC. Dicing the junctional zone into small pieces can be similarly carried out inside a Class 1 MSC. If contamination persists, avoid movement inside the room while performing the dissection. Alternatively, the dissection can be carried out in a Class 2 MSC. Use of additional antibiotics including anti-mitotic agents is the last resort.

Problem
Low tissue viability or high cellular stress in explants.

Potential solution
This may result from prolonged dissection time and/or the PBS used not being ice cold. PBS should be pre-cooled prior to use. It should then be kept in an ice-bath at all times. Spongiotrophoblast cells in the junctional zone are highly metabolically active. If the placenta is kept in PBS for a prolonged period because of slow procedures, this may cause nutrient deprivation, thereby provoking stress. The PBS being not sufficiently cold may also facilitate higher metabolic activity in tissues, contributing to stress. The cellular stress levels in the explants can be checked using western blotting analysis for cellular stress markers such as heat shock proteins, ER and mitochondrial chaperone levels on Time = 0 tissues. There are multiple ways to shorten the procedure time, such as dissecting fewer placentas. For a beginner, it may take 5-10 min to dissect out the junctional zone from a single placenta. However, once you become familiar with the technique, it usually takes 2-3 min. Ideally, limit the entire procedure time to under 2 h.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to, and will be fulfilled by the lead contact, Prof. Steve Charnock-Jones (dscj1@cam.ac.uk).

Materials availability
All reagents and consumables are commercially available.
Data and code availability Not applicable.