Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

All animal experiments were performed according to the Animal Protection Guidelines of Weizmann Institute of Science and approved by relevant Weizmann Institute IACUC (#01390120-1, 01330120-2, 33520117-2). Healthy pregnant women were asked to give their informed consent to collect blood from their umbilical cord, as approved by the Rambam Medical Center Helsinki Committee (#RMB-0452-15). Healthy adults were asked for their informed consent to have blood collected according to the guidelines of the Weizmann Institute of Science Helsinki Committee (#1566-3).

1. Media preparation

1. Prepare the dissection medium using 500 mL of Dulbecco's Modified Eagle Medium (DMEM) without phenol red and L-glutamine, supplemented with 10% fetal bovine serum, 5 mL of penicillin/streptomycin, and filter-sterilize using a 0.22 µm filter.
   NOTE: Keep the dissection medium at 4 °C for up to 1 month.
2. Prepare ex utero embryo culture medium (EUCM) freshly every culture day inside a biological hood using 25% of embryonic medium plus 50% rat serum and 25% human umbilical cord blood serum (HCS) or human adult blood serum (HBS).
3. To prepare embryonic medium, add 5 mL of glutamine, 5 mL of HEPES, and 5 mL of penicillin/streptomycin in 500 mL of DMEM without phenol red and L-glutamine. Prepare 10-15 mL aliquots and store them at 4 °C for up to two months.
   NOTE: Double the antibiotic concentration if experiencing any contamination.
4. Heat-inactivate the rat serum at 56 °C for 30-45 min and filter through a 0.22 µm polyvinylidene difluoride filter. Use the serum for culture on the same day of inactivation. Thaw HCS or HBS and use it immediately for experiments.
   ​NOTE: Commercial rat serum provides consistent results (a minimum of 4 lots were tested without evident variation). Alternatively, high-quality rat serum can be obtained in-house as described previously^8,14. If HCS is not available, replace it with in-house-collected HBS. Rat serum, HCS, and HBS can be refrozen once and kept at -20 °C for use on a different day. Thaw and combine the refrozen serum with a larger volume of freshly thawed serum and do not refreeze it.

2. Collection of human umbilical cord blood serum and human adult blood serum

1. To isolate HCS, collect umbilical cord blood from healthy pregnant women on the day of the scheduled caesarian delivery.
   1. Double-clamp the umbilical cord 5-7 cm from the umbilicus and transect between the clamps immediately after delivery of the infant.
   2. Manually draw blood using a large-bore 14 G needle and a 50 mL syringe directly from the umbilical vein while the placenta remains in situ to avoid any traces of hemolysis.
      NOTE: Collect blood after the infant is removed from the field of surgery, and umbilical blood has been drawn for clinical tests.
2. Dispense the collected blood to 5 mL procoagulant sterile test tubes and immediately transfer them to 4 °C for 15 min to allow complete coagulation. Centrifuge the coagulated test tubes at 2,500 × g for 10 min at 4 °C.
3. Discard tubes showing signs of hemolysis (pink-red serum). Collect the serum (yellow-color) using a 5 mL pipette and filter it through a 0.22 µm filter. Heat-inactivate the serum immediately in a 56 °C water bath for 45 min.
4. Prepare 1-1.5 mL aliquots of the inactivated serum and store them at -80 °C for up to six months. If needed, ship at -70 °C using dry ice.
5. For adult human blood serum collection, draw blood from healthy adults and immediately isolate serum following the same protocol described for umbilical cord blood serum. Store the HBS as heat-inactivated and filtered aliquots at -80 °C for up to six months.
   ​NOTE: Human umbilical cord serum and adult human serum prepared freshly in-house gave superior results to commercially available serum. Collect HCS from healthy women, over the age of 18 and under 40 years scheduled for cesarian section delivery. Exclude women who gave vaginal birth and women with any chronic illness or active medical conditions, including gestational diabetes or hypertension.

3. Ex utero roller culture of embryos from E7.5 to E11

1. Setting up the roller culture incubator and gas regulator
   1. Culture E7.5 or more advanced embryos using the roller culture incubator system. Turn on the rotator and the heating unit at 37 °C for at least 1 h before the embryo dissections. Add autoclaved water to the gas inlet bottle and the outlet test tube.
      NOTE: Place a thermometer adjacent to the rotating drum and frequently check the stability of the temperature inside the incubator. Open the incubator as quickly as possible as prolonged opening time will increase the temperature and affect embryo development. When necessary, add more autoclaved water to the gas inlet bottle and the outlet test tube to keep them to a minimum of half capacity during culture. Protect the embryos inside the incubator from light by covering the incubator with a cloth.
   2. Turn on the gas regulator module by pressing the main switch and subsequently turning on the Oxygen/Nitrogen and CO₂ controllers (Figure 1A). Set the oxygen and CO₂ values to 5% using the respective controllers. Open the gas regulator and set the gas pressure auf ~6.5-7 pounds per square inch (psi) by moving the voltage switch in the pressure transmitter (Figure 1B). Confirm the gas pressure value using a digital pressure gauge.
   3. Monitor the gas flow by checking the rate of bubbles created inside the outlet water-filled test tube allocated inside the precision incubator. Set the proper bubble rate by closing/opening the gas flow valve on the lid of the water bottle. Ensure that the gas flow allows the formation of bubbles at a rate of 2-4 bubbles per second or set the bubble flow at the first point where bubbling comes out to the water-filled outlet tube.
   4. Fill the culture bottles with 2 mL of culture medium and plug them into the hollowed rotating drum for preequilibration for 1 h. Use the hollowed silicon bungs to seal the bottles to the drum. Keep sealed the empty spaces in the rotating drum using the solid bungs.
      NOTE: The absence of bubbles in the outlet tube (no gas coming through the system) or an exceptionally high gas flow may affect embryo development. In the case of a lack of bubble flow to the outlet test tube, check that all silicon bungs are properly positioned in the rotating drum and sealing the system, and check that the water bottle is closed properly and all the tubing is connected correctly. A lack of bubbling coming into the water bottle may indicate a malfunction in the gas regulator.
2. Dissection of mouse embryos from pregnant mice
   1. Preequilibrate the dissection medium inside an incubator at 37 °C and 5% CO₂ for a minimum of 1 h. Leave the lid slightly open to allow gas exchange.
   2. Sacrifice the pregnant female by cervical dislocation, clean the abdomen of the female with 70% ethanol, and cut the skin and abdominal wall with scissors. Locate one end of the uterus and cut at the intersection between the ovary and the uterus. Next, cut along the uterus until the other end and transfer it to a 100 mm Petri dish filled with room temperature Dulbecco's phosphate-buffered saline (DPBS).
      NOTE: Use of non-hormone-primed, naturally mated females is recommended.
   3. Quickly wash the conceptuses in DPBS and cut in pairs to facilitate embryo handling. Move all the pairs of conceptuses to preequilibrated dissection medium in a 60 mm Petri dish and cut into individual conceptuses.
   4. Remove the uterine wall of the conceptuses by tearing the uterine tissue using a pair of gross forceps. Use fine microsurgical forceps to cut the tip of the pear-shaped decidua. Insert the forceps next to the embryo parallel to its long axis, and open the forceps to split the decidua into halves.
   5. Finally, leave the intact ectoplacental cone attached to the egg cylinder by grasping the embryo from the decidua and peeling the parietal yolk sac off the embryo using fine forceps.
      NOTE: To avoid affecting the embryo, perform dissections on a microscope equipped with a heating plate at 37 °C within a maximum of 30-40 min. Dissect one litter of embryos at a time.
   6. Immediately after dissection, transfer the embryos to a new plate filled with equilibrated dissection medium using a glass Pasteur pipette to prevent the embryos from sticking to the tissue debris.
   7. Select those embryos in the neural plate/early head fold stage showing no damage in the epiblast and transfer 5-6 embryos per bottle to the preequilibrated glass culture bottles.
      NOTE: To prepare the glass Pasteur pipette, cut the opening of the pipette to an adequate size to fit the embryos using a glass cutter, and keep it in ethanol to avoid contamination. Wash the glass pipette with PBS before transferring embryos. When transferring the embryos to the culture bottle, make sure to carry over as little volume of the dissection medium as possible to avoid diluting the EUCM.
   8. Place the bottles in the rotating culture system at 37 °C, in an atmosphere of 5% O₂ and 5% CO₂.
   9. Each day of culture, remove the bottles one by one from the incubator to evaluate embryo development under a stereomicroscope. Make sure to close the empty hole in the drum with a solid bung when removing a bottle. Cut the tip of a sterile plastic Pasteur pipette to fit the embryo size and move the embryos to a Petri dish to facilitate observation. Make sure that the embryos are always covered by medium.
      NOTE: Minimize the time for which the embryos are outside the incubator to avoid detrimental effects in the embryos due to a decrease in body temperature. Handle the embryos carefully to avoid rupture of the yolk sac blood vessels, affecting embryo survival.
   10. At culture day 1 (equivalent to E8.5), transfer groups of 3 embryos to a new bottle with 2 mL of fresh and preequilibrated EUCM containing extra 3 mg/mL of D-glucose and a gas mixture of 13% O₂, 5% CO₂.
   11. After 48 h (equivalent to E9.5), transfer groups of two embryos to a new bottle with fresh preheated EUCM plus 3.5 mg/mL of glucose in a gas atmosphere of 18% O₂ and 5% CO₂.
   12. At 72 h of culture (equivalent to E10.5), move each embryo to an individual bottle containing 1.5-2 mL of fresh medium supplemented with 4 mg/mL of glucose and a gas supply of 21% O₂ and 5% CO₂.
       NOTE: Embryos reach their maximum growth about midnight of culture day 4.
   13. Use fine forceps to remove the yolk sac and amnion, and detach the umbilical cord for proper observation of embryo morphology. Turn off the gas regulation module and precision incubator.
       ​NOTE: Preequilibrate the culture medium for 1 hour by incubation inside a glass bottle in the roller culture with an adequate gas atmosphere according to the embryo stage. Clean the culture bottles and all incubator glassware after every use by performing three washes with running distilled water followed by an overnight wash submerged in 70% ethanol. Rewash three times with running distilled water, let the bottles dry overnight, and sterilize by autoclave. Likewise, autoclave the silicon plugs regularly. Thoroughly clean all dissection tools with 70% ethanol and dry-sterilize them.

4. Extended embryo culture from E5.5/E6.5 to E11

1. Culture pregastrulation (E5.5) and early gastrulation (E6.5) embryos until the early somite stage (E8.5) in static plates.
2. Preequilibrate the dissection medium for 1 h in an incubator with 5% CO₂ at 37 °C.
   NOTE: To allow gas exchange, do not close the lid of the tube completely.
3. Prepare the culture plates by adding 250 µL of freshly prepared EUCM to each well. Place the plates inside a CO₂ incubator at 37 °C for preequilibration.
   NOTE: Although 8-well plates are well-suited for embryo growth, culture can be done in any other plate by adjusting the volume of the medium according to the size of the plate.
4. Dissect egg cylinder embryos out of the uterus following the technique described for E7.5. Remove the parietal yolk sac of the embryo and leave the intact ectoplacental cone attached to the egg cylinder.
5. Transfer individual embryos into each well of the 8-well plate using a micropipette and place the plate inside the incubator with 5% CO₂ at 37 °C.
6. Image the embryos under a stereomicroscope and select for culture only those with a well-formed amniotic cavity, with no evident damage and without the Reichert's membrane.
   NOTE: Use 20 µL pipette tips to transfer E5.5 embryos and 200 µL tips to transfer E6.5 embryos. In the case of cultures starting at E6.5, HCS can be replaced by freshly collected HBS.
7. Remove half of the medium and add 250 µL of freshly prepared prewarmed EUCM after 24 h of culture, ensuring that the embryos are always immersed in the culture medium. In the case of cultures starting at E5.5, after two days of culture (equivalent to E7.5), remove 200 µL and add 250 µL of new EUCM.
   NOTE: During static culture, embryos might attach to the plate (mainly when using plastic plates), which will severely compromise embryo development. Attachment of the embryo to the plate is more frequent on the first day of culturing E5.5 embryos. To prevent attachment, carefully push the embryos away from the plate surface using fine, sterile forceps. Verify that only the ectoplacental cone remains attached to the surface of the plate.
8. Transfer the embryos into the roller culture at the early somite stage (4-7 somites; after three days of culture for embryos explanted at E5.5 and two days for cultures started at E6.5), following the same indications described previously for E8.5 stage embryos.
   NOTE: Transferring the embryos to the roller culture at somite stages different than indicated above results in failure of further development. In contrast with E7.5 ex utero cultures, it is possible to maintain the embryos in a constant atmosphere of 21% oxygen and 5% CO₂, providing a slightly higher efficiency of embryo development than atmospheres with dynamic oxygen concentration.