This method will be fundamental for understanding gastrulation along a formation in mammals. By allowing experimentation in living embryos during embryonic stages that have long been hidden inside the maternal uterus. It allows, continues efficient and appropriate growth of mouse embryos outside the uterus for an extended period of up to 6 days.
Quick and adequate embryo dissection and pregastrulation of the gas concentration and pressure are the most critical steps for successful embryo growth. To begin culturing 7.5 day old or more advanced mouse embryos, turn on the rotator and the heating unit of the roller culture incubator system for at least 1 hour before the embryo dissections. Also, add autoclave water to the gas inlet bottle and the outlet test tube.
Next, turn on the gas regulator module by pressing the main switch, then use the respective controllers to set the oxygen and carbon dioxide values to 5%Open the gas regulator and move the voltage switch in the pressure transmitter to set the gas pressure to approximately 6.5 to 7 pounds per square inch. Confirm the gas pressure value using a digital pressure gauge. 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 or opening the gas flow valve on the water bottle's lid. Ensure that the gas flow allows the formation of bubbles at a rate of 2 to 4 bubbles per second or set the bubble flow at the first point where bubbling comes out to the water filled outlet tube. Next, fill the culture bottles with 2 milliliters of culture medium.
Use the hollowed silicon bungs to seal the bottles and plug them into the hollowed rotating drum for pre-equilibration for 1 hour. Keep the empty spaces in the rotating drum sealed using the solid bungs. Next, dissect the embryos from a euthanized pregnant female mouse.
Clean the abdomen with 70%ethanol and cut the skin and abdominal wall using scissors. Locate one end of the uterus and cut at the intersection between the ovary and the uterus. Continue cutting along the uterus until the other end and transfer to a 100 millimeter Petri dish filled with DPBS.
Quickly wash the conceptuses in DPBS and cut in pairs to facilitate embryo handling. Move all the pairs of conceptuses to pre-equilibrated dissection medium in a 60 millimeter Petri dish, then cut into the individual conceptuses. Next, using a pair of gross forceps, remove the uterine wall of the conceptuses by tearing the uterine tissue, then using fine, microsurgical forceps, 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. Finally, using fine forceps, grasp the the embryo from the decidua and peel the parietal yolk sack off the embryo, leaving the intact ectoplacental cone attached to the egg cylinder. Select the embryos in the neural plate, or early head fold stage, that show no damage in the epiblast.
Transfer 5 to 6 selected embryos per bottle to the pre-equilibrated glass culture bottles and place the bottles in the rotating culture system at 37 degrees Celsius, in an atmosphere of 5%oxygen and 5%carbon dioxide. To culture pregastrulation and early gastrulation embryos and static plates, add 250 microliters of freshly prepared ex-utero embryo culture medium to each well of an 8 well plate. Place the plates inside a carbon dioxide incubator at 37 degree Celsius for pre-equilibration.
After dissecting the embryos out of the uterus as demonstrated previously, transfer the individual embryos into each well of the 8 well plate using a micro pipette and place the plate inside the incubator with 5%carbon dioxide at 37 degrees Celsius. Image the embryos under a stereo microscope and select for culture only those with a well-formed amniotic cavity with no evident damage and without the Reichert's membrane. The roller culture conditions described in this protocol for 7.5 day old embryos support constant and normal embryo growth with an average efficiency close to 75%after 4 culture days.
The efficiency of embryo development varies across diverse mouse genetic backgrounds but is consistently robust. Supplementation with HBS instead of HCS yields an efficiency of approximately 68%after 4 days of culture. The development of 6.5 embryos and static plates is correctly recapitulated with an efficiency of more than 90%using ex-utero embryo culture medium with both HCS and HBS.
Cultures starting from pregastrulating embryos show a 46%efficiency of proper development to the early somite stage and nearly 17%of the embryos complete proper development after 6 days of culture. The morphogenesis and tissue development proceed properly until 42 somites. Representative developmental defects observed in embryos for cultures initiated from embryonic day 7.5, 6.5, and 5.5 are shown here.
Embryos with minor damage to the epiblast or embryos retaining the Reichert's membrane should be discarded. Early embryos will not grow properly or display severe developmental delays while attachment of the embryonic epiblast to the surface of the plate will cause the failure of further embryo development. The main abnormalities observed in defective embryos are the development of the posterior region outside the yolk sack or defects in the growth of the neural folds.
In cultures developed from 5.5 day old embryos are frequently observed developmental defect is the presence of a small, underdeveloped epiblast. Using this method, researchers are able to perform a variety of physical or chemical manipulations in developing post-implanted embryos. For instance, manipulation, cellular transplantation, or lineage tracing.
This method not only paves the way for studying mouse post-implantation developing in great detail, but also for implementing similar culture methods in embryos from other species, such as human.
