The overall goal of this procedure is to utilize a serum free oxygenated culture system to study and manipulate specific aspects of mouse embryonic development. This is accomplished by first isolating the uterus with embryos from the mother mouse. The second step is to separate the embryos with an intact yolk sack from the placental decidua after segmental dissection of the uterus.
Next, the embryos should be gently exteriorized from the yolk sac while maintaining the integrity of the embryonic vasculature. The final step is to culture the embryos in a serum free oxygenated rolling bottle culture apparatus at 37 degrees Celsius for a specific amount of time. Ultimately, this whole embryo culture system, which supports the development of different structures at both the morphological and molecular levels, can be used for the study of developing mouse embryos using a variety of techniques.
This method can answer key questions in the field of developmental biology, such as the role of different signaling mechanisms in sulfate specification and tissue morphogenesis in the developing mammalian embryo. Visual demonstration of this method is critical as the isolation of embryos with an intact yolk sac and subsequent ization steps or difficult to learn any damage to the embryonic vasculature or osac vasculature can affect the development of embryo in the culture. To begin disinfect all work surfaces with 70%alcohol and spray every item before placing it into the culture hood.
When ready, prepare the culture medium and all supplements needed for the procedure First, add the albumin powder to the knockout DMEM and mix thoroughly until completely dissolved. Next, add the N two supplement, KSR, and the antibiotics mix thoroughly and filter sterilize. Store the media in 50 milliliter conical tubes at four degrees Celsius until ready to use Before starting the dissection, preheat the necessary solutions to 37 degrees Celsius and keep them warm throughout their procedure.
At this point, the culture bottles should also be prepared and placed in the culture apparatus in order to pre gass and maintain them at 37 degrees Celsius. This will also save time in transferring the embryos later. To begin, place the euthanized animal onto a sterile work surface and spray the ventral abdominal surface with ethanol.
To keep hair from sticking to the instruments, open the abdominal cavity to locate the uterus. Lift the uterus with forceps and use light operating scissors to cut at the uterine body and at the tips of the uterine horns. To separate it quickly, rinse the entire uterus in warm PBS to remove any blood and immediately place in a Petri dish filled with warm DMEM.
Sterilize the instruments with 70%ethanol Before proceeding under a dissecting microscope, segmentally dissect the uterus with light operating scissors to produce small openings on either side. Gently insert a pair of blunted micro dissecting tweezers to widen the opening with the placental deciduous exposed. Gently tear it with a pair of micro dissecting tweezers to expose the parietal yolk sac with the riker's membrane.
Use one edge of the tweezers to gently pierce both and separate them from the underlying visceral yolk sack To expose the embryos embryos. With intact visceral yolk sacks should be transferred immediately to warm culture medium after separation from the uterus. To aid in the development of the embryo in culture, make a small opening in the yolk sack with a sharp pair of tweezers by gently piercing in an area adjacent to the head region, taking care to avoid major blood vessels.
Alternatively, two pairs of blunt tweezers can be used to hold the yolk sack and gently tear to make a small opening. Expand the opening with the tweezers to a size just enough to fit the embryonic head. Hold the amniotic membrane, wrapping the embryo gently away from the body and tear it with a pair of tweezers, the embryonic head, and later the whole embryo should be gently exteriorized from the yolk sack while maintaining the integrity of the embryonic vasculature.
Any damage to the yolk sack vasculature can affect development and such embryos should not be used for culture. Next, examine the embryos and group them by morphological criteria, including body and head size, limb and eye morphology. Stage them by counting the number of somites.
Transfer the embryos immediately to a Petri dish with fresh, warm culture medium, and take it to the culture hood where the embryos should again be transferred to a Petri dish With sterile culture media working in the culture hood, use a sterile plastic pipette to insert a mouse embryo into each bottle prepared earlier. Next, carry the culture bottles aseptically to the culture apparatus. Secure them to the rolling disc and turn on the rotation.
This allows the embryos to float freely in the media and also helps with free gas exchange. Gas flows from a nearby cylinder into the chamber and then into the bottles through the rubber cork culture. The embryos for 16 to 40 hours and regularly check for the gas outflow and culture chamber temperature.
The culture media should be completely replaced with fresh media at specific time intervals. It is best to let the embryos adapt to the culture conditions for at least 30 minutes before performing any manipulation embryos that perform poorly and show feeble heartbeats can be discarded, and the remaining embryos that show good heartbeat can be utilized for further studies. These images demonstrate that embryos in culture replicate in utero development.
About 58%of cultured embryos be exhibit comparable development to in utero developed embryos. A intermediate development is seen in about 28%of embryos C and 14%of embryos show poor development in culture D.Developmental differences occur when mouse embryos are co cultured embryos that appear similar at the start of co culture A and B show differences in their embryonic development. After 16 hours, A and B Once master, this technique can be done in 25 to 30 minutes for a later size of 10 to 12 embryos.
We have found that this embryo culture system is a valuable addition to our envivo studies of embryonic development.
