Recombinant limbs are a powerful model for a study in the process of cell differentiation and power formation under the influence of embryonic signals in an in vivo environment. This assay allows multiple variations permitting potential applications without being restricted to chicken limb developmental biology. It can be applied to other stem or progenitor from other organisms.
To begin, remove eggs from the incubator after three and a half days, swab with 70%ethanol, and leave them to air dry. Next, identify and locate the developing embryos by candling the egg and observing the blood vessels. Discard eggs that do not have an embryo.
Tap the blunt end of the egg shell with the end of a pair of blunt forceps to open a window and remove about one square centimeter of the shell using the forceps. Then transfer the eggs to a plastic or carton holder. Place the eggs one by one under the stereomicroscope.
Identify the air membrane and remove it by picking a small hole in the area without any vessels. Pull this area out with the aid of fine surgical forceps and remove pieces of eggshell in contact with the embryo. Tear open the amniotic sac with fine surgical forceps.
Carefully remove the embryo from the egg with blunt forceps and transfer it into a sterile Petri dish containing ice-cold 1x PBS. Wash the embryos once in ice-cold 1x PBS and withdraw any remaining membranes under the stereomicroscope. Then locate the hindlimb buds.
Snip each hindlimb bud longitudinally, cutting very close to the embryo flank with fine surgical forceps. Maintaining the embryos in 1x PBS. Then transfer the limb buds into a 1.5 milliliter micro centrifuge tube with a plastic transfer pipette.
Next, replace the PBS with 500 microliters of 0.5%trypsin solution. Incubate the limb buds in a thermoblock for seven minutes at 37 degrees Celsius. Then replace the trypsin solution with 500 microliters of two milligram per milliliter collagenase type four in HBSS and incubate it in the thermoblock for another eight minutes.
Remove as much collagenase as possible and replace it with one milliliter of cold high glucose DMEM with 10%FBS to inactivate the enzymes. Pipette the mixture gently around 10 times. Filter the suspension containing the cells with a 70 micrometer cell strainer to leave behind the ectoderms.
Pipette the suspension again around five to 10 times and centrifuge at 200 times g for five minutes at room temperature. Then discard the supernatant. Wash the excess collagenase with one milliliter of media, then centrifuge the suspension and discard the medium carefully.
Next, add 1.5 milliliters of media without disturbing the cells and incubate them for one to 1.5 hours at 37 degrees Celsius in a thermoblock, allowing the cells to form a compact pellet. After obtaining the hind limb buds, transfer them into an empty micro centrifuge tube with a plastic pipette. Remove any excess 1x PBS and replace it with 500 microliters of 0.5%trypsin in sterile 1x PBS.
Incubate the mixture for 30 minutes at 37 degrees Celsius in a thermoblock. Transfer the trypsin solution with the limb buds into a sterile Petri dish. Then remove as much of the excess trypsin as possible and flood the Petri dish with ice-cold 1x PBS with 10%FBS until all limb buds are covered.
Identify the limb buds under the stereomicroscope. Next, identify the ectoderm as a slightly detached transparent membrane from the limb mesodermal cells. Detach and separate the ectoderm with forceps holding the most proximal end of the limb bud with fine surgical forceps.
Take the mesodermal pellet from the previous steps and discard around 600 microliters of the medium. Then carefully detach the pellet with a pipette tip without smashing it. Turn the tube upside down to transfer the pellet into the Petri dish containing the empty ectoderms.
Cut a small piece of the pellet with a pair of fine surgical forceps and place it as close as possible to the ectoderm. Open the ectoderm with the surgical forceps and place the pellet as tightly as possible into the ectodermal hall. Open the amniotic sac near the forelimb to expose the right flank of the embryo.
Guided by the forelimb position, perform wound scratching with a tungsten needle to the length of two to three somites, slightly damaging the mesoderm until it bleeds. Individually transfer the recombinant limbs into the chick embryo, placing its base over a somite wound. Fix the recombinant limb correctly with two pieces of palladium wire.
Align the base of the recombinant limb with the wound. Seal the window with tape and return the egg to the incubator. In successful implantation, the recombinant limb was observed as a protuberance that was securely attached to the mesodermal wall of the donor embryo.
On the contrary, the recombinant limb was detached from the mesodermal wall or presented a rough morphology when either cell viability or the graft failed. Alcian blue staining demonstrated skeletal elements in a six day chicken, chicken recombinant limb. Before clearing the Alcian blue, images in ethanol were obtained for morphological and quantitative measurements.
H&E stained or unstained limbs were sliced to observe tissues and cells. This technique can develop limb organoids in vivo and study cell differentiation or morphogenetic process by using the stem cells from different sources or species.
