The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The overall goal of this experiment is to develop a reliable cam xenograft model of hepatocellular carcinoma. This is achieved by first incubating the eggs to prepare them for inoculation. As a second step, the choal and toic membrane or the cam of the egg is dropped and a window is cut into the eggshell.

Next, the egg is inoculated with human hepatocellular carcinoma cells through the eggshell window in order to establish tumor growth. The results show that three dimensional vascularized tumors were successfully grown using human hepatocellular carcinoma cell lines. Using this protocol, embryonic survival rates of up to 93%were achieved.

The main advantages of the CAM assay over other in vivo techniques such as mouse tumor models is the short experimental assay time, low overall cost of the experiment and ready. Reproducibility CAM assays can be used to study angiogenesis, tumor, cell invasion, and metastasis. They can also be used to determine the effect of small molecules and drugs on growth and development of tumors.

Visual demonstration of this procedure is important because the different steps that go into preparing the egg, including candling, identifying vasculature, making small punctures near the air sac and on top of the egg without draining it. Cutting the windows open without cracking the eggshell and making sure that the cam is intact through all the processes could be challenging for new users. To begin place.

Eight day old specific pathogen free embr native chicken eggs in a rotating egg tray with a stamp dens facing upward. Set the rotating tray inside of an egg incubator at 36 degrees Celsius with 50%humidity and incubate the eggs for 48 hours. After incubation, place the eggs in an egg rack with a stamped ends up and place them in a laminar flow hood.

Turn off the room and hood lights hold the stamped end of the egg lightly to the egg candler to expose the vasculature of the chick Chorio Allen Toic membrane known as the cam and the air sack. Make a mark between the two major blood vessels with a pencil. Next, make one hole at the tip of the egg above the air sack at the stamped end and a second hole at the pencil mark.

Using a sterile push pin, the hole should be about three millimeters deep. Squeeze the air from a safety bulb and press the open end firmly against the hole above the air sack. Slowly release the bulb to apply suction pulling air into the hole at the pencil mark and causing the cam to drop away from the shell.

Confirm that the air sack has moved from the stamped end of the egg to the pencil marked hole using the candler. If it is not, make both holes slightly deeper and reapply suction with the safety bulb. Next, hold the egg in one hand using a rotary tool with a 1516 inch cutoff wheel attachment.

Make two transfers cuts through the shell without touching the cam. The cut should be two centimeters in length and one centimeter apart. Then make longitudinal cuts at the ends of the two transverse cuts by lightly touching the cutoff wheel to the shell.

To remove the shell slide sterile forceps under and parallel to the shell piece. Grab the piece with the forceps and cleanly remove it. Hold one end of a piece of tape over itself, and then place the tape over the opening in the egg.

Shell place the egg in an egg tray and set the tray in the incubator without rotation for no more than two to three hours before inoculation. To prepare for inoculation, place a basement membrane protein mixture, such as matrigel on ice to prevent polymerization. Next, use four to five milliliters of a two millimolar solution of EDTA per flask to detach the tumor cells and place the flask in the tissue culture incubator for five minutes.

Reus, suspend the detached cells in the medium and count the cell number. Use between 500, 000 and 2 million cells for grafting onto the cam, depending on the cell line. Next, transfer the volume required to obtain the appropriate number of cells to a new tube and centrifuge for five minutes.

At 500 times gravity, discard the supernatant and resuspend the cell pellet. In 40 microliters of PBS containing calcium and magnesium known as PBS plus plus and 20 microliters of basement membrane mixture. At this point, drugs or other test agents can be added to the cell suspension.

Next, place the egg tray in the laminar flow hood. Peel the tape off of the egg and drop a sterile one millimeter silicone ring from a cryogenic vial through the opening and onto the cam pipe. Had the 60 microliter solution of cells, PBS plus plus and basement membrane mixture into the center of the silicone ring, retape the window and carefully move the egg tray back to the incubator.

Allow tumors to grow for five to seven days. For harvesting the tumors. Place an underpad and a biohazard bag in the hood.

Prepare 35 millimeter by 10 millimeter plates containing one milliliter of PBS plus plus and para formaldehyde containers as needed for histology. Insert the pointed end of sterile dissection, scissors into the egg at the hole near the stamped end, and cut around the length of the entire egg. The cam and the tumor with the ring should stick to the top half of the shell with the opening.

Extract the tumor from the cam using dissection, scissors, and forceps, and place the tumor in a 35 millimeter by 10 millimeter plate. Measure and weigh the tumor. Fix the tumor in paraform, aldehyde and paraffin embedded for use in histology and immunohistochemistry experiments.

After harvesting the tumor, mince the tumor in the plate with dissection scissors. Then pour the mince tumor into a 15 milliliter centrifuge tube and remove the PBS plus plus by pipetting. Add two milliliters of a one milligram per milliliter solution of collagenase in PBS plus plus and mix by inverting the tube several times.

Incubate the tube at 37 degrees Celsius for at least 30 minutes and up to several hours. Next, add five milliliters of medium to the tube and mix thoroughly by pipetting 20 to 40 times. This is critical for dissociating the cells completely.

After the sediment has settled, transfer the snat into a fresh tube, centrifuge the tube at 500 times, gravity for five minutes, and discard the supernatant leaving behind the dissociated cell pellet. When the cam was seated with HUH seven tumor cells, a vascularized tumor was visible inside the ring. After five days of growth, histology of the HUH seven tumor from the CAM assay showed that the tumor cells resemble undifferentiated hepatocellular carcinoma cells.

While working with this method, it is important to remember that certain optimizations might be required as far as different cells or different drugs are concerned. For instance, some cell lines might need higher or lower densities to form three dimensional tumors.