This procedure allows researchers to evaluate and select optimal nano-particles for studies in large animals and clinical trials. This simple method helps determine whether the nanoparticle can target the human cancer cells in vivo and if so, how efficient the targeting is. Demonstrating the procedures will be Dr.Xiodan Qin, a postdoc, and Mr.Andrew Lam, a research technician from our lab.
Fill two chamber mating tanks with fish water in the evening and separate the upper tanks using dividers. Place a male zebrafish into one side of the chamber and one or two females zebrafish into another side of the chamber. Remove the dividers at 8:00 AM the following morning.
Add artificial enrichment plants and tilt the top chamber slightly to create a shallow area of water. Allow the zebrafish to breed for three to four hours and collect the eggs from the bottom chamber by pouring the water through a mesh net. Transfer the eggs to a Petri dish with no more than 200 eggs per dish.
Remove any dead or unfertilized eggs. Fill the dish two thirds full with fresh fish water, and place it in the incubator. After 24 hours, remove the dish from the incubator and remove as much water as possible from the dish.
Add a few drops of one milligram per milliliter pronase solution and gently swirl the dish. When the chorions show signs of disintegration, pipette the embryos up and down a few times to break down the chorions and release the embryos. Once the majority of the embryos are out of the chorions, immediately add fresh fish water to terminate the process.
Rinse the embryos with fish water three times to remove the floating chorions Return the embryos to the incubator. Make a 3%agarose solution by adding three grams of electrophoresis-grade agarose to 100 milliliters of autoclaved fish water. Microwave the agarose and water until the agarose is completely dissolved.
To create the injection plate, pour hot agarose solution into a Petri dish until it is three quarters full and place the micro injection mold on the agarose. After the agarose has solidified, carefully remove the mold. Fill the resulting injection plate with autoclaved fish water and store it at four degrees Celsius.
To make the injection needles, pull borosilicate glass capillaries one millimeter outer diameter by 0.78 millimeters on a pipette puller. Store the injection needles on putty in a large Petri dish that has been wiped with an ethanol towel. Before harvesting the HeLa cells, rewarm the injection plate and fish water in the incubator at 35.5 degrees Celsius.
30 minutes to an hour before transplantation, harvest the HeLa cells. Working in a tissue culture hood, remove the medium from the flask by aspiration. Rinse the cells with sterile PBS to remove all traces of the medium.
Add three milliliters of sterile trypsin EDTA to the flask and incubate it at 37 degrees Celsius for three to five minutes. Observe the flask under the microscope to monitor the progress of enzymatic digestion. When approximately 80%of the cells are suspended, add six to eight milliliters of growth medium to the flask.
Collect the cells by pipetting gently and transfer them to a 15 milliliter sterile tube. Centrifuge the tube at 135 times G for five minutes. Aspirate the supernatant and resuspend the HeLa cells in three milliliters of complete growth medium.
After repeating the wash steps twice, resuspend the cells in one milliliter of complete growth medium. Then count the cells under the microscope using a hemocytometer. After centrifuging the cells again, remove the supernatant.
Resuspend the cells in a 1.5 milliliter microcentrifuge tube at a concentration of five times 10 to the seventh cells per milliliter. Keep the cells warm by holding the tube in one hand when transporting to the fish facility. Using a plastic pipette, place the zebrafish embryos on the injection plate.
Lay embryos on their sides with the anterior facing forward aligned to the grooves on the plate. Turn on the air source and the micro injector. After retrieving the HeLa cells from the incubator, pipette the cells up and down 20 to 30 times using a P200.
Using a gel loading tip with a cut end, immediately load three microliters of the HeLa cell suspension into one of the injection needles. Insert the needle into the needle holder and place the injection plate under the microscope. Inject the cell mixture into the embryo at the vascularized area under the perivitelline cavity by pressing the foot pedal.
Then pipette a few drops of sterile fish water onto the embryo. To continue the injection process, use the non-dominant hand to move the injection plate to the next embryo, and then use the dominant hand to extend and retract the injector while simultaneously pressing the foot pedal. Finish injection of all embryos on the plate and wash the embryos with sterile fish water into a sterile Petri dish.
Immediately incubate the dish at 35.5 degrees Celsius. After three hours, examine the injected embryos and remove the dead ones. On the next day, make injection needles for the nano-particles and store them on putty in a large Petri dish.
Working under a fluorescent microscope, carefully pick up embryos with tail metastases and place them on a new injection plate with sterile fish water. For each zebrafish with a tail metastasis, inject either nano-particle solution or water behind the eye. Also inject nano-particle solution behind the eyes of the zebrafish in the control group which did not receive HeLa transplants and incubate all the injected embryos at 35.5 degrees Celsius.
Immediately after injection and again at 30 to 60 minute intervals, transfer two to three embryos to a Petri dish and immobilize them by adding five drops of diluted MS222 solution. Once the embryos stop swimming, remove most of the water to allow the embryos to lie on their sides. Using a pipette with a thin soft brush attached to its end, align the embryos so they lie with the anterior facing forward and only one eye visible.
Using 2X magnification, capture images of the whole embryo on red, blue, and Brightfield channels. To capture the tail area, image the embryos at 6.4X magnification. Focus the embryo under the red channel to avoid the bleeding of nano-particles.
Immediately after imaging, add fresh fish water to the embryos and return them to the incubator. Injected and control zebrafish embryos were imaged using a fluorescent microscope. The red dots are metastatic human cervical cancer cells while the blue dots represent the nano-particles.
In one control group, zebrafish that were injected with cancer cells but not nano-particles, fluorescent HeLa cells were visible in the red channel and no specific fluorescent signals were detected in the blue channel. In the other control group, zebrafish were injected with nano-particles, but not with cancer cells. Blue fluorescent nano-particles were distributed throughout the circulatory system.
No specific red florescence was visible. When zebrafish with tail metastases were injected with nano-particles, overlaid images from the red and blue channels show co-localization of HeLa cells and nano-particles. After performing this method, we can analyze tumor burden and cancer cell survival, the animals with and without injecting nano-particles to adjust their ability of killing cancer cells.
This technique may facilitate the selection of the optimal nanoparticle that can specifically recognize the cancer cell in vivo leading to early detection of cancer and cancer metastasis.
