The overall goal of this procedure is to establish an orthotopic retinoblastoma tumor in an immunodeficient mouse, providing a convenient model for further experimentation and allowing propagation of the tumor. This is accomplished by first harvesting cells from a primary or xenograft tumor and culturing tumor spheres in vitro. The second step of the procedure is to inject the cultured tumor cells into the vitreous cavity or subretinal space of the eye of a rag two null immunodeficient mouse.
The third step of the procedure is to monitor the injected mice for signs of tumor formation, including Leukocoria GLO enlargement and periocular distension. The final step of the procedure is to harvest the xenograft tumor. Ultimately, results can be obtained that show orthotopic tumor formation in the murine eye through Histopathologic examination.
Visual demonstration of this technique is critical due to the delicate nature of the intraocular injection. Surrounding structures can be damaged easily, if not identified properly. Generally, individuals new to this method is struggle because the placement of the injection in the vitreous cavity requires precision to deliver the tumor cells in the correct location.
Now, this method can provide insight into the biology of retinoblastoma tumors. It can also be applied to other systems such as the interocular administration of vectors for gene transfer. First, prepare fresh defined stem cell media in a sterile tissue culture treated 10 centimeter dish.
Place the retinoblastoma tumor tissue and aspirate excess transport media, leaving enough to prevent drying, mechanically disaggregate the tissue with a scalpel using a cross-cutting technique immediately add 100 microliters of defined stem cell media to the tissue and gently spread the mince tissue using the scalpel. Next, add 10 milliliters of defined stem cell media to the dish. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide in a humidified incubator and inspect daily for turbidity and pH changes.
Some tumor spheres should arise from the disrupted tumor tissue almost immediately with increasing numbers over the course of seven to 10 days when sufficient new growth is observed. And weekly thereafter, centrifuge tumor spheres at 300 times G for five minutes. Resus, suspend them in freshly prepared defined stem cell media and pipette up and down 10 times to disrupt larger centrally necrotic spheres and to allow new, healthy spheres to form.
To prepare cultured tumor spheres for injection, gently transfer spheres into a 15 milliliter conical tube. Gently pipette up and down 10 to 15 times to disrupt the spheres. Count the cells to be injected using a hemo cytometer using trian blue to exclude nonviable cells.
Prepare for as many injections as needed based on the number of cells available and experimental requirements. Next, centrifuge the tubes at 300 times G for five minutes. Carefully aspirate the supernatant and resuspend the cells in five milliliters.
PBS repeat the centrifugation and aspiration steps once more. Lastly, resuspend the cells in 10 microliters sterile PBS per injection. To prepare the animals for the procedure begin by placing an anesthetized mouse on a heated pad.
To maintain body temperature, administer one drop of propane HCL to the right eye and wait for one minute. Next, administer one drop of phenylephrine HCL to the right eye and wait five minutes. If dilation of the pupil has not occurred, administer another drop and wait another five minutes.
Place the anesthetized animal on its side under the microscope, rest the head on gauze. Face the right side up and center the eye to obtain a red reflex of the right eye. Fundus when observed through the microscope, prop up the right globe by gently pushing down simultaneously with two fingers on the eyelids.
Hold this position steady for the remainder of the procedure. Using a sterile 30 gauge needle attached to a lure lock syringe. Pierce the globe laterally through the conjunctiva and sclera adjacent to the corneal limbus in the area of the pars plana, and just through the choroid into the vitreous cavity.
Remove the needle from the opening. Sterilize a Hamilton needle with an alcohol swab. Draw the cell suspension into the Hamilton syringe and insert the needle into the opening until the needle is visualized through the microscope behind the lens in the vitreous, near the retina.
With the help of a second person, hold the needle steady in this position while depressing the plunger, slowly remove the needle. If necessary, use a cotton swab to absorb any blood or fluid from the opening. Gently release the pressure to close the mouse eyelids.
Return the animal to a heated pad for recovery. Every day after the injection, examine the eye for leukocoria. That usually becomes apparent two to four weeks post-injection.
Once tumor growth is detected, euthanize the animal according to institutional guidelines. Under a stereoscopic microscope, make a circumferential incision at the limbus, removing the cornea and the lens, and carefully dissect bulk of the tumor mass from the other tissues using tweezers. Place the tumor in sterile RPMI 1640.
Media use tweezers to slowly pull the open globe from the socket until the optic nerve is exposed. While gently lifting the globe from the socket. Use scissors to cut the optic nerve, leaving as much nerve attached to the globe as possible in order to evaluate optic nerve invasion by the tumor place in 10%neutral buffered formalin for histology seen.
Here is an example of cultured tumor spheres. After three days, only a few tumor spheres are apparent among the disaggregated tumor tissue. However, at 10 days, the tumor spheres have increased in number.
The spheres exhibit a crisp outer membrane as shown in the insert in the upper right several weeks after injection. The injected mouse exhibits leukocoria, the white pupillary reflex is due to tumor located in the back of the eye. In these examples of h and d stained eye sections, notice the xenograft retinoblastoma tumor filling the vitreous cavity on the left, and the absence of such a growth in the mock injected eye on the right.
After watching this video, you should have a good understanding of how to prepare cultured tumor cells, properly perform an intraocular injection in a mouse eye, and how to look for tumor progression Once mastered, the injection technique can be accomplished in approximately 30 minutes per animal. If done properly, Don't forget that tumor cells can be hazardous and precautions such as wearing adequate PPE, including lab coat and masks should always be taken while performing this procedure. Human cancer cells should always be manipulated under bsl two conditions.
