The overall goal of this procedure is to selectively target stem cell-derived teratomas in mice for evaluation of the in vivo effectiveness of small animal external beam radiation therapy. The main advantage of external radiation beam therapy is that it is a targeted therapy for stem cell associated teramtomas that avoids the off-targets adverse effects of systemically delivered therapies. Advances in external beam radiation therapy have enabled the specific targeting of small teratomas while avoiding damage to normal tissues, making this therapy ideal for treating radiation sensitive teratomas.
This method can not only provide insight into optimizing strategies for cell therapies, it also has the potential to become clinically applicable in human therapies. Begin by taking a six-well plate containing iPSCs. To induce tetratoma formation in immunodeficient animals add one milliliter of recombinant cell dissociation enzyme mix per well of the 6-well plate containing human induced pluripotent stem cells transduced with a double fusion reporter gene for a five minute incubation at room temperature.
At the end of the incubation disperse the cells by pipetting and stop the enzymatic reaction by adding an equal volume of cell culture medium to each well. Pull the single cell suspensions in a 15 milliliter conical tube for counting. After centrifugation is complete, aspirate the supernatant and resuspend the pellet in 30 microliters of Matrigel matrix solution and place the tube on ice.
If utilizing double fusion reportagen transfected cells, these double positive affect cells should also be suspended in 30 microliters of matrix. Next, load each cell population into a one milliliter syringe equipped with 28.5 gauge needle and confirm a lack of response to toe pinch in each anesthetized eight to 10 week old athymic nude recipient mouse. Then, subcutaneously inject one cell matrix mixture into the dorsal flank of each recipient animal.
At the appropriate experimental time point after inoculation inject 375 milligrams per kilogram of deluciferon intraperitoneally into each recipient. After 10 minutes image the bioluminescence signal in each anesthetized reporter probe injected animal for 30 minutes using one minute acquisition windows at five minute intervals, according to standard bioluminescence imaging protocols. For teratoma irradiation, at the appropriate experimental time point, first place an anesthetized recipient animal onto the bed of an image-guided preclinical irradiator and acquire a set of 400 projection micro-computed tomography images over 360 degrees using a 40 peak kilovoltage, 2 milliamp x-ray beam, and reconstruct the image into volumetric images with an isotropic pixel size of 0.2 millimeters.
It is critical to reconstruct volumetric images of the teratomas. These images will help to plan the radiation treatments for the specific delivery of the therapy to the teratomas only, while minimizing any off-target radiation of the surrounding tissues. Then use the micro-CT images and the RT image software package to plan a radiation treatment protocol.
Administer the treatment for three consecutive days to deliver a total of 18 grays to the target tumor. The injected mice typically demonstrate teratoma growth formation four to eigth weeks after tumor cell injection as confirmed by bioluminescence imaging. When irradiated with a cumulative does of 18 grays one month after cell delivery the tumors will shrink dramatically resulting in a significant coincident decrease in luciferase signal.
Importantly, as these images illustrate, normal tissue biopsies harvested five milliliters from the irradiated site do not appear to sustain any significant damage. After watching this video, you should have a clear understanding of how to create stem cell derived teratomas in vivo and to effectively and safely treat them with external beam radiation. This simple approach requires the acquisition of high resolution CT images of a subject after which a series of radiation beam therapies could be prescribed to irradiate the target tumors, while avoiding adjacent tissue.
Don't forget that working with radiation can be extremely hazardous and that precautions, such as using the appropriate shields, should always be taken while performing this procedure.
