This protocol describes the technique to generate mouse models bearing primary osteosarcoma and the pulmonary metastasis lesions. By intratibial injection of osteosarcoma cells, this model best mimics the clinical development characteristics of tumor osteosarcoma. Accurate numbers of osteosarcoma cells are directly injected into the tibia using a microvolume syringe, allowing for identical tumor formation rates and tumor volumes.
On the day of osteosarcoma cell injection, wash the cells twice with PBS. Trypsinize the cells with 1.5 milliliters of 0.25%trypsin for three minutes. Then add six milliliters of 10%serum-containing MEM media.
Collect the cells in a 15-milliliter centrifuge tube. Aspirate 20 microliters of cell suspension into the chamber of the cell counting plate, and calculate cell concentration using an automatic cell counter. Centrifuge the cells at 800 times g for five minutes at room temperature.
Aspirate the supernatant with a pipette, resuspend the cell pellet in an 8.5-milligram-per-milliliter basement membrane matrix, and place them on ice. Place the mouse in the supine position. Hold the ankle of the mouse using the thumb and index finger, and disinfect the injection site of the tibia with a 70%ethanol swab.
Rotate the ankle joint of each mouse outward to move the tibia and fibula, and bend the knee joint to a suitable position until the proximal tibia plateau is visible through the skin. Attach the needle to a one-milliliter syringe, and point the needle tip toward the injection site, parallel to the axis of the tibia. Percutaneously insert the needle to drill a hole through the tibia platform toward the distal end of the tibia.
Load the osteosarcoma cell suspension previously placed on ice into a microvolume syringe, and replace the one-milliliter syringe in the tibia with the osteosarcoma cell-loaded microvolume syringe. Slowly inject approximately 10 microliters of osteosarcoma cell suspension into each athymic mouse's tibia without applying much pressure. Then remove the microvolume syringe from the injection site, and press the site with the cotton swab for about 30 seconds.
Place the mouse back into a clean cage, and observe its recovery for 10 minutes. This figure depicts the mouse model with orthotopic osteosarcoma developed from intratibial osteosarcoma cell injection and its isolated form. A gradual increase in the tumor volume is observed every week.
The X-ray images obtained from the first through the sixth week after injection of luciferase-labeled osteosarcoma cells showed a gradual increase in volume. Furthermore, the image obtained after the injection into the mouse tibia showed orthotopic osteosarcoma growth in vivo. The pulmonary metastasis caused by intratibial injection of the osteosarcoma cells was successfully tracked in vivo using a bioluminescence live imaging system.
Observation under a stereomicroscope revealed metastatic colonies in the isolated lung tissues. HE staining on paraffin-embedded lung tissues showed metastatic lesions. The needle should be percutaneously inserted into the joint capsule through the patellar ligament to drill a hole through the tibia platform toward the digital tibia by rotating the syringe.
