The overall goal of this procedure is to evaluate the efficacy of the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells in a rat knee ostearthritis, or a KOA model, using in vivo fluorescent imaging. In vivo stem cell tracking can help answer key question in the regenerative math-ing field about irritation and distribution of human adipose derived in mesenchymal stem cells in KOA animal models. The main advantage of using DiD is that its rat-shifted emission spectrum allows deep tissue imaging in live animals with no cytotic effects or functional damage to the dye-labeled cells.

Begin by placing an anesthetized 250 to 300 gram, eight to 12 week old male Sprague Dawley rat in the left lateral position on a heated pad. Use a razor to thoroughly shave the right knee, and disinfect the surgical area with 10%povidone iodine solution and 70%ethanol. Cover the nonsurgical area with a surgical pad.

And use a scalpel to make a two centimeter incision laterally along the patellar tendon to expose the joint capsules. Next, use a surgical scalpel to transect the medial collateral ligament, reflecting the meniscus toward the femur, and grasp the medial meniscus with forceps. Use a scalpel to cut through the meniscus at its narrowest point from the tibial attachment.

To obtain a reliable and feasible KOA onset, the femur side of the meniscus should be completely cut through in each experimental animal. Then, close the joint capsule with a four-O observable suture and monitor the rat until it is fully recovered. Eight weeks after surgery, detach human adipose-derived mesenchymal cells from an 80%confluent culture with two milliliters of trypsin plus EDTA for three minutes at 37 degrees celsius.

After a few minutes, neutralize the trypsin with four milliliters of complete culture medium and collect the cells by centrifugation. Resuspend the pellet at a one times 10 to the sixth cells per milliliter concentration in five milliliters of serum-free culture medium. And label the cells with 50 microliters of DiD cell labeling solution for 50 minutes at 37 degrees celsius.

At the end of the incubation, collect the cells by centrifugation and wash them two times in five milliliters of PBS per wash. After the last centrifugation, dilute the cells to a 2.5 times 10 to the sixth viable cells per 100 microliters of PBS concentration and load the cells into a one milliliter syringe equipped with a 26 gauge needle. Next, reshave the arthritic knee of the first experimental animal to expose the joint area, and disinfect the exposed skin with 70%ethanol.

Inject the labeled cells into the center of the triangle formed by the medial side of the patellar ligament, the medial femoral condyle, and the medial tibial condyle, and open the imaging software. Initialize the in vivo bioluminescence imaging system and position the animals in the supine position within the central stage of the imaging system. With the chamber door closed, check the fluorescent box and select the appropriate excitation and emission fluorescent filter sets.

Set the field of view to D, the pixel bending to eight, the F Stop to two, and the exposure time to auto. After obtaining the images, allow the animals to recover on a heat pad with monitoring until full recovery. Then, in the appropriate image analysis software, select the units of radiant efficiency for the measurement of the fluorescence and the regions of interest for analysis and quantify the fluorescent signals from each image.

In this representative experiment, serial sections of the knee joint of a knee osteoarthritis induced rat were evaluated by H&E and safranin O/Fast Green staining eight weeks after the surgery. The thickness of the articular cartilage is thinner in the arthritic knees than in the contralateral joints without surgery as observed in the H&E stained sections. Further, the proteoglycan is decreased and the fibrillated collagen is increased in the arthritis joint as visualized by safranin O/Fast Green staining, indicating the progression of the degenerative knee osteoarthritis phenotype induced by the surgery.

One hour after staining, DiD labeled human adipose-derived mesenchymal cells exhibit a round shape in vitro. After 24 hours, cultured DiD labeled mesenchymal cells demonstrate a long spindle shape confirming that DiD does not change the adherence ability of the cells. Imaging the knees of the osteoarthritic animals eight weeks after surgery reveals the presence of the DiD labeled cells from day zero until day 70 post injection.

After its development, this technique paved the way for researchers in the stem cell therapy field to determine the optimal routine and dosage for the safe and feasible injection of mesenchymal stem cells for the treatment of knee osteoarthritis in animals. After watching this video, you should have a good understanding of how to label human mesenchymal stem cell with DiD for the injection and in vivo tracking in the surgically induced rat KOA model.