In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

The overall goal of this protocol is to non-invasively follow the distribution and fates of labeled bone marrow mesenchymal stem cells after their injection by gamma scintigraphy. The fate of stem cells after the injection is poorly understood. The main advantages of this technique are that it allows the stem cells to be non-invasively tracked to multiple organs after administration, and that the short half-life of the A osteo ensures its clearance by 48 hours.

Although this has provided insight into the use of stem cells in the therapy of naturally occurring tendinopathies, it could also be used for the investigation of bone and cardiac disease therapies. Generally, the challenges for individuals new to this method are the coordination of the cells and the aset for the labeling and the setting up of the patient. For immediate application of the cells Begin by applying acoustic coupling gel to the clipped for limb and use a linear transducer to methodically scan the complete Palmer metacarpal region to obtain serial on incidents transverse in longitudinal images to identify the maximal injury zone.

After local anesthetic administration, scrub the implantation site with a chlorhexidine surgical scrub solution for at least five minutes, followed by a wipe with an ethanol soaked gauze. First dawn, a pair of gloves when the animal has been prepped, pellet a one times 10 of the seventh cell aliquot of bone marrow mesenchymal stem cells in one milliliter of bone marrow supernatant by centrifugation. Then aspirate the supernatant with a two milliliter syringe, equipped with an 18 or 19 gauge needle, leaving approximately 100 microliters of solution in the tube.

Transfer the supernatant into a micro centrifuge tube on ice, and then use gentle flicking to resuspend the cells in the residual 100 microliters of supernatant. Taking care that the cells are completely resuspended and that there are no visible tissue clumps. Then set the cells at room temperature.

Now mix one milliliter of the technetium Protec nutate into a vial of H-M-P-A-O. After five minutes, use a one milliliter syringe and needle to treat the mesenchymal stem cells with the entire aliquot of the TECHNETIUM 99 M-H-M-P-A-O. Then cap the tube, mix the cells with gentle flicking and place the tube at room temperature behind a lead shield.

After 30 minutes, open the tube with a forceps and use a tube milliliter syringe equipped with a 21 gauge needle to add one milliliter of PBS to the cells. Recap the tube and mix the cells. Next, centrifuge the cells and use the two milliliter syringe to carefully transfer the supernatant to a alleged shielded tube labeled wash one.

Then wash the pellet in another milliliter of PBS as just demonstrated. Transferring the second wash to alleged shielded tube labeled W2 after the second wash. Resus suspend the cells thoroughly in the residual PBS and at the aliquot of ice cold bone marrow supernatant, holding the transducer in parallel with the needle.

Slowly introduce a 1.5 or two inch 20 gauge needle into the maximum injury zone in the center of the lesion in a longitudinal orientation. Next, using the appropriate shielding, aspirate the cells into a two milliliter lure, lock syringe equipped with a 20 gauge needle and discard the needle, taking care not to lose any fluid. After attaching the syringe to the pre placed needle in the tendon, arrange the ultrasound probe under the needle so that the needle tracked in the tissue is clearly visible and inject the cells at a slow but steady pace into the lesion.

Confirm that the fluid ejecting into the lesion is visible on the ultrasound image as koic fluid containing hyper echogenic air bubbles when all of the cells have been administered, withdraw the needle. Use the acquire study selection and bone static options in the processing software to acquire static images for 60 seconds each. Next, obtain lateral images from the lesion area, from the carpus to the distal extremity, the equivalent area of the contralateral limb, the left lung field, and the left thyroid at the indicated times.

Then select the region of interest and ellipse tools and reshape the selection mask over the lesion area as appropriate. Finally, choose the add two statistics option to obtain the counts over the region of interest. Taking care of that identically sized regions of interest are recorded for each animal at the different time points.

After intralesional delivery, the cells will be observed as a focal area of signal with a limited spread of the cells into the surrounding tendon tissue over time. By contrast, when the cells are administered by regional perfusion via the digital Palmer vein, a gradual reduction of the signal is observed at the injection site. Over time, the lung fields are usually negative after intralesional injection, but occasionally a very focal signal that becomes more generalized over time may be detected at early time points.

The thyroid remains negative by this administration root as well. Regional perfusion also demonstrates a focal signal in the lungs, which becomes more diffuse over time, although the signal is significantly more intense compared to that observed with the intralesional root. With a thyroid also often exhibiting a focal signal, the total initial radioisotope count of the cells at times zero pre injection, and the counts from the initial gamma gram at the region of interest.

At times zero post injection can be used to calculate the percent decrease in the counts at each time point after injection to determine the predicted decay of the TC 99 M from the initial counts. The persistence of the cells remaining at the injection site at each time after injection can then also be calculated. Following this procedure, other routes of cell menstruation can be applied to answer additional questions about the homing ability of the mesenchymal stem cells after its development.

This technique has paved the way for researchers in the field of regional medicine to explore methods for improving cell retention within tend injuries. After watching this video, you should have a good understanding of how to label cells and record their distribution and concentration in different organ systems. Don't forget that working with ionizing radiation can be extremely hazardous and the precautions such as personal protective equipment, radiation monitors, and lead shielding and the proper disposal of contaminated waste are essential while performing this procedure.