Name: In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy (Video) | JoVE Journal
Uploaded: 2015-12-09T13:00:00.000+00:00
Duration: 7 min 52 s
Description: Learn to non-invasively track technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy using gamma scintigraphy. This method enables monitoring of cell distribution and survival post-implantation.

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5 minutes

The authors would like to acknowledge funding from the Horserace Betting Levy Board U.K. (grant number 721) and VetCell BioScience Ltd, U.K. and by Consejer&#236;a de Innovaci&#242;n, Ciencia y Empresa, Junta de Andaluc&#236;a, Spain.

The cases described herein were performed following Ethical permission granted by the Animal Ethics and Welfare Committee of the Colegio de Veterinarios de Malaga, Spain, and the Royal Veterinary College, North Mymms, U.K. The procedures used on the horses are based on approved protocols that are used in clinic on horses receiving stem cell based therapies which includes sedation, bone-marrow aspiration, intra-tendinous injection, regional perfusion, intravenous injection, post-procedural treatment and pain management and monitoring after implantation.
1. Key Arrangements to be Made in Advance
<ol>
	<li>Seek institutional ethics and animal welfare approval and adhere to local legal requirements prior to starting work.</li>
	<li>Seek institutional approval to work with ionizing radiation as dictated by local and legal regulations including the appropriate level of protection of personnel and the environment and the disposal of contaminated waste.</li>
	<li>Use the following inclusion criteria: horses present with a non-traumatic overstrain injury (tendinopathy or desmopathy) to the superficial flexor digital tendon of the forelimb and lesions resulting in a defect within the tendon and the defect is surrounded by intact tendon and/or paratenon. 
	Note: The examination and diagnosis for such injuries and the clinical preparation of the horses for cell implantation has been detailed elsewhere6,15. 
	Note: The isolation and expansion in culture of BMMSC derived from the bone marrow of horses has been detailed previously6,16. The current protocol for injecting BMMSC for SDFT injuries in the horse uses 10 million cells per ml4 of autologous bone marrow supernatant (BMS)16.</li>
	<li>Use cells that have undergone no more than 4 passages for in vivo implantation to avoid potential cellular phenotype or genetic alterations associated with high passage number cells.</li>
	<li>Deliver the required number of cells in the BMS (within 24 hr in a chill box at 4 &#8211; 8 oC) to the veterinary clinic where the cell implantation is to be performed. 
	Note: The clinical use of mesenchymal stem cells in bone marrow supernatant is a patented procedure owned by ReCellerate Inc (U.S.A.) and its use may require authorization. 
	Note: Tc-99m is a gamma emitter (140 keV) with a relatively short half-life (6.0058 hr, therefore nearly 94% of it decays to its stable form of 99Tc in 24 hr). The gamma energy makes it suitable for detection by gamma cameras (scintigraphy) and the short half-life results in very limited radiation exposure time to both the horse and the animal handling personnel. This protocol describes the on-site labeling of cells using Tc-99m-HMPAO [HMPAO labeled with Tc-99m (as sodium pertechnetate)].</li>
	<li>Order the Tc-99m fresh from the supplier such that it is delivered and used for labeling the cells within 2 hr of elution from the Tc-99m generator. Ensure that the Tc-99m is supplied as 1 GBq in a 1 ml solution (supplier&#8217;s standard buffer).</li>
	<li>Use all buffers at RT. Perform the labeling procedure, implantation of the cells and imaging (gamma scintigraphy) in an isotope containment room with all equipment cleaned with alcohol wipes prior to the start of work.</li>
</ol>
2. Preparation of the Horse and Ultrasonography of the Forelimb Tendon

<ol>
	<li>Record ultrasound scans at first admission for injury (when bone marrow is aspirated) and then at the time point when radiolabeled cells are injected.</li>
	<li>Perform ultrasonography and cell implantation with the horse bearing weight evenly in a stall and under standing sedation (rather than general anesthesia). Administer sedation using a mixture of detomidine HCl and butorphanol tartrate, each at 0.01 to 0.02 mg/kg IV. 
	Note: Recovery is rapid and post-surgical treatment requires no special considerations.&#160;</li>
	<li>After perineural anesthesia is applied (step 2.6) in the affected limb, keep the horse at the station under sedation to reduce movement of the horse during the treatment. Visually check the appropriate level of sedation judged from the lowered head position and the demeanour of the horse and that the horse is comfortable during cell injection and the movements of the gamma camera during the acquisition of scintigraphic images. 
	Note: It is not necessary to apply vet ointment on eyes (to prevent dryness) because under sedation blinking is not affected and the horse is able to maintain hydration of the eyes. The monitoring after the implantation of the cells is non-invasive (ultrasound and gamma scintigraphy).</li>
	<li>Clip closely the hair overlying the tendon (with horse hair clippers) then clean the clipped area using a combination of a chlorhexidine surgical scrub and finally alcohol.</li>
	<li>Apply acoustic coupling gel to the area and scan methodically to record scans of the complete palmar metacarpal region, sequentially labeled levels 1 - 717, with a linear transducer (12 MHz or similar) to obtain serial on-incidence transverse and longitudinal images. Store the images digitally so that the cross-sectional area and the maximal injury zone4 can be obtained with the aid of image analysis software.</li>
	<li>Prepare the area overlying the palmar nerves immediately distal to the carpus for aseptic injection of local anesthetic to ensure complete desensitization of the skin overlying the tendon and the superficial digital flexor tendon. Inject 2 ml of mepivicaine both subcutaneously and adjacent to the palmar nerves (in their sub-fascial location) between the flexor tendons and the suspensory ligament immediately distal to the carpus on both sides of the limb.</li>
	<li>Once the preliminary scan has been performed prepare the implantation site by scrubbing the area with a chlorhexidine surgical scrub solution for at least 5 min and finally alcohol with soaked gauze. Perform all subsequent steps in an aseptic fashion.</li>
</ol>
3. Tc-99m-labeling of Equine Mesenchymal Stem Cells
Note: The cell labeling steps can be performed during ultrasonography of the forelimb as this will minimize isotope decay between cell labeling and implantation of labeled cells into the tendon.
<ol>
	<li>Remove the BMS from the cells as it interferes with the uptake of isotope. To do this, retrieve the vial of BMMSC (10 million cells in 1 ml of BMS) from the chill box and pellet the cells in a microcentrifuge at 350 x g for 5 min at RT. Carefully remove the supernatant with an 18G or 19G needle (attached to a 2 ml syringe), leaving a small drop ( &#8804;0.1 ml) in the tube to aid cell resuspension. Save the supernatant (the BMS) in a sterile microcentrifuge tube and keep on ice for later re-use.</li>
	<li>Resuspend the cells in the residual droplet of supernatant by flicking the tube gently with fingers (rather than vortex) and leave the tube in a rack at RT. Ensure the cells are completely resuspended and that there are no visible cell clumps.</li>
	<li>Prepare the Tc-99m as follows. Transfer 1 ml of technetium pertechnetate into a vial of HMPAO using a 1 ml syringe and needle. Mix gently but thoroughly and leave for 5 min behind lead shielding. Note: This step can be done while the cells are spinning in step 2.1.</li>
	<li>Add all of the Tc-99m-HMPAO mixture using a 1 ml syringe and needle to the cells and mix gently. Incubate for 30 min at RT behind lead shielding.</li>
	<li>For the next steps, use a forceps (or a piece of tissue paper) to open the lid of the microcentrifuge tube to minimize isotope contamination of the (gloved) thumb and subsequently other equipment. Use a 2 ml syringe and 21 G needle to add or remove wash buffer. 
	Note: The use of a field isotope monitor is encouraged to monitor contamination of surfaces and equipment.</li>
	<li>Add 1 ml PBS to the Tc-99m-HMPAO-cell mixture, close the lid, mix gently and spin to pellet the cells as in step 3.1.</li>
	<li>Carefully remove the PBS to a new tube (save this behind lead shielding and label as W1). Resuspend the cell pellet in 1 ml of fresh PBS. Centrifuge the tube and remove the supernatant as above (save this and label as W2).</li>
	<li>Calculate the efficiency of labeling by measuring the counts in W1 and W2 tubes and in the cell pellet. Do this by placing each tubes in a Well isotope dose calibrator instrument and recording the radioactivity as counts per minute (CPM). Calculate the labeling efficiency as (Radioactivity as CPM of cells) / (Radioactivity of cells + supernatant) x 100.</li>
	<li>Resuspend the cells thoroughly in the residual PBS and then add the BMS saved from step 3.1. The cells are ready for intralesional implantation.</li>
</ol>
4. Implantation of Tc-99m-HMPAO-labeled Cells
<ol>
	<li>Intralesional implantation into the tendon under ultrasound guidance
	<ol>
		<li>Slowly introduce a 1.5 or 2 inch (50 mm) 20 G needle into the maximum injury zone of the lesion in a longitudinal orientation with the ultrasound transducer, covered in a sterile drape, in line with the needle so that the length of the needle can be identified ultrasonographically and the tip if the needle is accurately located in the center of the lesion.</li>
		<li>Using appropriate shielding, collect the cells into a 2 ml syringe (Luer-lock, to minimize the risk of external contamination and in a lead-shielded syringe cover) using an 20 G needle. Discard the needle being careful not to lose any fluid. Now attach the syringe with the labeled cells onto the needle pre-located in the tendon.</li>
		<li>Place the ultrasound probe under the needle so that the needle tract in the tissue is clearly visible. Inject the cells at a slow but steady pace into the lesion. Ensure that there is no resistance to injection. If this is encountered carefully reposition the needle under ultrasound guidance. Check that the fluid ejecting into the lesion is visible on the ultrasound image as anechoic fluid containing hyperechogenic air bubbles.</li>
		<li>Withdraw the needle and immediately place pressure over the needle hole to prevent loss of injected cells and to minimize the spread of isotope over the external surface of the limb. Immediately dress the limb with one layer of autoadhesive bandage. The horse is now ready for gamma scintigraphy. Perform this immediately. 
		Note: After injecting the cells into the tendon, acquire a count of the empty syringe and needle to assess possible direct loss of cells that may remain in the syringe.</li>
	</ol>
	</li>
	<li>Regional perfusion of Tc-99m-HMPAO-labeled cells
	<ol>
		<li>Follow step 2.4.</li>
		<li>Apply a 100 mm rubber tourniquet bandage on the proximal metacarpus with two 100 mm cotton roll gauze bandage positioned medially and laterally.</li>
		<li>Aseptically prepare the skin over the lateral palmar digital vein at the level of the lateral proximal sesamoid bone by scrubbing the area with a chlorhexidine surgical scrub solution for at least 5 min and finally with gauze soaked in alcohol.&#160; Perform all subsequent steps in an aseptic fashion.</li>
		<li>Prepare a 100 mm extension set with a 21 G x &#190;&#8221; (0.8 x 19 mm) butterfly needle for venipuncture by filling with heparinized sterile saline and then introduce the needle into the lateral palmar digital vein. Once the vein is entered blood will partially fill the extension set. At the Luer lock connector attach a Luer lock rubber cap for facilitating subsequent injection of labeled cells.</li>
		<li>Dilute the MSCs in 19 ml of PBS by collecting the cells in a 20 ml syringe preloaded with the PBS.&#160; Apply the cells via the rubber cup using an 18 G (1.2 x 40 mm) hypodermic needle at a slow but steady rate (30 &#8211; 40 sec).&#160; Flush the catheter with 1 ml of PBS preloaded in a syringe.</li>
		<li>Remove the needle and immediately apply pressure at the skin over the needle hole with a layer of five 4 x 4 cotton gauzes. Then place a light bandage with elastic adhesive over the proximal sesamoid area&#160;and leave the tourniquet in place for 30 min after injection.</li>
		<li>Release the tourniquet after 30 min. The horse is now ready for gamma scintigraphy. 
		Note: After injecting the cells, acquire a count of the empty syringe and needle to assess possible direct loss of cells that may remain in the syringe.</li>
	</ol>
	</li>
</ol>
5. Gamma Scintigraphy
<ol>
	<li>Scintigraphy images
	<ol>
		<li>Acquire planar scintigraphy images with a gamma camera (set at 140 KeV photoelectric peak using a 20% symmetrical window) equipped with a low energy, parallel hole collimator.
		<ol>
			<li>Obtain all images after time 0 on standing horses sedated as in step 2.2. After the time 0 scintigram, replace the light bandage over the implantation site with a padded bandage. Remove this bandage prior to each scintigraphy exam.</li>
			<li>Acquire static images for 60 sec each (256 by 256 matrix) using the processing software with the &#8220;acquire&#8221; &#8211; &#8220;study selection&#8221; - &#8220;bone static&#8221; options. It is essential that the horse does not move during the image acquisition period because the static mode option does not have a module for correction of movement.</li>
			<li>For intralesional injection, 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. During the acquisition of forelimbs images, position a lead screen between the legs to avoid imaging of the limb contralateral. Acquire the images at 5 min after injection (time 0), and then at 1, 3, 6, 12, 24, 36 and 48 hr.</li>
			<li>For regional perfusion, obtain lateral and dorsal gamma images as for 5.1.1.3. Obtain images 5 min after tourniquet release. This will be time 0. It can be useful to image the limb immediately after injecting the cells to assess counts prior to tourniquet release. Obtain gamma images at 1, 3, 6, 12, 24, 36 and 48 hr after administration of the cells.</li>
		</ol>
		</li>
		<li>After the images have been acquired, process the images manually using the &#8220;processing&#8221; option of the software with options selected for &#8220;Sokoloff&#8221; color and &#8220;Invert&#8221; color. Next select the &#8220;region of interest&#8221; (ROI) and &#8220;ellipse&#8221; as this allows reshaping of the selection mask and movement over the lesion area. Obtain the counts over the ROI by choosing the &#8220;add to statistics&#8221; option. Ensure that identically sized regions of interest (ROI) are recorded for each animal at the different time-points.</li>
		<li>Next, correct the counts for the predicted decay of the technetium at each time point. Use the following equation to correct for decay: A = Aoe-(0.693t/T1/2), where Ao is the initial activity of the isotope, A is the decay corrected radioactivity at time zero, t is the elapsed time and T1/2 is the half-life of the isotope. Then express the corrected counts at each time point as a percentage of the ROI at time 0 to work out the percentage of cells remaining, using the following formula:</li>
	</ol>
	</li>
</ol>
%cells remaining (t) = 100 &#8211; {[(predicted decay(t) &#8211; decay(t))/ predicted decay(t)]}&#160;x 100
decay(t) = ROI(t) / ROI(0) x 100.

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<li>Nixon, A. J., Dahlgren, L. A., Haupt, J. L., Yeager, A. E., Ward, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+adipose-derived+nucleated+cell+fractions+on+tendon+repair+in+horses+with+collagenase-induced+tendinitis%5BTitle%5D)+AND+%22American+Journal+of+Veterinary+Research%22%5BJournal%5D)">Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis.</a> American Journal of Veterinary Research. 69, 928-937 (2008).</li>
<li>Schnabel, L. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+and+insulin-like+growth+factor-I+gene-enhanced+mesenchymal+stem+cells+improve+structural+aspects+of+healing+in+equine+flexor+digitorum+superficialis+tendons%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons.</a> Journal of Orthopaedic Research. 27, 1392-1398 (2009).</li>
<li>Smith, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beneficial+effects+of+autologous+bone+marrow-derived+mesenchymal+stem+cells+in+naturally+occurring+tendinopathy%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy.</a> PloS one. 8, e75697(2013).</li>
<li>Sole, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+and+persistence+of+technetium-99+hexamethyl+propylene+amine+oxime-labelled+bone+marrow-derived+mesenchymal+stem+cells+in+experimentally+induced+tendon+lesions+after+intratendinous+injection+and+regional+perfusion+of+the+equine+distal+limb%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Distribution and persistence of technetium-99 hexamethyl propylene amine oxime-labelled bone marrow-derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb.</a> Equine Veterinary Journal. 45, 726-731 (2013).</li>
<li>Godwin, E. E., Young, N. J., Dudhia, J., Beamish, I. C., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantation+of+bone+marrow-derived+mesenchymal+stem+cells+demonstrates+improved+outcome+in+horses+with+overstrain+injury+of+the+superficial+digital+flexor+tendon%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon.</a> Equine Veterinary Journal. 44, 25-32 (2012).</li>
<li>Crevier-Denoix, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+properties+of+pathological+equine+superficial+digital+flexor+tendons%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Mechanical properties of pathological equine superficial digital flexor tendons.</a> Equine Veterinary Journal. 29, Suppl S23. 23-26 (1997).</li>
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<li>Alexander, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy-saving+mechanisms+in+walking+and+running%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Energy-saving mechanisms in walking and running.</a> The Journal of Experimental Biology. 160, 55-69 (1991).</li>
<li>Guest, D. J., Smith, M. R., Allen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+the+fate+of+autologous+and+allogeneic+mesenchymal+progenitor+cells+injected+into+the+superficial+digital+flexor+tendon+of+horses%3A+preliminary+study%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study.</a> Equine Veterinary Journal. 40, 178-181 (2008).</li>
<li>Guest, D. J., Smith, M. R., Allen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equine+embryonic+stem-like+cells+and+mesenchymal+stromal+cells+have+different+survival+rates+and+migration+patterns+following+their+injection+into+damaged+superficial+digital+flexor+tendon%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon.</a> Equine Veterinary Journal. 42, 636-642 (2010).</li>
<li>Sole, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scintigraphic+evaluation+of+intra-arterial+and+intravenous+regional+limb+perfusion+of+allogeneic+bone+marrow-derived+mesenchymal+stem+cells+in+the+normal+equine+distal+limb+using+%2899m%29+Tc-HMPAO%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Scintigraphic evaluation of intra-arterial and intravenous regional limb perfusion of allogeneic bone marrow-derived mesenchymal stem cells in the normal equine distal limb using (99m) Tc-HMPAO.</a> Equine Veterinary Journal. 44, 594-599 (2012).</li>
<li>Carvalho, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+mesenchymal+stem+cell+migration+after+equine+tendonitis+therapy%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Evaluation of mesenchymal stem cell migration after equine tendonitis therapy.</a> Equine Veterinary Journal. 46, 635-638 (2014).</li>
<li>Welling, M. M., Duijvestein, M., Signore, A., van der Weerd, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+biodistribution+of+stem+cells+using+molecular+nuclear+medicine+imaging%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">In vivo biodistribution of stem cells using molecular nuclear medicine imaging.</a> Journal of Cellular Physiology. 226, 1444-1452 (2011).</li>
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<li>Kasashima, Y., Ueno, T., Tomita, A., Goodship, A. E., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimisation+of+bone+marrow+aspiration+from+the+equine+sternum+for+the+safe+recovery+of+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells.</a> Equine Veterinary Journal. 43, 288-294 (2011).</li>
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</ol>

JD and RKS are Scientific Advisory Board members to ReCellerate Inc.

In addition to bone marrow, stem cells isolated from sources such as adipose tissue are suitable for labeling with this protocol. Furthermore, cells from a frozen state may be revived and expanded in culture to the desired numbers for labeling studies12.
A critical factor that determines the efficiency of labeling the BMMSC is the time between elution of the Tc-99m from the molybdenum generator at the radiopharmacy, preparation of Tc-99m-HMPAO and use of the radiopharmaceutical in t...

Regenerative strategies for the repair of diseased or damaged tissues are based on multipotent stem cells derived from a variety of tissue and implanted into the affected area. Recent advances in the application of autologous BMMSC for the treatment of tendon and ligament injuries in the horse have shown improved outcome measures in both experimental1-5 and clinical studies6. The horse is a particularly attractive model for assessing the efficacy of stem cell related treatments because it suffers from age and overstrain related injuries to the tendons of the distal forelimb, it is an athletic animal, and it is a large, facilitating bone marrow recovery and accurate implantation. Tendon injuries heal naturally with fibrosis but the healed tendon is functionally inferior7 and has a high risk of re-injury8. The superficial digital flexor tendon (SDFT) is most commonly affected as it has evolved to act as an elastic energy store and experiences high loading stresses to achieve energy efficient and high speed locomotion. Restoring function after injury is therefore critical. These injuries are similar to those affecting the Achilles tendon in humans which performs a similar function9. There are no good treatment options to treat or achieve good repair for such injuries, therefore cell-based regenerative strategies offer an attractive opportunity to improve outcomes and to reduce re-injury.
In most studies 5 &#8211; 20 million autologous BMMSC are injected directly into the lesion which usually occur within the core of the tendon body which therefore acts as a receptacle for the cells. The fate of the cells once injected is not clear and different cell labeling methods to track the cells have been described recently. Cells labeled with a fluorescence tag were shown to survive only in relatively small numbers (&#60;10%)10,11. Fluorescence labels necessitate tissue extraction and sectioning for histological analysis which is time consuming and does not readily facilitate temporal analysis in a large animal model or in clinical cases. In more recent work we have used the radioisotope 99mTc to label cells and follow their fate by gamma scintigraphy1. This method allows rapid comparisons to be made between different routes of cell delivery, including intralesional, intravenous via the jugular vein1 or regional perfusion via intra-arterial12 or intravenous1,12 injections. The persistence and distribution of cells can then be imaged by gamma scintigraphy of various organs. This has demonstrated that only 24% of the cells injected intralesionally remained in the lesion by 24 hr1 and this is supported by another study using experimentally created lesions and using the same radiolabel5. Furthermore, the cells show limited ability to home into tendon lesions when delivered by regional perfusion or intravenously but are dispersed into the lungs by the latter routes4.
BMMSC labeled with iron nanoparticles is an alternative method to track cells implanted into forelimb tendons13. Although iron nanoparticle labeled cells allow cell tracking in vivo by MRI, temporal studies in a large animal are limited by the number of times anesthesia can be administered at each time point for performing the MRI scans. Furthermore, iron nanoparticles are hypointense on MRI which limits the information on the migration of labeled cells into the tendon body. Other radioisotopes that can be used include Indium-111 but this suffers the disadvantage of a longer half-life than Tc-99m (2.8 days vs 6.0 hr) and higher gamma ray emission energy. In addition, cell viability has been reported to be reduced when labeled with Indium-11114. Tc-99m, on the other hand, is routinely used in both equine and human nuclear medicine to label peripheral blood mononuclear cells and follow their distribution in vivo by scintigraphy. It can be relatively easily taken up by cells using HMPAO as a linker molecule to bind the technetium, as Tc-99m-HMPAO, to cells. Tc-99m-HMPAO labeled BMMSC show good viability and can proliferate in vitro4. This protocol details the labeling and tracking of equine autologous BMMSC implanted into naturally occurring lesions in the forelimb SDFT.
It is important to note that the protocol is intended only to be used as a research tool. Its use as a clinical therapeutic modality is not recommended as the effect of the radiolabel on cellular phenotype has not been fully elucidated.

<table><tbody><tr><td>Technetium&lt;sup&gt;99m&lt;/sup&gt;&amp;nbsp;</td><td></td><td></td><td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&amp;nbsp; The isotope must be used within 2 hr of elution from the molybdenum-99 generator</td></tr><tr><td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&amp;nbsp;</td><td>GE HealthCare</td><td></td><td>Please enquire directly with GE HealthCare</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Microfuge, Minispin/Minispin Plus</td><td>Ependorf</td><td>22620100</td><td></td></tr><tr><td>18 G and 19 G Needles</td><td>Terumo Medical</td><td>NN-1838R (18 G);&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; NN1938R (19 G)</td><td></td></tr><tr><td>Syringes 1 ml and 2 ml</td><td>Scientific Laboratory Supplies Ltd</td><td>SYR6200 (1 ml); SYR6003 (2 ml)</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 ml</td><td>Greiner Bio-One Ltd</td><td>616201</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>LifeTechnologies</td><td>14190</td><td></td></tr><tr><td>Sterile Gauze Swabs</td><td>Shermond Ltd</td><td>UNG602</td><td></td></tr><tr><td>CoflexVet self adhering bandage</td><td>Andover Healthcare, Inc.</td><td>3540RB-018</td><td></td></tr><tr><td>Ultrasound imaging software</td><td>Scion Image, Scion Corporation, USA</td><td></td><td></td></tr><tr><td>MicasXplus Scintigram processing software</td><td>Bartec Technologies Ltd</td><td></td><td>http://www.bartectechnologies.com/veterinaryscintigraphy.html</td></tr><tr><td>Field isotope counter for monitoring isotope</td><td>John Caunt U.K.</td><td>GMS1800a</td><td>http://www.johncaunt.com/</td></tr><tr><td>Well counter for isotope measurements, dose calibrator</td><td>Capintec Southern Scientific</td><td>CRC-25R</td><td></td></tr></tbody></table>

in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy

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

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

Tc-99m-HMPAO incorporation into BMMSs does not adversely affect their ability to adhere to tissue culture plastic and while they show proliferation ability to form monolayers (Figure 1) we have not fully determined whether proliferation rates or other cellular phenotypes are affected. Their morphology is similar to unlabeled cells with a typical spindle shape. The cellular labeling efficiency (i.e., uptake of label) typically varies from about 1.5% to 25%. The main reason for the low labeling ef...

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

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.

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Research

JoVE Journal

Medicine

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Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

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Learn to non-invasively track technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy using gamma scintigraphy. This method enables monitoring of cell distribution and survival post-implantation.

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

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