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

Summary

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.

Abstract

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 (<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 (t_1/2 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.

Introduction

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 experimental^1-5 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 re....

Protocol

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

1. Seek institutional ethics and anima....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Technetium99m			Please enquire with local ionisation radiation supplier in accordance with legal requirements.  The isotope must be used within 2 hr of elution from the molybdenum-99 generator
Ceretec - Hexamethylpropyleneamine oxime (HMPAO)	GE HealthCare		Please enquire directly with GE HealthCare
Microfuge, Minispin/Minispin Plus	Ependorf	22620100
18 G and 19 G Needles	Terumo Medical	NN-1838R (18 G);         NN1938R (19 G)
Syringes 1 ml and 2 ml	Scientific Laboratory Supplies Ltd	SYR6200 (1 ml); SYR6003 (2 ml)
Microcentrifuge tubes 1.5 ml	Greiner Bio-One Ltd	616201
PBS - Phosphate-Buffered Saline	LifeTechnologies	14190
Sterile Gauze Swabs	Shermond Ltd	UNG602
CoflexVet self adhering bandage	Andover Healthcare, Inc.	3540RB-018
Ultrasound imaging software	Scion Image, Scion Corporation, USA
MicasXplus Scintigram processing software	Bartec Technologies Ltd		http://www.bartectechnologies.com/veterinaryscintigraphy.html
Field isotope counter for monitoring isotope	John Caunt U.K.	GMS1800a	http://www.johncaunt.com/
Well counter for isotope measurements, dose calibrator	Capintec Southern Scientific	CRC-25R