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1Department of Radiology and Biomedical Imaging, Medical Center, University of California San Francisco
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Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.
Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects. J. Vis. Exp. (38), e1793, doi:10.3791/1793 (2010).
The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.
1. Labeling of hMSCs with Endorem
2. Preparing Agarose
3. Creating MASI
Figure 1. Coronal T2 weighted SE images (TR 4000 ms/ TE 18.27 ms) of a patella specimen with implanted hMSCs in cartilage defects.
The described protocol provides a reproducible way to label MSCs with iron oxide nanoparticles and implant these labeled MSCs into cartilage defects. This technique permits non-invasive depiction of stem cell transplants in cartilage defects with MR imaging, which allows an early detection of MASI failure. A dislocation or efflux of the labeled cells can be diagnosed based on a disappearance of the label from the transplantation site on MR images. An early diagnosis of these events is desirable and would prevent the patient from unnecessary alternative invasive diagnostic procedures. The described non-invasive method for diagnosing MASI outcomes could aid in the successful development of cell based therapies for cartilage regeneration in osteoarthritis. Our protocol is currently applied in preclinical in-vivo studies, would be in principle clinically applicable and could serve as non-invasive outcome measure for the assessment of MASI therapies in clinical practice.
This work was supported by a grant from the National Institute of Arthritis and Musculoskeletal Skin Diseases, NIH RO1AR054458.
|D-MEM High Glucose||Reagent||Sigma-Aldrich||D5648|
|PBS (Ca++, Mg++ free)||GIBCO, by Life Technologies||14190-144|
|Penicillin/Streptomycin||GIBCO, by Life Technologies||15140-122|
|Agarose Typ VII||Sigma-Aldrich||A9045||Dilute Agarose in PBS to final concentration of 4%|
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