![]() When imaging cells at discrete daily intervals, it is extremely difficult to find exactly the same cells that were being imaged 16. Apart from unpractical to perform continuous 3D live cell imaging for more than a day, the increased probability of photo-damaging cells or photobleach the fluorophore over time is often the limiting factor. As MSC are slowly moving cells when in 3D, tracing the same cells through time requires imaging performed for several days and it is usually not feasible to keep the cells under the microscope for long periods. There are some reports on the use of fluorescent proteins or dyes to track stem cells, but most use labeling in order to find the cells at one fixed time point and not to follow them by time-lapse analysis 6, 15. Most of these bioimaging techniques are well established but are more oriented to localize the final homing site of transplanted cells or to roughly quantify cell survival after implantation, being rather limited for studying the effect of cell-ECM interactions on MSC dynamical behavior. ![]() Some studies have suggested that cell transfection with fluorescent proteins 5, 6, 7 will be required for long term cell tracking and others are pursuing magnetic nanoparticles 8, bioluminescent probes 9, 10, 11, quantum dots 12, 13 and radioactive isotope alternatives 14 for stem cell labeling. To this end, a number of cell labeling techniques have been developed. A tool to longitudinally follow MSC in 3D is essential to better understand cell migration and organization in 3D microenvironments. Regardless of the approach, to improve the effectiveness of such applications it is critical to understand the determinants of stem cell migration in 3D microenvironments.ĭespite the worldwide effort to modulate and direct stem cell fate for tissue regeneration, the influence of 3D extracellular matrix (ECM) features on MSC motility still remains largely unknown. Most strategies being currently explored involve either stem cell transplantation to patients 1, implantation of the triad scaffolds/stem cells/growth factors 2, 3, or the use of materials that stimulate endogenous stem cell recruitment 4. ![]() MSC are an attractive cell source for regenerative cell-based therapies due to its well established multipotency, immunomodulatory and paracrine properties, combined with their ability to migrate into damaged tissues. This platform thus provides a straightforward approach to characterize MSC dynamics in 3D and has applications in the field of stem cell biology and for the development of biomaterials for tissue regeneration. Comparison of cells mobility within matrices with tuned physicochemical properties revealed that MSC embedded in Matrigel migrated 64% more with 5.2 mg protein/mL than with 9.6 mg/mL and that MSC embedded in RGD-alginate migrated 51% faster with 1% polymer concentration than in 2% RGD-alginate. This strategy provided reliable tracking in 3D microenvironments with different properties, including the hydrogels Matrigel and alginate as well as chitosan porous scaffolds. Human MSC were transfected to express a fluorescent photoswitchable protein, Dendra2, which was used to highlight and follow the same group of cells for more than seven days, even if removed from the microscope to the incubator. Here, a simple and reliable imaging technique was developed to study MSC dynamical behavior in natural and bioengineered 3D matrices. ![]() Mesenchymal Stem/Stromal Cells (MSC) are a promising cell type for cell-based therapies - from tissue regeneration to treatment of autoimmune diseases - due to their capacity to migrate to damaged tissues, to differentiate in different lineages and to their immunomodulatory and paracrine properties.
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