Hence, this system represents a good model for studying nerve regeneration of a peripheral nerve
Hence, this system represents a good model for studying nerve regeneration of a peripheral nerve. The behavior of M-OECs entrapped in Matrigel? was tested by placement on the membrane used for organotypic co-cultures, with the regeneration process being monitored by staining motor neurons with NF-200 antibody (see Experimental section for details). studies aimed at exploring the potential of OECs in GSK-J4 the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) by MRI to determine their migration details in normal and injured spinal cords, including the possibility that OECs can cross a complete spinal cord injury zone . MNPs are largely employed in biomedicine. The use of MNP has been established for many clinical diagnostic/therapeutic uses, e.g., MRI contrast agents in magnetic resonance imaging , for cell tracking via MRI , magnetic hyperthermia , gene therapy , vectors for drug delivery , model involving organotypic culture, the present study explored the BST2 utility of magnetizing OECs by labelling them with homemade MNPs, as this would enable precise shepherding of the transplanted cells to a nerve lesion. The results indicate that the labelling does not harm the OECs, and that migration of the magnetized OECs can be directed precisely by a magnetic field. Lastly, magnetized OECs can be transplanted in organotypic slices of spinal cord and peripheral nerve. In these organotypic co-cultures, magnetized OECs survive and are able to integrate within the tissue. Altogether, the method described represents a new approach for controlling the migration of the OECs and appears useful for future studies aimed at effective regeneration of spinal cord injuries. 2. Results 2.1. Magnetization of OECs via MNPs OECs were cultured with MNPs (M-OECs) for 24 h and analyzed by light microscopy (Figure 1a,b) and electron microscopy (Figure 1c,d). Few particles could be detected on the cell surface, whereas clusters of particles were found inside cell cytoplasm. EDS (energy-dispersive X-ray spectroscopy) spectra clearly showed that these agglomerates are composed of iron, reflecting the intracellular presence of the MNPs (Figure 1e,f) and confirming the efficient labelling process of the OECs. Open in a separate window Figure 1 (a,b) Light microscopy photomicrographs of olfactory ensheathing cells (OECs) treated with 10 g/mL (a) or 25 g/mL (b) of MNPs (M-OECs) (arrows in a and b); (c,d) dual beam SEM/FIB images of M-OECs treated with 10 g/mL of MNPs. The internalization of the MNPs can be seen in the cross section of single OEC cells (light grey spots in e) and confirmed by the Fe-content from EDS analysis of these areas (spectral analysis in f). 2.2. M-OECs Viability Cell viability of M-OECs was tested in both a time- and dose-dependent manner by using propidium iodide (PI) dye exclusion assay. Flow cytometer analysis GSK-J4 showed that the treatment induces a negligible toxicity after 72 h of treatment with all the concentrations of MNPs tested. In particular, GSK-J4 at the highest concentration tested (25 g/mL), the viability was found ~94.25% 3.12%, and not far from the control (~97.02% 5.05%) (Figure 2f). These results were confirmed by PI staining via fluorescent microscopy (Figure 2aCe); cells treated with MNPs exhibited permeability to PI similar to the control, except for 25 g/mL, where few red spots are noticeable. Immunoblot analysis was performed to document any differences in the expression of proteins involved in cell replication (AKT, protein kinase B), apoptosis (P53) and cell death (ERK, extracellular-signal-regulated kinases) between the control and the cell GSK-J4 treated with 10 g/mL of MNPs over time. At any time tested, no differences were found in the protein expression and their phosphorylation level confirming the negligible GSK-J4 toxicity induced on OECs by MNP labelling (Figure 3a). Open in a separate window Figure 2 (aCe) Fluorescent microscopy of cells treated with MNPs 0, 1, 5, 10 and 25 g/mL, respectively. Blue: Hoechst nuclear staining. Red: PI staining (white arrows show PI positive cells); (f) PI staining via flow cytometry.