He nanoparticles were washed out by using HBSS (Invitrogen, Paisley, Scotland, UK), and then the cells were harvested as described earlier.AnimalsTransplantationTen-week-old male Wistar rats were obtained, and their body weight ranged between 270 g and 300 g to minimize differences in body size to achieve standardized spinal cord lesions. To complete this study, 83 animals were used. The numbers of animals used for all parts of the study are summarized in Additional file 1: Table S1. All experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) regarding the use of animals in research and were approved by the Ethics Committee of the Institute of Experimental Medicine ASCR, Prague, Czech Republic.Spinal cord injuryA balloon-compression lesion was performed in a total of 79 male Wistar rats, as described by Urdzikova et al. . In brief, the animals were anesthetized with 2 isoflurane (Forane; Abbott Laboratories, Queenborough, UK) and shaved on the back from C7 to Th1. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 Under sterile conditions, the skin was cut in the midline from Th7 to Th12. The soft tissue was removed, as well as the spinous processes of vertebrae Th8 to Th11. A 2F Fogarty catheter was inserted into the epidural space and advanced cranially for 1 cm, so that the center of the balloon rested at the Th8 to Th9 level of the spinal cord. The balloon was rapidly inflated with 15 l of saline for 5 minutes. The catheter was then deflated and removed. The soft tissue and skin were sutured with unresorbable thread, and the animals were allowed to feed and drink ad libitum. During the surgical procedure, the body temperature of the animal was maintained at 37 with a heating pad, and 3 uurane in air was administered at a flow rate of 0.3 L/min. After being returned to their cages, the rats were assisted in feeding and urination until they had recovered sufficiently to perform these functions on their own. The animals received gentamicin sulfate (5 mg/kg) for 3 days to prevent postoperative infections.The animals were transplanted 1 week after SCI. This time point is generally accepted as a suitable therapeutic window, as the inflammatory reaction (creating a hostile environment for cell-transplant survival) decreases during the first 7 days, and the glial scar that Nelfinavir (Mesylate) prevents grafthost tissue communication is not yet developed . The animals were secured in a stereotaxic apparatus with a rat-specific vertebra holder (Cunningham spinal adaptor; Stoelting Co., Wood Dale, IL, USA). The spinal cord was exposed at T8. In total, 5 ?105 SPC-01 cells/5 l (either unlabeled or labeled with nanoparticles) were injected through a glass pipette into the center of the lesion at a depth of 1 mm below the dorsal surface at a rate of 1 l/min by using a Nano-Injector (Stoelting). Cells were harvested as described earlier, and the cell suspension was prepared just before the transplantation procedure. The number of cells was determined based on the results obtained from our pilot study, in which 1 ?105 SPC-01 cells/1 l were also injected into the proximal, central, and distal parts of the lesioned spinal cord. However, the survival rate of the transplanted cells was worse than when injecting 5 ?105 SPC-01 cells/5 l into the lesion. The glass pipette was kept in place after injection for a further 5 minutes to prevent leakage of the cell suspension. The control group received 5 l of saline. Triple-drug immunosuppression was used.