Journal: Bioactive Materials
Article Title: Integrated cryopreservation-thawing-transplantation platform for neural stem cell-based spinal cord injury repair
doi: 10.1016/j.bioactmat.2026.01.024
Figure Lengend Snippet: Comprehensive functional validation of CTT Platform after cryopreservation and simulated transport. A) Schematic illustration of the experimental workflow. Fresh or cryopreserved PM@NSC (at −80 °C or −196 °C for 3 months) were thawed and subjected to a 4-h simulated transport at 4 °C prior to in vitro analysis or in vivo transplantation for SCI repair. B) Representative confocal microscopy images assessing post-thaw cell cytoskeletal integrity of NSCs loaded onto PM. Phalloidin (green) for F-actin; DAPI (blue) for nucleus. Scale bar: 50 μm. C) Western blot bands of Nestin, Sox2, and Ki67 show no significant differences in protein expression levels among control (fresh), cryopreserved (−80 °C/-196 °C), and cryopreservation (−80 °C/-196 °C)-transport groups. D) Representative confocal microscopy images assessing post-thaw cell viability of NSCs loaded onto PM. Calcein AM (green) for live cells; PI (red) for dead cells. Scale bar: 50 μm. E) Western blot bands of Cleaved-Caspase3, Bcl-2, and Bax protein expression in protein expression levels among control (fresh), cryopreserved (−80 °C/-196 °C), and cryopreservation (−80 °C/-196 °C)-transport groups. F) Quantitative analysis of cell survival rate of NSC in each group (n = 5). G) Quantitative analysis of Nestin/GAPDH, Ki67/GAPDH, and Sox2/β-Actin ratios in each group (n = 3). H) Quantitative analysis of Cleaved-Caspase3/GAPDH, Bcl-2/GAPDH and Bax/GAPDH ratios in each group (n = 3). I) Representative photographs of rat hindlimb motor functions in each group, 8 weeks after SCI. J) MEP results show variations in latency and amplitude in the left hind leg of each group 56 days after SCI K) H&E staining of gastrocnemius muscles indicated variations in muscle fiber morphology among the groups 56 days after SCI. Scale bar: 200 μm L) Sagittal and axial T2-weighted MRI images of rats in each group 56 days after SCI. M) Footprint analysis with print views, footfall patterns, 3D footprints, and 2D footprints revealing differences in gait patterns among separate groups. N) BBB scores demonstrate comparable locomotor functional recovery across all groups, including the Control group and the Cryopreserved-Transport treated group (n = 5). O) Quantitative analysis of gastrocnemius muscle fiber cross-sectional area, indicating similar muscle functional recovery (n = 5). P) Quantitative analysis of MEP latency and amplitude in the left hind leg in each group (n = 5). Q) Quantitative analysis of T2 density in sagittal and coronal planes in spinal cord lesions among groups (n = 5). R) Quantitative footprint analysis on day 56 post-injury included the maximum footprint intensity, footprint positioning, and the regularity index of the left hindlimb (n = 10). All data are presented as the mean ± SEM. Statistical analysis showed no significant differences (n.s.) among the experimental groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Article Snippet: The primary antibodies used in this research are listed below: CD68 (Abcam, Cambridge, UK), CD206 (Abcam, Cambridge, UK), GFAP (Bioss, Beijing, China), iNOS (Abcam, Cambridge, UK), Tuj-1 (Abcam, Cambridge, UK), NF-200 (Invitrogen, CA, USA), MBP (Abcam, Cambridge, UK), HIF-1α (Abcam, Cambridge, UK), VEGFA (Abcam, Cambridge, UK), P-CaMKII (Abcam, Cambridge, UK), CaMKII (Abcam, Cambridge, UK), P-CREB (Cell Signaling Technology, USA), CREB (Cell Signaling Technology, USA), P-PI3K (Cell Signaling Technology, USA), PI3K (Cell Signaling Technology, USA), P-AKT (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), Cleaved-Caspase3 (Cell Signaling Technology, USA), Bcl-2 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), GAPDH (Proteintech, IL, USA).
Techniques: Functional Assay, Biomarker Discovery, In Vitro, In Vivo, Transplantation Assay, Confocal Microscopy, Western Blot, Expressing, Control, Staining, Muscles
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