Comparative Analysis of Different Fixation and Dehydration Methods for Cells Seeded on Gel-Based Scaffolds using Scanning Electron Microscopy

MatrigelTM, a complex mixture of ECM proteins, and Collagen type 1 (Col-1), a major component of the natural extracellular matrix, are widely utilised in both cell culture and tissue engineering. High magnification by scanning electron microscopy allows detailed microstructural analysis of the cells and scaffolds. However, the efficiency of the techniques may be directly affected by different fixation and dehydration methods. Optimisation of the fixation and dehydration protocol for cell growth on scaffold is important as it influences the morphological integrity and structural preservation of cells within gel-based scaffolds. This study compared the osmium tetroxide hexamethyldisilane (HMDS) and critical point drying (CPD) method to fix and dehydrate cells which were seeded on Col-1 and MatrigelTM scaffolds by SEM analysis. Col-1 and MatrigelTM scaffolds were prepared and seeded with dental pulp stem cells (DPSC) for 3 days; followed by fixation using with McDowell-Trump fixative, osmium tetroxide hexamethyldisilane (HMDS) or the critical point drying (CPD) method and analysed by SEM. Both HMDS and CPD displayed good compatibility with cells and scaffolds for SEM analysis. No significant differences were observed on the morphology of DPSC prepared using either HMDS or CPD (Mann–Whitney U test, p = 0.10 for both). However, each of the fixation methods substantially provided different preservative effects on Col-1 and MatrigelTM microstructures. In conclusion, both HMDS and CPD methods were found suitable for fixation and dehydration of DPSC plated on Col-1 and MatrigelTM scaffolds for analysis by SEM.