Methodological Insights from Low-Vacuum SEM for Morphological Analysis of Schwann Cells on Electrospun Scaffolds

Schwann cells (SCs) are critical effectors of peripheral nerve regeneration, and their interaction with biomaterial scaffolds is a key parameter in neural tissue engineering. This pilot study described and evaluated protocols for a morphological, quantitative, and morphometric analysis of SCs seeded on electrospun polyhydroxybutyrate (PHB) scaffolds using variable-pressure scanning electron microscopy (VP-SEM) under a low vacuum, without a metal coating. Six protocols were compared, varying the number of seeded cells (50,000 or 100,000) and the method used to label the seeded face of the scaffold: no marking, graphite pencil, or permanent ink (Sharpie). Confocal microscopy confirmed SC viability and adhesion. The VP-SEM analysis revealed that seeding 100,000 cells significantly increased the number of detectable cells on the scaffold surface. Graphite labeling was associated with higher cell counts and a more stellate morphology, consistent with the biocompatibility of carbon-based materials reported in the literature. Conversely, ink labeling appeared to inhibit SC adhesion. A refined protocol for measuring SC extensions using ImageJ's ROI Manager and segmented line tools was also established. These findings provide practical methodological insights to improve the reliability and reproducibility of SC morphological analyses on ultra-thin polymeric scaffolds, with implications for peripheral nerve regeneration research.