ObjectiveTo observe the morphological characteristics of rat submandibular gland cells (RSMGs) co-cultured with silk fibroin-chitosan, (SFCs) by inverted microscopy, scanning electron microscopy (SEM), fluorescence microscopy and laser scanning confocal microscopy (LSCM), and provide technical support for observation and evaluation of cell growth on three-dimensional (3D) scaffolds. MethodsThe submandibular glands were excised from SD rats at age of 0 to 8-days and RSMGs were primarily cultured, purified and subcultured. The origin of cells were identified by immunocytochemistry (CK8 and amylase). Then RSMGs were chosen for the further studies when cells reached 75% to 80% confluence. Meanwhile SFCs blend membranes (5 mm x 5 mm x 2 mm) were prepared. RSMGs cultured on the scaffolds were monitored for 3,5 to 7 days. The morphological appearance of the seeded scaffolds was observed by inverted microscopy, SEM, fluorescence microscopy and LSCM. ResultsLiving cells that were seeded on SFCs medium of 3D scaffolds could be observed directly under the invert microscope. SEM showed the surface ultrastructure of the complex growth situation more clearly. After fluorescent staining, cells anchoring on scaffolds were observed by fluorescence microscopy and LSCM. The fluorescent signals of cell nuclei were evenly distributed in the pores of scaffolds. Through layer scanning and 3D LSCM reconstruction technique, images of thick specimens could be obtained. By rotating the images, 3D profile or whole structure could be observed from different angles and more accurate information of positioning could be obtained. ConclusionsAll four kinds of microscopic techniques can be used in examining the co-cultivation of RSMGs and SFCs. Images can be observed more distinctly through 3D LSCM reconstruction technique combined with fluorescent staining technique. It has a wide application value in this research field.