Serum-free endothelial cell culture medium for vascular smooth muscle cells sheet formation.

Background: Cell sheet technology has been identified as a promising approach for the construction of tissue-engineered vascular grafts (TEVGs). However, concerns regarding immunogenicity and ethical issues, which are raised by the use of fetal bovine serum (FBS) in traditional culture systems, limit its potential for clinical translation. Serum-free medium (SFM) has emerged as a safer and more controllable alternative, but further validation is required to determine its effectiveness and superiority in generating high-quality cell sheets.

Methods: This study systematically compared cell sheets generated under SFM and 10% FBS culture conditions in terms of structure, cellular phenotype, and functional properties. The expression levels of α-SMA and SM22, markers of vascular smooth muscle cells(VSMCs), were evaluated using immunofluorescence staining, qRT-PCR, and Western blot analysis to assess cellular phenotype. Histological staining and mechanical testing were employed to compare the morphology and mechanical properties of the cell sheets, while extracellular matrix (ECM) deposition and biochemical characteristics were also analyzed.

Results: Under SFM conditions, cells exhibited significantly higher α-SMA and SM22 expression levels (qRT-PCR showed a 1.8-fold and 2-fold increase, respectively; ****p < 0.0001) with clearer cytoskeletal arrangement. Cell sheets formed in SFM displayed comparable area(ns, p > 0.05), thickness(**p < 0.01), and mechanical properties to those cultured in 10% FBS, while ECM deposition was significantly enhanced (collagen content increased by approximately 40%, **p < 0.01). Furthermore, histological analysis revealed that cell sheets generated under SFM conditions were more compact and uniform, exhibiting superior structural organization.

Conclusion: SFM facilitates the generation of cell sheets that exhibit structural and functional properties analogous to those cultured in FBS. Additionally, SFM promotes cellular phenotype transition and ECM deposition. Consequently, SFM provides a safer, more controllable, and clinically translatable solution for cell sheet construction.