Abstract: BACKGROUND: In the field of tissue engineering, the use of smooth muscle cells for three-dimensional culture to construct vascular grafts holds significant importance. However, the current widespread use of culture media containing fetal bovine serum presents potential ethical, safety, and compositional complexity issues that may affect experimental outcomes and hinder the clinical application of vascular grafts.
OBJECTIVE: To identify a serum-free medium with well-defined components to replace traditional serum-containing culture medium for smooth muscle cell culture and evaluate the feasibility of using serum-free culture medium for constructing tissue-engineered blood vessels.
METHODS: Bovine thoracic aortic smooth muscle cells were isolated and cultured using a tissue explant method, and cells from passages 3 to 8 were used for subsequent experiments. Serum-free culture medium was prepared: DMEM F12 medium containing 5 mg/mL bovine serum albumin, 50 µg/mL L-ascorbic acid, 0.4 µg/mL fetuin, 15 ng/mL basic fibroblast growth factor, 15 ng/mL platelet-derived growth factor BB, and 10 µg/mL insulin, 5.5 µg/mL transferrin, and 67 ng/mL sodium selenite. (1) Two-dimensional culture: Vascular smooth muscle cells were divided into four groups and cultured in serum-free medium or DMEM F12 medium containing 0%, 5%, or 10% fetal bovine serum, respectively. Cell morphology was observed, and cell proliferation was assessed using CCK-8, EdU, a CytoCube cell counter, and flow cytometry. Total collagen content was determined by hydroxyproline quantification. Expression of smooth muscle cell contractile markers, calmodulin 1 and α-smooth muscle actin, was assessed by immunofluorescence staining and qRT-PCR. Expression of type I and type III collagen was assessed by immunoblotting. The scratch test was used to detect cell migration. (2) Three-dimensional culture: Vascular smooth muscle cells were seeded on polyglycolic acid scaffolds and cultured in four groups and cultured in serum-free medium or DMEM F12 medium containing 0%, 5%, or 10% fetal bovine serum, respectively. Scanning electron microscopy was used to analyze external tissue morphology. Hematoxylin-eosin staining and Masson staining were used to observe tissue morphology. Immunofluorescence staining was used to detect type I and type III collagen expression. Total collagen content was measured using the hydroxyproline quantification method.
RESULTS AND CONCLUSION: (1) Two-dimensional culture: Inverted microscopy revealed that the 0% serum culture group had the lowest cell density and larger cell size. Compared with the 5% and 10% serum culture groups, the cells in the serum-free culture group exhibited a more elongated, spindle-shaped structure and more regular cell arrangement. CCK-8, EdU, CytoCube cell counter, and flow cytometry assays showed that cell proliferation in the serum-free culture group was less than that in the 10% serum culture group, but greater than that in the 0% and 5% serum culture groups. Calmodulin 1 and α-smooth muscle actin expression levels were lower in the 5% and 10% serum culture groups, and serum-free culture group than in the 0% serum culture group, while cell migration was stronger than that in the 0% serum culture group. Total collagen content was higher in the serum-free culture group than in the other three groups. Type III collagen expression was higher in the 10% and serum-free culture groups than in the 0% and 5% serum culture groups. (2) Three-dimensional culture: Scanning electron microscopy revealed that smooth muscle cells in each group formed early tissue-engineered vascular scaffolds on the polyglycolic acid scaffolds, with the serum-free culture group displaying the greatest extracellular matrix deposition. The serum-free culture medium group showed significantly higher total collagen content and type I and type III collagen expression than the other three groups. Hematoxylin-eosin staining and Masson staining revealed the formation of tissue-engineered vascular scaffolds in all three groups except the 0% serum culture group. (3) The results indicate that serum-free culture medium promotes the proliferation of vascular smooth muscle cells in vitro and the formation of tissue-engineered vascular grafts.

Key words: tissue-engineered vascular graft, vascular smooth muscle cell, serum-free medium, collagen, proliferation, fetal bovine serum, biomaterial