中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (20): 5093-5102.doi: 10.12307/2026.163

• 组织工程血管材料 tissue-engineered vascular materials •    下一篇

促进组织工程血管移植物早期阶段形成的无血清培养基

陈  颖1，2，孙盱衡3，刘  青1，肖  聪1，蒋虹婧1，林展翼1，2，3，4   

  1. 1华南理工大学医学院，广东省广州市   510006；2南方医科大学附属广东省人民医院(广东省医学科学院)，广东省广州市   510080；3广东省心血管病研究所，广东省人民医院，广东省医学科学院，广东省广州市   510145；4先进制造科学与技术广东省实验室(季华实验室)，广东省佛山市   528200
  • 接受日期:2025-05-28 出版日期:2026-07-18 发布日期:2025-11-21
  • 通讯作者: 林展翼，博士，主任医师，教授，华南理工大学医学院，广东省广州市 510006；南方医科大学附属广东省人民医院(广东省医学科学院)，广东省广州市 510080；广东省心血管病研究所，广东省人民医院，广东省医学科学院，广东省广州市 510145；先进制造科学与技术广东省实验室(季华实验室)，广东省佛山市 528200
  • 作者简介:陈颖，女，1999年生，广东省佛山市人，汉族，华南理工大学在读硕士，主要从事小口径血管组织工程研究。
  • 基金资助:
    先进制造科学与技术广东省实验室(季华实验室)科研项目(X210111TD210)：动静脉造瘘血管移植体外工程化培养的装置研制，项目负责人：林展翼

A serum-free culture medium for the early-stage formation of tissue-engineered vascular grafts

Chen Ying1, 2, Sun Xuheng3, Liu Qing1, Xiao Cong1, Jiang Hongjing1, Lin Zhanyi1, 2, 3, 4   

  1. 1School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong Province, China; 2Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou 510080, Guangdong Province, China; 3Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510145, Guangdong Province, China; 4Guangdong Provincial Laboratory of Advanced Manufacturing Science and Technology (Ji Hua Laboratory), Foshan 528200, Guangdong Province, China
  • Accepted:2025-05-28 Online:2026-07-18 Published:2025-11-21
  • Contact: Lin Zhanyi, MD, Chief physician, Professor, School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong Province, China; Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou 510080, Guangdong Province, China; Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510145, Guangdong Province, China; Guangdong Provincial Laboratory of Advanced Manufacturing Science and Technology (Ji Hua Laboratory), Foshan 528200, Guangdong Province, China
  • About author:Chen Ying, Master candidate, School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong Province, China; Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou 510080, Guangdong Province, China
  • Supported by:
    Guangdong Provincial Laboratory of Advanced Manufacturing Science and Technology (Ji Hua Laboratory) Research Project, No. X210111TD210 (to LZY) 

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摘要：

文题释义：
胰岛素-转铁蛋白-硒：为一种细胞培养补充剂，含有胰岛素、转铁蛋白、硒和乙醇胺元素，作为无血清培养基添加剂可促进多种细胞的增殖。胰岛素促进细胞的增殖与代谢，转铁蛋白则通过提供铁离子支持细胞的生长和分化，硒有助于减少细胞内的自由基损伤，从而提高细胞的存活率和增殖能力。 
聚乙醇酸：作为一种生物可降解的高分子材料，具有良好的生物相容性和力学性能，为细胞提供稳定的支撑平台。聚乙醇酸的降解速度可以与新生血管的生长速度相匹配，用于构建组织工程血管，帮助维持血管形态和结构的稳定。

背景：在组织工程领域，利用平滑肌细胞进行三维培养来构建血管移植物具有重要意义。然而，当前广泛采用的含胎牛血清培养基存在道德、安全以及成分复杂性等潜在问题，可能影响实验结果，不利于血管移植物的临床应用。
目的：选定一种成分明确的无血清培养基替代传统含血清培养基用于平滑肌细胞培养，评估无血清培养基用于构建组织工程血管的可行性。
方法：采用组织块培养法分离培养牛胸主动脉平滑肌细胞，取第3-8代细胞用于后续实验。配制无血清培养基：含5 mg/mL牛血清白蛋白、50 µg/mL L-抗坏血酸、0.4 µg/mL胎球蛋白、15 ng/mL碱性成纤维细胞生长因子、15 ng/mL血小板源性生长因子BB和10 µg/mL胰岛素-5.5 µg/mL转铁蛋白-67 ng/mL亚硒酸钠的DMEM F12培养基。①二维培养：将血管平滑肌细胞分4组培养，分别加入无血清培养基与含体积分数0%，5%，10%胎牛血清的DMEM F12培养基，观察细胞形态，通过CCK-8、EdU、CytoCube细胞计数仪、流式细胞仪检测细胞增殖情况，羟脯氨酸定量法检测总胶原蛋白含量，免疫荧光染色与qRT-PCR检测平滑肌细胞收缩表型标志物钙调蛋白1、α-平滑肌肌动蛋白表达，免疫印迹检测Ⅰ型胶原、Ⅲ型胶原表达，划痕实验检测细胞迁移。②三维培养：将血管平滑肌细胞接种于聚乙醇酸支架上分4组培养，分别加入无血清培养基与含体积分数0%，5%，10%胎牛血清的DMEM F12培养基，扫描电镜分析组织外部形态，苏木精-伊红与Masson染色观察组织形态，免疫荧光染色检测组织Ⅰ型/Ⅲ型胶原表达，羟脯氨酸定量法检测总胶原蛋白含量。
结果与结论：①二维培养：倒置显微镜下可见0%血清培养组细胞密度最低、细胞体积较大，对比5%，10%血清培养组，无血清培养组细胞呈现更细长的梭形，细胞排列更规则。CCK-8、EdU、CytoCube细胞计数仪、流式细胞仪检测显示，无血清培养组细胞增殖少于10%血清培养组，多于0%，5%血清培养组。5%，10%血清培养组与无血清培养组钙调蛋白1、α-平滑肌肌动蛋白表达均低于0%血清培养组，细胞迁移能力强于0%血清培养组。无血清培养组总胶原蛋白含量高于其他3组。10%血清培养组、无血清培养组Ⅲ型胶原表达高于0%，5%血清培养组。②三维培养：扫描电镜下可见各组平滑肌细胞聚乙醇酸支架上形成了组织工程血管支架的早期组织，其中无血清培养基组组织中细胞外基质沉积最多。无血清培养基组组织中总胶原蛋白含量与Ⅰ型、Ⅲ型胶原表达显著高于其他3组。苏木精-伊红与Masson染色显示，除0%血清培养组外，其余3组均可见组织工程血管支架的形成。③结果表明，无血清培养基可促进体外血管平滑肌细胞的增殖、促进组织工程血管移植物的形成。
https://orcid.org/0009-0000-8012-8386 (陈颖) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 组织工程血管移植物, 血管平滑肌细胞, 无血清培养基, 胶原蛋白, 增殖, 胎牛血清, 生物材料

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

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