中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (26): 6873-6879.doi: 10.12307/2026.113

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

用于细胞三维培养的即用型海藻酸钠@纸材料

余金烨1，2，蒋  南1，赵一浔2，黄梦静2，杨  洁2，孙  瑞2，冯所兰2，蒋  卉2，3，4，杨  军1，2，3，4   

  1. 1南京医科大学药学院，江苏省南京市   211166；2南京市食品药品监督检验院，江苏省南京市   211198；3国家市场监督管理总局重点实验室(食源性致病微生物检测及溯源技术)，江苏省南京市   211198；4江苏省市场监管重点实验室(食源性致病菌检测及溯源技术)，江苏省南京市   211198
  • 接受日期:2025-09-08 出版日期:2026-09-18 发布日期:2026-03-12
  • 通讯作者: 杨军，研究员级高级工程师，南京医科大学药学院，江苏省南京市 211166；南京市食品药品监督检验院，江苏省南京市 211198；国家市场监督管理总局重点实验室(食源性致病微生物检测及溯源技术)，江苏省南京市 211198；江苏省市场监管重点实验室(食源性致病菌检测及溯源技术)，江苏省南京市 211198 蒋卉，高级工程师，南京市食品药品监督检验院，江苏省南京市 211198；国家市场监督管理总局重点实验室(食源性致病微生物检测及溯源技术)，江苏省南京市 211198；江苏省市场监管重点实验室(食源性致病菌检测及溯源技术)，江苏省南京市 211198
  • 作者简介:余金烨，男，1999年生，四川省达州市人，汉族，南京医科大学在读硕士，主要从事细胞培养材料和细胞传感方面的研究。
  • 基金资助:
    国家重点研发计划“食品营养与安全关键技术研发”重点专项(2022YFF1101002)子课题，子课题项目负责人：蒋卉；国家自然科学基金项目(32202176)，项目负责人：蒋卉；国家市场监督管理总局科技创新人才计划项目(QNBJ202328)，项目负责人：蒋卉；
    江苏省重点研发计划项目(BE2022684)，项目负责人：杨军；江苏省市场监督管理局重大科技项目(KJ2024017)，项目参与人：
    蒋卉、余金烨

Ready-to-use sodium alginate@paper material for three-dimensional cell culture

Yu Jinye1, 2, Jiang Nan1, Zhao Yixun2, Huang Mengjing2, Yang Jie2, Sun Rui2, Feng Suolan2, Jiang Hui2, 3, 4, Yang Jun1, 2, 3, 4   

  1. 1School of Pharmacy, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China; 2Nanjing Institute for Food and Drug Control, Nanjing 211198, Jiangsu Province, China; 3Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Microorganisms, State Administration for Market Regulation, Nanjing 211198, Jiangsu Province, China; 4Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Bacteria for Jiangsu Province Market Regulation, Nanjing 211198, Jiangsu Province, China
  • Accepted:2025-09-08 Online:2026-09-18 Published:2026-03-12
  • Contact: Yang Jun, Researcher-level senior engineer, School of Pharmacy, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China; Nanjing Institute for Food and Drug Control, Nanjing 211198, Jiangsu Province, China; Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Microorganisms, State Administration for Market Regulation, Nanjing 211198, Jiangsu Province, China; Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Bacteria for Jiangsu Province Market Regulation, Nanjing 211198, Jiangsu Province, China Jiang Hui, Senior engineer, Nanjing Institute for Food and Drug Control, Nanjing 211198, Jiangsu Province, China; Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Microorganisms, State Administration for Market Regulation, Nanjing 211198, Jiangsu Province, China; Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Bacteria for Jiangsu Province Market Regulation, Nanjing 211198, Jiangsu Province, China
  • About author:Yu Jinye, MS candidate, School of Pharmacy, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China; Nanjing Institute for Food and Drug Control, Nanjing 211198, Jiangsu Province, China
  • Supported by:
    National Key Research and Development Program of China “Key Technology Research and Development for Food Nutrition and Safety”, No. 2022YFF1101002 (to JH); National Natural Science Foundation of China, No. 32202176 (to JH); Science and Technology Innovation Talent Program of State Administration for Market Regulation, No. QNBJ202328 (to JH); Key Plan of Research and Development of Jiangsu Province, No. BE2022684 (to YJ); Science and Technology Project of Jiangsu Provincial Market Supervision and Administration Bureau, No. KJ2024017 (to JH and YJY) 

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摘要：

文题释义：
cells-in-gels-in-paper(CiGiP)：是一种利用纸作为支撑材料，将细胞嵌入水凝胶中进行培养的方法。纸提供细胞生长的三维支架，而水凝胶则模拟了细胞外基质的物理和化学特性，为细胞提供了三维生长的基质。
三维细胞培养：是一种模拟体内微环境的体外细胞培养方法，通过构建具有空间结构的细胞生长体系，使细胞在立体环境中增殖、分化和相互作用。

背景：CiGiP是一种使用水凝胶将细胞封装在纸纤维中的三维培养技术，为细胞三维培养发展提供了一个良好的思路，但是水凝胶需要提前制备后再添加到纸材料上，缺乏一定的便捷性，阻碍了CiGiP的广泛应用。 
目的：制备一种即用型海藻酸钠@纸材料，分析该材料在细胞三维培养中的应用。
方法：①将 2%海藻酸钠溶液滴加到滤纸上，使其均匀渗透在纸材料中，得到现配现用海藻酸钠@纸材料。在现配现用海藻酸钠@纸材料上滴加1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合液，再分别滴加不同浓度(5%，10%，15%，20%)的聚乙二醇-二胺，冷冻干燥后得到即用型海藻酸钠@纸材料。通过溶胀率筛选出15%聚乙二醇-二胺用于制备后续即用型海藻酸钠@纸材料。②将人胚胎肾细胞HEK 293培养于即用型海藻酸钠@纸材料中，以二维培养的细胞为对照，通过乳酸脱氢酶释放检测评估即用型海藻酸钠@纸材料的细胞毒性。将SYTOTM 9绿色荧光核酸染料标记的HEK 293细胞分别培养于即用型海藻酸钠@纸材料、现配现用海藻酸钠@纸材料中，在激光共聚焦显微镜、扫描电镜下观察细胞黏附情况。③将即用型海藻酸钠@纸材料置于干净的培养皿中室温储存0，40，80，120 d，检测材料微观形貌、化学结构与孔隙率变化。HEK 293细胞分别培养于储存0，40，80，120 d后的即用型海藻酸钠@纸材料中，通过乳酸脱氢酶释放检测评估即用型海藻酸钠@纸材料的细胞毒性。将SYTOTM 9绿色荧光核酸染料标记的HEK 293细胞分别培养于储存0，40，80，120 d后的即用型海藻酸钠@纸材料中，激光共聚焦显微镜下观察细胞黏附情况。
结果与结论：①乳酸脱氢酶释放检测显示即用型海藻酸钠@纸材料无细胞毒性。激光共聚焦显微镜显示HEK 293细胞均匀黏附在两组材料上，两组材料的细胞黏附效果无明显差异；扫描电镜下可见HEK 293细胞在两组材料中维持细胞间的相互作用，两组间无明显差异。②储存120 d后，即用型海藻酸钠@纸材料中的海藻酸钠水凝胶没有从纸纤维上脱落，孔隙率未见明显变化。乳酸脱氢酶释放检测显示，储存40，80，120 d后的即用型海藻酸钠@纸材料无细胞毒性。激光共聚焦显微镜显示，HEK 293细胞均匀黏附在储存40，80，120 d后的即用型海藻酸钠@纸材料上，与在未储存即用型海藻酸钠@纸材料上的黏附效果无明显差异。以上结果说明即用型海藻酸钠@纸材料具有良好的稳定性。
https://orcid.org/0009-0001-1878-4101 (余金烨) 

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: 即用型, 海藻酸钠水凝胶, 纸, 三维培养, 冷冻干燥, 交联剂, 细胞毒性, 细胞黏附性, 孔隙率, 生物材料

Abstract: BACKGROUND: Cells-in-gels-in-paper (CiGiP) is a three-dimensional cell culture technique that uses hydrogels to encapsulate cells within paper fibers. It offers a promising approach for the development of three-dimensional cell culture. However, the hydrogels must be prepared in advance and then added to the paper material, which lacks certain convenience and hinders the widespread application of CiGiP.
OBJECTIVE: To prepare a ready-to-use sodium alginate@paper material and to analyze its application in three-dimensional cell culture.
METHODS: (1) 2% sodium alginate was incorporated into paper materials, allowing it to evenly penetrate the paper material to obtain a ready-to-use sodium alginate@paper material. A mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was then dripped onto the ready-to-use sodium alginate@paper material. Polyethylene glycol-diamine at different concentrations (5%, 10%, 15%, and 20%) was then added dropwise. The prepared sodium alginate@paper material was freeze-dried to obtain the ready-to-use sodium alginate@paper material. 15% polyethylene glycol-diamine was selected based on swelling ratio for subsequent preparation of ready-to-use sodium alginate@paper materials. (2) Human embryonic kidney (HEK) 293 cells were co-cultured with the ready-to-use sodium alginate@paper materials. Cells cultured alone were used as controls. The cytotoxicity of the ready-to-use sodium alginate@paper materials was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTOTM 9 green fluorescent nucleic acid dye were plated onto the ready-to-use sodium alginate@paper materials and freshly prepared alginate@paper materials, and cell adhesion was observed under laser confocal microscopy and scanning electron microscopy. (3) The ready-to-use sodium alginate@paper materials were stored in clean culture dishes at room temperature for 0, 40, 80, and 120 days, and the micromorphology and porosity of the materials were examined. HEK 293 cells were seeded to ready-to-use sodium alginate@paper materials after storage for 0, 40, 80, and 120 days and the cytotoxicity of the ready-to-use sodium alginate@paper materials was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTOTM 9 green fluorescent nucleic acid dye were seeded onto ready-to-use sodium alginate@paper materials after storage for 0, 40, 80, and 120 days, and cell adhesion was observed under laser confocal microscopy.
RESULTS AND CONCLUSION: (1) Lactate dehydrogenase release assays demonstrated that the ready-to-use sodium alginate@paper materials were non-cytotoxic. Laser confocal microscopy revealed that HEK 293 cells adhered uniformly to both materials, with no significant difference in adhesion between the two groups. Scanning electron microscopy revealed that HEK 293 cells maintained cell-cell interactions in both materials, with no significant difference between the two groups. (2) After 120 days of storage, the sodium alginate hydrogel in the ready-to-use sodium alginate@paper materials did not detach from the paper fibers, and the porosity did not change significantly. Lactate dehydrogenase release assays demonstrated that the ready-to-use sodium alginate@paper materials were non-cytotoxic after storage for 40, 80, and 120 days. Laser confocal microscopy revealed that HEK 293 cells adhered uniformly to the ready-to-use sodium alginate@paper material after storage for 40, 80, and 120 days, with no significant difference in adhesion compared with unstored ready-to-use sodium alginate@paper. These results demonstrate the excellent stability of the ready-to-use sodium alginate@paper material.

Key words: ready-to-use, sodium alginate hydrogel, paper, three-dimensional culture, freeze-drying, crosslinking agent, cytotoxicity, cell adhesion, porosity, biomaterial

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