用于生物人工肝反应器构建三维支架材料的选择与优化  

Screening and optimization of three-dimensional scaffolds for the construction of bioartificial liver bioreactor

作　　者：陈黎 黄蕾 余梦瑶 张国英 张世昌 Chen Li;Huang Lei;Yu Mengyao;Zhang Guoying;Zhang Shichang(Department of Obstetrics,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu Province,China;Department of Laboratory Medicine,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu Province,China;Department of Infectious Diseases,Southwest Hospital,Third Military Medical University,Chongqing 400038)

机构地区：[1]南京医科大学第一附属医院产科,江苏省南京市210029 [2]南京医科大学第一附属医院检验学部,江苏省南京市210029 [3]第三军医大学西南医院传染科,重庆市400038

出　　处：《中国组织工程研究》2021年第28期4429-4434,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81671836),项目负责人:张世昌。

摘　　要：背景:生物反应器是生物人工肝的核心部分,其性能直接关系到生物人工肝支持治疗的效果和作用。将支架材料应用于中空纤维性反应器中有望解决中空纤维型反应器对肝细胞培养支持不足的问题。目的:筛选可用于生物人工肝反应器构建的三维支架材料。方法:检测聚氨酯、壳聚糖、聚乳酸-聚羟乙酸酯支架材料的孔隙率。将HL-7702人肝细胞分别接种于3种支架上,培养4 h时,检测细胞接种率;培养24 h时,检测细胞活性与细胞内乳酸脱氢酶漏出量;培养3 d时,检测细胞内白蛋白合成量。根据上述实验结果选择聚氨酯进行以下实验:鼠尾胶原被覆组,以鼠尾胶原溶液浸泡材料后接种HL-7702人肝细胞悬液;纤维蛋白凝胶组,以含纤维蛋白原的培养液重悬HL-7702人肝细胞,随后接种于支架上;聚氨酯对照组,直接接种HL-7702人肝细胞悬液。培养1,3,5,7 d后,检测细胞活性、细胞内乳酸脱氢酶漏出量与白蛋白合成量。结果与结论:①3种支架均为多孔结构,聚氨酯和壳聚糖有较好的弹性,聚乳酸-聚羟乙酸酯质地较硬,聚氨酯和壳聚糖支架材料的孔隙率显著高于聚乳酸-聚羟乙酸酯(P<0.05);②聚氨酯内的细胞接种率高于聚乳酸-聚羟乙酸酯(P<0.05),低于壳聚糖(P<0.05);聚氨酯、壳聚糖内的细胞活性与白蛋白合成量高于聚乳酸-聚羟乙酸酯(P<0.05);3组间的细胞内乳酸脱氢酶漏出量比较差异无显著性意义(P>0.05);③纤维蛋白凝胶组的细胞分布较聚氨酯对照组、鼠尾胶原被覆组均匀,培养3,5,7 d的细胞活性与白蛋白含量高于聚氨酯对照组、鼠尾胶原被覆组(P<0.05),培养3,5,7 d的乳酸脱氢酶漏出量低于聚氨酯对照组、鼠尾胶原被覆组(P<0.05);④结果表明,聚氨酯支架材料经纤维蛋白凝胶优化培养可用于生物人工肝反应器的构建。BACKGROUND:Bioreactor is the core part of bioartificial liver,and its performance determines the effect of bioartificial liver support therapy.The application of scaffolds in hollow fiber bioreactor is expected to solve the problem of insufficient support for hepatocyte culture.OBJECTIVE:To screen three-dimensional scaffolds for the development of bioartificial liver reactor.METHODS:The porosity of polyurethane,chitosan and poly(DL-lactide-co-glycolide)scaffolds was detected.HL-7702 human hepatocytes were seeded on three kinds of scaffolds respectively,and the cell inoculation rate was detected at 4 hours;the cell viability and lactate dehydrogenase leakage were detected at 24 hours;and the intracellular albumin synthesis was detected at 3 days.According to the above experimental results,polyurethane was selected in the following experiment.In the rat tail collagen coating group,material was inoculated with rat tail collagen solution and then inoculated with HL-7702 human hepatocyte suspension.In the fibrin gel group,HL-7702 human hepatocytes were resuspended in fibrinogen culture medium,and then inoculated on the scaffold.In the polyurethane control group,material was inoculated with HL-7702 human hepatocyte suspension directly.After 1,3,5,and 7 days of culture,cell viability,lactate dehydrogenase leakage and albumin synthesis were detected.RESULTS AND CONCLUSION:(1)The three kinds of scaffolds were porous.Polyurethane and chitosan scaffolds had good elasticity,but poly(DL-lactide-coglycolide)scaffold was hard.The porosity of polyurethane and chitosan scaffolds was significantly higher than that of poly(DL-lactide-co-glycolide)scaffold(P<0.05).(2)The inoculation rate of cells in polyurethane scaffold was significantly higher than that in poly(DL-lactide-co-glycolide)scaffold(P<0.05),and significantly lower than that in chitosan scaffold(P<0.05).The cell viability and albumin synthesis of cells cultured in polyurethane and chitosan scaffolds were significantly higher than that of poly(DL-lactide-co-glycolide)scaf

关 键 词：材料 支架材料 三维支架 聚氨酯 生物反应器 生物人工肝 生物相容性 

分 类 号：R459.9[医药卫生—治疗学] R314[医药卫生—临床医学]