以胶原水凝胶为支架构建肝细胞三维培养系统

In vitro establishment of a three-dimensional hepatocyte culture system using collagen hydrogel as scaffolds

背景:肝细胞体外培养时快速失去功能限制了肝细胞疗法的发展。目的:以胶原水凝胶为支架建立能够长时间有效维持肝细胞功能的肝细胞三维培养系统。方法:将SD大鼠肝细胞与预混肝细胞生长因子及DMEM的液态Ⅰ型胶原混合,形成肝细胞/胶原凝胶复合物,将该复合物接种至培养板,待形成胶冻状凝胶后再加培养液进行培养。通过光学显微镜、苏木精-伊红染色及透射电子显微镜对肝细胞的形态学特征及超微结构进行观察;并通过糖原染色、免疫荧光染色、实时定量PCR进一步对肝细胞特异表型及功能进行检测。收集培养上清,检测细胞白蛋白及尿素合成能力。结果与结论:①肝细胞/胶原水凝胶复合物形成后,可见肝细胞呈圆形分布于水凝胶中,培养14d后仍保持肝细胞形态并呈类似肝脏结构的肝索样聚集排列,超微结构观察显示肝细胞之间形成紧密连接。②培养14d后,糖原染色、白蛋白及肝细胞核因子4α免疫荧光染色呈阳性,证实肝细胞具有糖原及白蛋白合成能力并仍具有分化潜能。③三维培养中,肝细胞的白蛋白及尿素合成能力及分泌水平明显高于二维培养,且至少能够在较高水平维持15d。④培养7d后,Albumin、HNF-4α、Claudin-3、CYP1A1、CYP3A1以及G6P等肝细胞特异性基因的表达水平明显高于二维培养。以上结果证实,以胶原水凝胶为支架构建的三维培养系统使肝细胞在结构上更类似肝脏组织,并能在更长时间内有效的维持肝细胞合成代谢等各方面功能。

BACKGROUND：Rapid loss of liver-specific functions of the cultured hepatocytes limits the development of hepatocyte-based therapies. OBJECTIVE：To establish a three-dimensional culture system based on col agen hydrogel that enables to enhance liver-specific functions for a long period during culture of hepatocytes. METHODS：Hepatocytes were isolated from Sprague-Dawley rats and then encapsulated into liquid type Ⅰcol agen solution that was premixed with hepatocyte growth factor and Dulbecco＇s modified Eagle’s medium to create hepatocyte/col agen hydrogel compounds. The compounds were inoculated into a 96-wel plate. After gelation, culture medium was added. Light microscope, hematoxylin-eosin staining and transmission electron microscopy were used to examine the morphological characteristics and ultrastructure of the cultured hepatocytes. The cellsupernatant was col ected and tested for albumin secretion and urea synthesis. Periodic acid-schiff staining, immunofluorescence staining and quantitative real-time PCR were also used to further clarify liver-specific phenotype or function of the hepatocytes.RESULTS AND CONCLUSION：（1） Light microscope revealed that hepatocytes were round shape and distributed uniformly in col agen hydrogel. The three-dimensional hepatocyte culture system exhibited similarities to liver-like structure and tight junction were formed between hepatocytes after 14 days of culture. （2） Within the three-dimensional culture system, hepatocytes remained positive for periodic acid-schiff staining, albumin and hepatocyte nuclear factor-4αpositive after 14-day culture, which provided the convincing evidence of highly differentiated primary hepatocytes with functions of glycogen and albumin synthesis. （3） The albumin and urea productions in the three-dimensional culture system had a significantly higher level than in the two-dimensional culture, and could remain at a high level at least for 15 days. （4） The expression levels of hepatocyte-specific genes including Albumin, HNF-4α, Claudin-3, CYP1A1, CYP3A1 and G6P were significantly improved in the three-dimensional culture as compared with the two-dimensional culture. The col agen hydrogel based three-dimensional culture system provides a valuable model to enhance the hepatocyte functional maintenance and lay the foundation for the development of hepatocyte-based therapy for liver disease.