In vitro establishment of a three-dimensional hepatocyte culture system using collagen hydrogel as scaffolds / 中国组织工程研究

ABSTRACT

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.