Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (29): 5323-5330.doi: 10.3969/j.issn.2095-4344.2013.29.010

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

Wang Min1, Lan Ya2, Hu Hao1, Shi Yong-quan1, Han Ying1, Zhou Xin-min1   

  1. 1State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University of PLA, Xi’an  710032, Shaanxi Province, China; 2Department of Gastroenterology, Yan’an University Affiliated Hospital, Yan’an  716000, Shaanxi Province, China
  • Received:2013-03-19 Revised:2013-04-14 Online:2013-07-22 Published:2013-07-22
  • Contact: Zhou Xin-min, M.D., Professor, Chief physician, State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University of PLA, Xi’an 710032, Shaanxi Province, China zhouxm128@fmmu.edu.cn
  • About author:Wang Min★, Studying for master’s degree, State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University of PLA, Xi’an 710032, Shaanxi Province, China wangminfmmu@126.com
  • Supported by:

    the Science and Technology Innovation Engineering Plan of Shaanxi Province, No. 2012KTCL03-04*

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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 collagen 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 Ⅰcollagen solution that was premixed with hepatocyte growth factor and Dulbecco's modified Eagle’s medium to create hepatocyte/collagen hydrogel compounds. The compounds were inoculated into a 96-well 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 cell supernatant was collected 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 collagen 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-4α positive 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 collagen 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.

Key words:  biomaterials, biomaterials and controlled drug release, extracellular matrix materials, hepatocyte, collagen type Ⅰ, hydrogels, cell culture, synthesis and metabolism, three-dimensional culture, liver tissue engineering, provincial grants-supported paper

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