Hepatocyte Culture in Autologous Decellularized Spleen Matrix

abstract

Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 ± 6.34 μg/10⁷cell/24h in dynamic culture in DSM compared to 62.43 ± 4.59 μg/10⁷cell/24h in sandwich culture (P < 0.01) and to 87.54 ± 5.25 μg/10⁷cell/24h in DLM culture (P > 0.05); urea release was 32.14 ± 8.62 μg/10⁷cell/24h in dynamic culture in DSM compared to 20.47 ± 4.98 μg/10⁷ cell/24h in sandwich culture (P < 0.05) and to 37.38 ± 7.29 μg/10⁷cell/24h cultured in DLM (P > 0.05). Conclusion: The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture.

Keywords:

• decellularized spleen matrix
• hepatocytes culuture
• liver
• spleen
• tissue engineering

Funding

This study was supported by grants from the Research Fund for the Doctoral Program of Higher Education of China (20110201130009) and the Science and Technology Research and Development Program of Shaanxi Province (2012KW-39–01).