Experimental approaches to creating a tissue-specific matrix for a bioartificial liver

Abstract

Shortage of donor organs for liver transplantation in the treatment of end-stage liver disease dictates the need to develop alternative methods that include technologies on tissue engineering and regenerative medicine. Objective: to study the ability of a tissue-specific matrix from decellularized human liver fragments (DHLF) to maintain adhesion and proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and HepG2 under static conditions and in a flow-through bioreactor. Materials and methods. Treatment with surfactants (SAS) – sodium dodecyl sulfate, Triton X-100 – followed by exposure to DNase was used for decellularization of human liver fragments (no more than 8 mm3). Biochemical screening included the determination of DNA quantity in the test samples. Efficiency of surfactant washing was assessed by the cytotoxicity of the matrix in the NIH 3T3 fibroblast culture. Viability and metabolic activity of cells were assessed via vital staining with a complex of fluorescent dyes LIVE/DEAD ® and PrestoBlue™ (Invitrogen, USA). Morphological examination of the liver cell-engineered constructs was carried out through histological staining and scanning electron microscopy with lanthanide contrast. Results. It was shown that the liver decellularization method used allows to obtain a biocompatible matrix with a residual DNA quantity <1%, which is capable of maintaining adhesion and proliferation of hAT-MSCs and HepG2. On day 7 of cultivation in the bioreactor, there was formation of a single conglomerate of the DHLF matrix with numerous groups of viable cells with a high nuclear-cytoplasmic ratio. The urea content in the culture medium is 1.5 ± 0.1 mmol/L, exceeding that of samples obtained under static conditions. This indicates the metabolic activity of HepG2 in the composition of the obtained culture systems. It was shown that constant flow of the culture medium in the perfusion bioreactor increased the proliferative activity of HepG2 and allowed to provide a more uniform colonization by matrix cells in comparison with static cultivation conditions. Conclusion. The conditions for uniform colonization of DHLFs in a flow-through bioreactor with cell cultures were established. The ability of the matrix to maintain adhesion and proliferation of hADSCs and HepG2 for 11 days indicates that it could be used in liver tissue engineering.

Keywords

• liver
• decellularization
• mesenchymal stem cells
• hepg2
• cell-engineered construct
• tissue engineering
• bioreactor