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Introduction: In the last two decades research has been focused on the development of biologically-derived matrices for reconstructive medicine[1]. Decellularized tissues from a xenogeneic donor represent a growth-friendly environment for cells of different species, including human, lacking most of the immunogenic components[2]. Decellularized porcine liver resembles the human counterpart and could be considered for reconstruction of human liver tissue in vitro. Thus there is a high need for bioreactor development where the effective cell repopulation can be achieved using a good quality scaffold.
Methodology: A bioreactor system was designed and constructed for perfusion cell culture in pig liver scaffold pieces with approx. volume 3 cm3. Two types of custom-made chambers with inserts holding four scaffolds individually fed with medium were developed. The circulation of oxygenated medium from a special bottle was enabled by peristaltic pumps. Porcine livers were decellularized with consecutive cycles of 1% Triton X-100 and 1% sodium dodecyl sulphate (SDS) solutions[3]. Then the scaffold was cut and tested in the bioreactor for human cell adhesion and proliferation. HepG2 cells were injected in the scaffold matrix at seeding density of 10^6/cm3, and then cultivated in the bioreactor for 1, 3 and 7 days. Fixed scaffold sections were stained with hematoxylin-eosin to monitor the efficiency of the cell repopulation.
Results: Completely decellularized porcine liver scaffolds with a well preserved matrix showed presence of HepG2 cells in all tested recellularization experiments. This includes both bioreactor chambers as well as all three time points. After one day of incubation, the cells were visible only at the injection site and in adjacent vascular ECM structures. After 3 and 7 days of culture the cells proliferated and started to migrate into the area surrounding the injection site. Some cells were also detected on the surface of the scaffold; these cells were probably released from scaffold into medium during the early perfusion and adhered later. Living cells detected inside the matrix were also indicative of the removal of any residual SDS content after the decellularization. It is known that even very low SDS concentrations can hamper the repopulation efforts, thus washing out the detergent residues is important.
Conclusions: We proved that both bioreactors can support cell adhesion and proliferation in perfusion 3D culture, thus they can be used for the repopulation of small decellularized liver pieces. Moreover, our models can be adapted to different types of tissues to verify the initial potential of the matrix to support cell growth. The ability to repopulate these small decellularized pieces is the first step to proceed firstly with lobules, in the case of decellularized liver scaffold, and then with the entire organ.
References/Acknowledgements:
[1] Song J.J. and Ott H.C., Trends Mol. Med., 17(8), 424–432 (2011).
[2] Massaro M.S. et al., Mater. Sci. Eng. C, 127 (2021).
[3] Moulisová V. et al., J. Tissue Eng., 11 (2020).
The work was supported from European Regional Development Fund-Project „AMTMI” (No. CZ.02.1.01/0.0/0.0/17_048/0007280), grant UNCE/MED006 Center of Excellence (Charles University "Center of Clinical and Experimental Liver Surgery").
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