Repopulating a decellularized liver scaffold with liver-derived organoids in a perfusion-based bioreactor

Abstract

Over the last decade new methods are explored in the field of tissue engineering to minimize the donor organ shortage. Engineering organs with a complex structure and large vascular network, such as the liver, remain a challenge. Luckily, the decellularization of an organ creates a scaffold that consists of the extracellular matrix (ECM) with important growth factors, bifunctional molecules such a fibronectin and multiple collagen types. This ECM provides the biophysical and biochemical cues needed for cells to adhere, proliferate and differentiate. However, there is no optimal method yet to recellularize such a decellularized liver scaffold. This project shows that it is possible to use a perfusion-based bioreactor for repopulating a porcine liver scaffold with liver-derived organoids. In the bioreactor, the Harvard Apparatus (Hugo Sachs Elektronik), decellularized porcine liver segments were infused with a HepG2 cell line and liver-derived organoids in seperate experiments. A setback in the project was the proneness to infections in the Harvard Apparatus (HA), which shortened the duration of experiments and influenced the results. The experiments were analyzed by histological and immunochemical staining and by qPCR. The HepG2 cell line validated the set up and recellularization with the HA, the cells engrafted throughout the scaffold and showed viability and signs of proliferation. The liver-derived organoids were successfully cultured and expanded in spinnerflasks, and were found engrafted and alive after 10 days in the scaffold. The qPCR data showed variability between the different organoid lines and between the different phases of the organoid culture. The results combined of this project are promising for future research, especially regarding the use of liver-derived organoids for recellularization.