Organization of liver organoids using Raschig ring-like micro-scaffolds and triple co-culture: Toward modular assembly-based scalable liver tissue engineering

In order to address the remaining issues of fragile structure and insufficient mass transfer faced in modular assembly-based liver tissue engineering, a Raschig ring-like hollowed micro-scaffold was proposed and fabricated using poly-ε-caprolactone with 60% porosity and 11.4 mm² effective surface area for cell immobilization. The method of cell inoculation, the types of cells for co-culture and the scalability of the proposed hollowed micro-scaffold in perfusion were all investigated to obtain an optimized organoid made of tissue modules. Extracellular matrix was found necessary to establish a hierarchical co-culture, and the triple co-culture of Human Hepatoma Hep G2 cells, liver sinusoid cell line TMNK-1 cells and fibroblasts (Swiss 3T3 cells) was recognized to be the most efficient to obtain higher cell attachment, proliferation and hepatic function. The equipped intersecting hollow channels provided in the micro-scaffold functioned as flow paths to promote mass transfer to the immobilized cells after the modules have been randomly packed into a bioreactor for perfusion culture, and resulted in enhanced albumin production and high cellular viability. Cell density comparable to those found in vivo were obtained in the perfused construct, which also maintained its rigid structure. Those results suggest that modular tissues made with hollowed micro-scaffold-based organoids hold great potential for scaling up tissue engineered constructs towards implantation.