Effect of Heparin-conjugated Collagen Gel on the Formation of Hepatic Tissue by the Bottom-up Method Using Spheroids

Regenerative medicine involves the regeneration and repair of cells, tissues, and organs to restore health. To meet the requirements of clinical applications, it is necessary to construct high-density tissues with sufficient oxygen and nutrient supplies. We propose a bottom-up approach that uses hepatocyte spheroids coated with a heparin-conjugated collagen gel, using heparin as a growth factor immobilization agent, and co-cultures the spheroids with human umbilical vein endothelial cells (HUVECs) to form a vascular network structure for oxygen and nutrient supply. Briefly, we used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to crosslink heparin onto the collagen fiber, coated hepatocyte spheroids with the heparin-conjugated collagen gel, seeded the coated hepatocyte spheroids together with HUVECs in hollow fibers, and co-cultured them for 10 days. The heparin-conjugated collagen gel coated showed higher immobilization and slower basic fibroblast growth factor (bFGF) release rates than the collagen-only coated gel, confirming the capability of heparin as a growth factor binder and releaser. Hematoxylin-eosin staining of the tissue showed that this bottom-up approach facilitated formation of vascular-like structure. According to evaluations by the number of nuclei, albumin secretion and urea secretion, use of heparin-conjugated collagen gel in the formation of hepatocyte‐endothelial cell composite tissue contributed to the enhancement of cell survival rate and liver-specific functions of the tissue. The addition of a Rho-associated protein kinase (ROCK) inhibitor in the first 24 h of culture showed a notable reduction in apoptosis, but the ROCK inhibitor showed no significant effect on functional expression and induced different histological structures on day 10 of culture, indicating that ROCK inhibition is unsuitable for this bottom-up approach. These results are expected to contribute to the development of a bottom-up approach that allows simpler and more expeditious tissue engineering.