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  • Modular assembly-based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering.

Modular assembly-based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering.

Annals of translational medicine (2020-12-15)
Jianyu He, Yuan Pang, Huayu Yang, Kevin Montagne, Marie Shinohara, Yilei Mao, Wei Sun, Yasuyuki Sakai
ABSTRACT

In liver tissue engineering, co-culturing hepatocytes with typical non-parenchymal hepatic cells to form cell aggregates is available to mimic the in vivo microenvironment and promote cell biological functions. With a modular assembly approach, endothelialized hepatic cell aggregates can be packed for perfusion culture, which enables the construction of large-scale liver tissues. Since tightly packed aggregates tend to fuse with each other and block perfusion flows, a loosely packed mode was introduced in our study. Using an oxygen-permeable polydimethylsiloxane (PDMS)-based microwell device, highly dense endothelialized hepatic cell aggregates were generated as hepatic tissue elements by co-culturing hepatocellular carcinoma (HepG2) cells, Swiss 3T3 cells, and human umbilical vein endothelial cells (HUVECs). The co-cultured aggregates were then harvested and applied in a PDMS-fabricated bioreactor for 10 days of perfusion culture. To maintain appropriate interstitial spaces for stable perfusion, biodegradable poly-L-lactic acid (PLLA) scaffold fibers were used and mixed with the aggregates, forming a loosely packed mode. In a microwell co-culture, Swiss 3T3 cells significantly contributed to the formation of hepatic cell aggregates. HUVECs developed a peripheral distribution in aggregates for endothelialization. In the perfusion culture, compared with pure HepG2 aggregates, HepG2/Swiss 3T3/HUVECs co-cultured aggregates exhibited a higher level of cell proliferation and liver-specific function expression (i.e., glucose consumption and albumin secretion). Under the loosely packed mode, co-cultured aggregates showed a characteristic histological morphology with cell migration and adhesion to fibers. The assembled hepatic tissue elements were obtained with 32% of in vivo cell density. In a co-culture of HepG2, Swiss 3T3, and HUVECs, Swiss 3T3 cells were observed to be beneficial for the formation of endothelialized hepatic cell aggregates. Loosely packed aggregates enabled long-term perfusion culture with high viability and biological function. This study will guide us in constructing large-scale liver tissue models by way of aggregate-based modular assembly.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling, Distributed for Phanos Technologies
Sigma-Aldrich
PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling, Distributed for Phanos Technologies
Supelco
GC Stationary Phase, phase Triton X-100, bottle of 50 g
Sigma-Aldrich
Triton X-100, laboratory grade