Tissue vascularization in vitro is necessary for cell transplantation and is a major challenge in tissue engineering. To construct large and regularly vascularized tissue, we focused on the integration of endothelial cell-covered spheroids. Primary rat hepatocytes were cultured on a rotary shaker, and 100-150 mum spheroids were obtained by filtration. The hepatocyte spheroids were coated with collagen by conjugation with a type 1 collagen solution. Collagen-coated hepatocyte spheroids were cocultured with human umbilical vein endothelial cells (HUVECs), and monolayered HUVEC-covered hepatocyte spheroids were constructed. Without a collagen coat, many HUVECs invaded hepatocyte spheroids but did not cover the spheroid surface. To construct regularly vascularized tissue, we packed HUVEC-covered hepatocyte spheroids in hollow fibers used for plasma separation. Packed spheroids attached to each other forming a large cellular tissue with regular distribution of HUVECs. At day 9 after packing, HUVECs invaded the hepatocyte spheroids and a dense vascular network was constructed. Collagen coating of spheroids is useful for the formation of endothelial cell-covered spheroids and subsequent regular vascularized tissue construction.

A scaffold-free tissue construct was formed by assembling endothelial cell-covered spheroids, and medium perfusion through the tissue construct was investigated using hydrostatic pressure-driven culture circuit. Primary rat hepatocyte spheroids covered by human umbilical vein endothelial cells (HUVECs) were assembled in culture chambers with a cylindrical culture space of 2 mm in diameter, and then medium was perfused through the assembled spheroids for 48 hours. The medium flow rate through the culture chamber was measured over the perfusion culture time, which decreased during the first several hours, then increased or remained low depending on the amount of spheroids in the culture chamber. Histochemical analyses showed single tissue construct formation by spheroid fusion when cultured from 2×10 5 nuclei spheroids, with the loss of boundaries between the spheroids. Moreover, a viable cell region was found at the center of the tissue construct in several locations. Poor adhesion was found between spheroids cultured from 4×10 5 nuclei spheroids. The total nuclei density in cultured tissue constructs was estimated to be about half of that in HUVEC-covered hepatocyte spheroids. This study demonstrated the possibility of medium perfusion through scaffold-free tissue constructs by assembling endothelial cell-covered spheroids, promising for a 3 large tissue construct culture in vitro.

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Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

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