Spontaneous hepatocyte migration towards an endothelial cell
Department of Surgery, Nagasaki University
Graduate School of Biomedical Sciences,
Department of Surgery, A. N. Syzganov's
National Scientific Center of Surgery, Almaty,
Susumu Eguchi, Department of Surgery,
Nagasaki University Graduate School of
Biomedical Sciences, 1‐7‐1 Sakamoto,
Nagasaki 852‐8501, Japan.
The Uehara Memorial Foundation to Y. Sakai;
Grants‐in‐Aid for Young Scientists (A) to Y.
Sakai (no. 16H05908) from the Ministry of
Education, Culture, Sports, Science, and Tech-
nology of Japan; Grants‐in‐Aid for Scientific
Research (C) to S. Eguchi (no. 26461916) from
the Ministry of Education, Culture, Sports,
Science, and Technology of Japan
A crucial part of the engineering liver tissue is contribution of nonparenchymal cells and maintenance
of a complex three‐dimensional (3D) structure in vitro for their normal physiology and function. We
generated 3D hepatic tissue using primary isolated rat hepatocytes and an endothelial cell tube net-
work from human endothelial vein epithelial cells (HUVECs). To create the 3D hepatic tissue, cocul-
ture of primary hepatocytes and tube‐structured HUVECs was performed on a Matrigel®. After the
HUVECs formed the tube structures, primary isolated rat hepatocytes were inoculated onto the
HUVEC tube‐structured layer and cultured for 24 hr. We investigated the cell migration, cellular
interaction, and distributions of HUVEC tube structures and hepatocytes using multi cell‐imaging
incubator, confocal microscopy, and electron microscopy analyses. During the culture time, time‐
lapse imaging showed spontaneous migration of the hepatocytes in the gel, and after the 24‐hr cul-
ture period, the vast majority of the hepatocytes had moved and adhered to the surface of the
HUVEC tube structures. A confocal microscopy assay confirmed this unique 3D cellular interaction
between hepatocytes and HUVEC tube structures. The hepatocytes were able to maintain their
spherical shape, as well as HUVECs (tube‐like form with tubular cavity). We speculate that
coculturing of hepatocytes and endothelial cells replicates part of their normal physiology and may
help induce migration in vitro and the growth of complex biological tissue structures.
endothelial cells, hepatic tissue, hepatocytes, tube structure
Liver transplantation is the only established treatment for patients
with end‐stage liver failure. However, due to the shortage of donors,
alternative treatments are needed. Hepatic tissue engineering has
been established as a promising alternative to liver transplantation
(Kim, Utoh, Ohashi, Kikuchi, & Okano, 2015; Lu et al., 2005).
However, there remain unresolved issues before this type of
treatment can be widely introduced in clinical practice (Zhang et al.,
In the current study, we generated three‐dimensional (3D) hepatic
tissue using primary isolated rat hepatocytes and an endothelial cell
tube network from human endothelial vein epithelial cells (HUVECs).
Interestingly, we observed unique migration and attachment patterns
of the hepatocytes to the formed HUVEC tube structures. Our
approach may advance tissue engineering strategies for studying 3D
vascularized hepatic tissues.
MATERIALS AND METHODS
We used male Wistar rats (7–8 weeks, weighing 160–200 g) purchased
from Kyudo (Tosu, Japan) as donors of primary hepatocytes. The rats
were housed in a temperature‐controlled environment with a 12‐hr
light/dark cycle and given free access to standard rat chow and water.
In all procedures, the rats were anesthetized with either 2%–2.5%
isoflurane inhalation or the intraperitoneal injection of pentobarbital
sodium (1 ml/kg). All experiments were performed according to the
Guidelines for Animal Experimentation at Nagasaki University.
An HUVECs (Cell Applications, Inc., San Diego, CA, USA) cell suspen-
sion was prepared from cryopreserved vials immediately after thawing
Received: 5 March 2017 Revised: 28 August 2017 Accepted: 12 September 2017
J Tissue Eng Regen Med. 2018;12:e1767–e1771. Copyright © 2017 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/term e1767