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Niche, 2012; 1: 8-11 doi : 10.5152/niche.2012.02

A Modified Method for Isolation of

Rat Hepatocyte: Saving Time
Increases Viability
Meltem Ate1, Ferda Alpaslan Pnarl2, Glnur Take Kaplanolu1, Meral Tiryaki2,
Sercan Mercan2, Davut Erdoan2, Glbahar Byk2, Zehra Frat2, Nilnur Eyerci2,
Oya Topalolu3, Ahmet Yeilyurt2
Original Investigations

Abstract
Objective: Hepatocyte transplantation is a potential alternative to liver replacement in humans. Several methods for
hepatocyte isolation in animal models have been published, many of these require extensive handling and can therefore compromise the viability and function of the isolated cells. The aim of this study is to isolate excessive amount of rat
hepatocytes with high viability in a short time period by modifying the standard isolation method of Seglen.
Methods: The hepatocyte isolation was performed using thetwo-step enzymatic method of Seglen in 5 rats. The hepatocyte
isolation in the remaining 5 rats was realized using a method modifying the amount of solutions, infusion methods and separation systems shortening process steps. The cells were counted, stained with trypan blue, FDA and PI for viability.
Results: We observed an increase in cell count and viability of the hepatocytes in a shorter time period with our modifi
ed method compared to the standard two-step enzyme method. We suggest that the increased cell viability is related
to the shorter isolation duration.
Conclusions: We propose using our modifi ed isolation method in a hepatocyte isolation procedure in which obtaining
excess cells with high viability is of critical importance.
Key words: Hepatocyte Isolation, modified method, rat

Introduction

1
Department of Histology
and Embryology, Faculty of Medicine,
Gazi University Ankara, Turkey
2
Clinic of Cell Research and Genetic
Diagnosis Center, Dkap Yldrm Beyazt
Training and Research Hospital,
Ankara, Turkey
3
Department of Endocrinology and
Metabolism, Dkap Yldrm Beyazt Training
and Research Hospital, Ankara, Turkey

Submitted : 04.03.2012
Accepted : 14.04.2012
Correspondence: Dr. Ferda Alpaslan Pnarl
Clinic of Cell Research and Genetic Diagnosis
Center, Dkap Yldrm Beyazt Training and
Research Hospital, Ankara, Turkey
Phone: +90 312 596 20 00
E-mail: ferdapinarli@yahoo.com
Copyright 2012 by Cellular Therapy and
Regenerative Medicine Society
Available on-line at www.nichejournal.org

The liver performs diverse functions required by

the organism, namely metabolism, detoxification
and synthesis. These complex processes occur to a
large extent in parenchymal cells. Severe liver failure
is associated with a poor prognosis and only liver
transplantation can compensate hepatic functions.
However, liver transplantation is a high cost, time
consuming and complicated procedure because of
limited donor availability and requirements for sophisticated technology and experienced support
teams in advanced centers (1). Furthermore, the need
for life-long immunosuppression, with its known side
effects, is a medical limitation of this therapy.
Hepatocyte transplantation is a potential alternative to liver replacement in humans. The reconstruction of functional parenchyma by transplanted hepatocytes supports the function of the
injured liver. Several experimental studies involving transplantation of normal mature hepatocytes
have achieved important therapeutic goals in a
variety of metabolic liver diseases (2). However, the
reconstruction of functional parenchyma by transplanted hepatocytes requires time, during which

donor cells proliferate, differentiate into fully functioning cells in vivo and then establish a normal parenchymal architecture (3).
Although hepatocyte transplantation has not yet
been established as a reliable alternative to liver
transplantation, animal studies seriously contributed to the understanding of the process of proliferation, engraftment, and regeneration after hepatocyte transplantation. Primary mouse hepatocytes
are an important tool in the biomedical research
field for the assessment of hepatocyte function.
The use of freshly isolated cells provides an environment in which the cells are more comparable
to their in vivo state. Although several methods
for hepatocyte isolation in animal models have
been published, many of these require extensive
handling and can therefore compromise the viability and function of the isolated cells. It is of critical
importance to have robust methods that produce
excessive amount of cells with high viability, good
purity and which function in a similar manner to
that in their in vivo state (4). The aim of this study
is to isolate excessive amounts of rat hepatocytes
with high viability in a short time period by modifying the standard isolation method of Seglen (5).

Niche 2012; 1: 8-11

Method
Ten adult male Wistar Albino rats (180-200g) obtained from the
Dkap Yldrm Beyazt Training and Research Hospital, Cell Research and Genetic Diagnosis Center were used in this study. The
rats were housed at a constant room temperature of 22C and
had free access to standard laboratory diet and tap water. Collagenase, NaCl, KCl, CaCl2-H2O, NaHCO3, HEPES, Bowin Serum Albumin, DMEM, and D-glucose were purchased from Sigma Chemicals Co. (Poole, UK). RT- PCR kit was obtained Qiagen (Germany).
The hepatocyte isolation was performed using the two-step
enzymatic method of Seglen in 5 rats. The hepatocyte isolation
in the remaining 5 rats was achieved using a method modifying the amount of solutions, infusion methods and separation
systems shortening process steps. The cells were counted in an
automated cell counter (Countess, Invitrogen, USA) and stained
with trypan blue, FDA and PI for viability. Three different suspensions were used in the study: The washing solution was obtained
with 3.5g/500 mLNaCl; 0.02g/500 mL KCl; 0.48g/500 mL CaCl2H2O; 5 mL HEPES; 1g/500 mL Bowin Serum Albumin dissolved in
DMEM. The perfusion solution was prepared with 0.9g/100 mL
NaCl; 0.04g/100 mL KCl; 0.09g/100 mL D-glucose; 0.21g/100 mL

Ate et al. A Modified Method for Isolation of Rat Hepatocyte

NaHCO3; and 2 mL HEPES DMEM. The enzyme solution consisted

of 0.007g/30 mL type 4 collagenase and perfusion solution.
The abdomens of the rats were opened under general anesthesia, a cannula was inserted into the portal vein and the perfusion
solution was slowly infused at 37C. After the discoloration of the
liver (from dark red to pink), the liver was displaced with the cannula and enzyme solution was infused via the portal vein in a separate environment. After the incubation period of 30 min. with
enzyme solution at 37C, the liver was cut into small pieces by a
surgical blade and filtrated from 210 m, 70 m and 40 m pores.
The supernatant was discarded after the samples were centrifugated with washing solution at 150 g for 3 min. and the pellet
was washed. The separation of the dead cells from the living ones
was performed with 10.8 mL percoll and 15mL DMEM gradient
and centrifugation at 2000 rpm for 20 min. The medium layer was
collected and washed twice at 150 g for 3 min., in order to purify
from percoll. The isolated hepatocytes were almost completely
(97%) morphologically hexagonal with a centrally located big
nucleus or double nuclei (Fig. 1).
Viability
Viability of cell suspension was determined using the trypan blue
dye exclusion test. A sample of hepatocytes suspension (0.25 mL)
was mixed with trypan blue (0.1 mL; 0.4% dye solution). A Cell
Countess system (Invitrogen, USA) was used to count the number of viable and non-viable cells and the percentage viability
was calculated. Suspensions with a viability of 95% were used for
tests (Figure 2A). The other viability test FDA and PI were used in
the fluorescent microscope (Fig. 2B).
PAS Staining
PAS staining was performed to show cell activity. Isolated cells
were washed with PBS three times and fixed within 4% formaldehyde for 30 min., oxidized within periodic acid for 5 min., processed with Schiffs reagent for 15 min., and washed with dH2O
for 10-15 min. before microscopic examination (Fig. 3).

Figure 1. Isolated hepatocytes morphologically hexagonal with a centrally located big nucleus or double nuclei

Seglen Method
The details of the physical and chemical parameters of this technique were described by Seglen in 1976 (5). This procedure, described in a simple summary form here, still remains the gold
B

Figure 2. A) Viability of cell suspension was trypan blue dye (-), B) FDA and PI were used in the fluorescent microscope

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Ate et al. A Modified Method for Isolation of Rat Hepatocyte

standard. The liver is resected with sterile surgical tools, transferred into a sterile beaker and 20 mL of ice-cold perfusion buffer (Ca-free) is added. The liver is minced with sterile, long, loose
scissors. The scissors should not be tight, since they may damage
cells as they are released. The mechanical mincing by the loose
scissors releases the hepatocytes (singlet or duplets). After the
liver is minced for 1-2 min, the suspension of the cells is filtered
by pouring over a beaker covered by a Nitex filter of 100 in
pore diameter (Polyamide nylon mesh filter, Tecto, Briarcliff, NY).
Hepatocytes and other cells enter through the pores, but undigested tissue is retained. The hepatocyte suspension is kept on
ice throughout the whole process. The suspension is centrifuged
in (typically) 50-mL sterile plastic conical tubes at very low gravity conditions (50 g). If not centrifuged, hepatocytes settle to the
bottom of the tube within about 10 min in unit gravity, because
of their large size and weight compared to the other cells. The
hepatocyte pellet is collected on the bottom of the conical tube,
but the supernant (containing the much smaller nonparenchymal cells, e.g. endothelial cells, Ito cells, bile duct cells, and cells
from the mesothelial capsule) is decanted. This process is repeated three times altogether. The final cell pellet predominantly contains hepatocytes (90%, as originally described). Hepatocytes are
78% viable The hepatocyte pellet contains approximately 40 million rat hepatocytes/cc of packed pellet (at 50g centrifugation).
This is the most commonly used approach to count hepatocytes,
since the isolated cells are present mostly as cell doublets and
triplets, and rarely as single cells, thus eliminating the use of automated procedures such as single sorting.

Results
The mean cell count was 4x107/mL and the mean viability was 78%
for the first 5 rats treated with the standard two-step enzyme isolation method in 4 hours. PAS staining confirmed the presence of
the hepatocytes in this method. The mean cell count was 6x107/
mL and the mean viability was 95% for the remaining 5 rats treated
with our modified method in 3 hours. The hepatocyte characterization was again performed with PAS staining (Table 1).

Discussion
The hepatocyte isolation studies was started with Howard and
Pesch who obtained living functional hepatocytes with collagenase from adult liver 25 years previously (5). After many studies,
the two-step collagenase perfusion method described by Seglen
became the gold standard and was then modified by Dunn (5, 6).
The hepatocytes maintain their specific liver functions in the culture environment. Cultured adult and fetal hepatocytes can be
used in the understanding of the liver differentiation and regeneration mechanisms and drug toxicity studies in vitro, as well as
in acute and chronic liver failure for life support until transplantation, as an artificial liver (6).
Most traditional methods published for isolating hepatocytes
use crude and partially purified enzyme preparations including
various types of collagenase and other proteases. More recently,
the use of better characterized collagenase preparations such as
Worthington Types 1 and 4 (CLS-1, 4) have provided better results. All crude collagenase preparations can contain lot-variable

Niche 2012; 1: 8-11

Figure 3. PAS staining (+) hexagonal cells

Table 1. The cell count, viability, purity and isolation time of the hepatocytes with the Seglen method and our modified method

Cell Count Viability PAS Isolation Purety

(mean) (mean) Stain Time (mean)

Seglen method

4x107/mL 78% (+) 4h 90%

Our method

6x107/mL 95% (+) 3h 97%

contaminating proteases, esterases and other enzymes requiring

researchers to pre-screen several lots of enzyme and/or continually modify isolation parameters and protocols.
The technique for hepatocyte isolation is based on the two-step
collagenase perfusion technique first developed by Berry and
Friend for the isolation of rat hepatocytes (7, 8). The perfusion
medium that was found most suitable by Howard and Pesch
comprised calcium free Hanks solutions containing 0.05% collagenase type 1 and 0.10% hyaluronidase (9). In some experiments,
an additional step which appeared to bring further improvement
in cell yield was included. After perfusion of the liver until it was
of soft consistency, the enzyme medium was replaced with a medium comprising calcium and magnesium-free Hanks solution
containing 2 nm EDTA, pH 7.4. EDTA and collagenase could not be
perfused simultaneously, since the enzyme was strongly inhibited by EDTA (9, 10). Preparation of isolated cells by continuously
recirculation of the enzyme medium through the liver followed
by perfusion with EDTA invariably gave yields at least 6 times as
great as those obtained by Howard and Pesch and sometimes
represented a conversion of over 50% of the liver to isolated
cells. The viability of the cells was also substantially higher than
that reported by Howard and Pesch, as demonstrated by the low
percentage stained by trypan blue (9). In other studies, the liver
perfusion and collagenase infusion were achieved with a pump
mechanism (10). In our study, we slowly injected the solution via
a syringe instead of using the pump mechanism. We showed that
the liver was perfectly perfused and the collagenase process was
carried out without any problem with this method.

Conclusion
We observed an increase in cell count and viability of the hepatocytes in a shorter time period with our modified method com-

Niche 2012; 1: 8-11

pared to the standard two-step enzyme method. We suggest that

the increased cell viability is related to the shorter isolation duration (p<0.05). We propose using our modified isolation method in
a hepatocyte isolation procedure in which obtaining excess cells
with high viability is of critical importance.
Conflict of interest: No conflict of interest was declared by the
authors.

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Effective Hepatocyte Transplantation Using Rat Hepatocytes with
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