A Modified Method for Isolation of Hepatocyte

8/10/2019 A Modified Method for Isolation of Hepatocyte

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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 there-

fore 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 separa-

tion 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

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

A Modied Method for Isolation of

Rat Hepatocyte: Saving Time

Increases ViabilityMeltem Ate1, Ferda Alpaslan Pnarl2, Glnur Take Kaplanolu1, Meral Tiryaki2,

Sercan Mercan2, Davut Erdoan2, Glbahar Byk2, Zehra Frat2, Nilnur Eyerci2,Oya Topalolu3, Ahmet Yeilyurt2

1Department of Hstologyand Embryology, Faculty of Medcne,

Gaz Unversty Ankara, Turkey

2Clnc of Cell Research and GenetcDagnoss Center, Dkap Yldrm Beyazt

Tranng and Research Hosptal,Ankara, Turkey

3Department of Endocrnology andMetabolsm, Dkap Yldrm Beyazt Tranng

and Research Hosptal, Ankara, Turkey

Submtted : 04.03.2012Accepted : 14.04.2012

Correspondence: Dr. Ferda Alpaslan PnarlClnc of Cell Research and Genetc Dagnoss

Center, Dkap Yldrm Beyazt Tranng andResearch Hosptal, Ankara, Turkey

Phone: +90 312 596 20 00E-mal: [email protected]

Copyright 2012 by Cellular Therapy andRegenerative Medicine Society

Available on-line at www.nichejournal.org

Introduction

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 so-

phisticated technology and experienced support

teams in advanced centers (1). Furthermore, the need

for life-long immunosuppression, with its known sideeffects, is a medical limitation of this therapy.

Hepatocyte transplantation is a potential alter-

native to liver replacement in humans. The re-

construction of functional parenchyma by trans-

planted hepatocytes supports the function of the

injured liver. Several experimental studies involv-

ing 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 trans-

planted hepatocytes requires time, during which

donor cells proliferate, differentiate into fully func-tioning cells in vivoand then establish a normal pa-

renchymal architecture (3).

Although hepatocyte transplantation has not yet

been established as a reliable alternative to liver

transplantation, animal studies seriously contribut-

ed to the understanding of the process of prolifera-

tion, engraftment, and regeneration after hepato-

cyte 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 envi-

ronment in which the cells are more comparableto 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 viabil-

ity 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 modi-

fying the standard isolation method of Seglen (5).


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9Ate et al. A Modied Method for Isolation of Rat HepatocyteNiche 2012; 1: 8-11

Method

Ten adult male Wistar Albino rats (180-200g) obtained from the

Dkap Yldrm Beyazt Training and Research Hospital, Cell Re-

search 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. Colla-genase, NaCl, KCl, CaCl

2-H

2O, NaHCO

3,HEPES, Bowin Serum Albu-

min, DMEM, and D-glucose were purchased from Sigma Chemi-

cals 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 modify-

ing 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 suspen-

sions were used in the study: The washing solution was obtained

with 3.5g/500 mLNaCl; 0.02g/500 mL KCl; 0.48g/500 mL CaCl2-H2O; 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

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 anesthe-

sia, 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 can-nula and enzyme solution was infused via the portal vein in a sep-

arate 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 centrifu-

gated 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 num-

ber 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% formal-dehyde for 30 min., oxidized within periodic acid for 5 min., pro-

cessed with Schiffs reagent for 15 min., and washed with dH2O

for 10-15 min. before microscopic examination (Fig. 3).

Seglen Method

The details of the physical and chemical parameters of this tech-

nique were described by Seglen in 1976 (5). This procedure, de-

scribed in a simple summary form here, still remains the goldFigure 1. Isolated hepatocytes morphologically hexagonal with a cen-

trally located big nucleus or double nuclei

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

A B


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10 Ate et al. A Modied Method for Isolation of Rat Hepatocyte Niche 2012; 1: 8-11

standard. The liver is resected with sterile surgical tools, trans-

ferred into a sterile beaker and 20 mL of ice-cold perfusion buf-

fer (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 filteredby 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 undi-

gested 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 grav-

ity 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 nonparenchy-

mal cells, e.g. endothelial cells, Ito cells, bile duct cells, and cells

from the mesothelial capsule) is decanted. This process is repeat-

ed three times altogether. The final cell pellet predominantly con-

tains hepatocytes (90%, as originally described). Hepatocytes are

78% viable The hepatocyte pellet contains approximately 40 mil-

lion 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 au-

tomated procedures such as single sorting.

Results

The mean cell count was4x107/mL and the mean viability was 78%

for thefirst 5 ratstreated with the standard two-step enzyme isola-

tion 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 characteriza-

tion 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 collage-

nase 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 cul-ture environment. Cultured adult and fetal hepatocytes can be

used in the understanding of the liver differentiation and regen-

eration mechanisms and drug toxicity studies in vitro,as well as

in acute and chronic liver failure for life support until transplanta-

tion, 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 re-

sults. All crude collagenase preparations can contain lot-variable

contaminating proteases, esterases and other enzymes requiring

researchers to pre-screen several lots of enzyme and/or continu-

ally 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% colla-genase 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 me-

dium 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 inhib-

ited 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 thanthat 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 hepa-

tocytes in a shorter time period with our modified method com-

Figure 3. PAS staining (+) hexagonal cells

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%

Table 1. The cell count, viability, purity and isolation time of the hepa-

tocytes with the Seglen method and our modified method


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

the increased cell viability is related to the shorter isolation dura-

tion (p

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A Modified Method for Isolation of Hepatocyte