Optimization of transfection methods for Huh7 and Vero cells: a comparative study
Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electrop...
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| Дата: | 2012 |
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Інститут клітинної біології та генетичної інженерії НАН України
2012
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Optimization of transfection methods for Huh7 and Vero cells: a comparative study / A. Hashemi, F. Roohvand, M.H. Ghahremani, M.R. Aghasadeghi, R. Vahabpour, F. Motevali, A. Memarnejadian // Цитология и генетика. — 2012. — Т. 46, № 6. — С. 19-27. — Бібліогр.: 37 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860268465094393856 |
|---|---|
| author | Hashemi, A. Roohvand, F. Ghahremani, M.H. Aghasadeghi, M.R. Vahabpour, R. Motevali, F. Memarnejadian, A. |
| author_facet | Hashemi, A. Roohvand, F. Ghahremani, M.H. Aghasadeghi, M.R. Vahabpour, R. Motevali, F. Memarnejadian, A. |
| citation_txt | Optimization of transfection methods for Huh7 and Vero cells: a comparative study / A. Hashemi, F. Roohvand, M.H. Ghahremani, M.R. Aghasadeghi, R. Vahabpour, F. Motevali, A. Memarnejadian // Цитология и генетика. — 2012. — Т. 46, № 6. — С. 19-27. — Бібліогр.: 37 назв. — англ. |
| collection | DSpace DC |
| container_title | Цитология и генетика |
| description | Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± ± 2.36 and 73.9 ± 1.6 % of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was respectively 14.2 ± 0.69 and 28 ± 1.11 % for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.
Наличие эффективного протокола трансфекции является первым условием успешных исследований по переносу генов в клетки млекопитающих, что достигается экспериментально для каждого конкретного типа клеток. Здесь мы приводим данные сравнительного исследования по оптимизации условий трансфекции клеток Huh-7 и Vero с помощью электропорации и химическими методами. Для двух химических соединений, jetPEI™ и Lipofectamine™ 2000, были оптимизированы сочетания различных клеток, соотношения ДНК/реагент и общие объемы трансфекции. Кроме того, для улучшения эффективности электропорации было изучено влияние силы электрического поля и длины импульса. Трансфекция клеток с помощью вектора pEGFP-N1, определение экспрессии GFP с помощью FACS и флюоресцентная микроскопия были использованы для оценки эффективности трансфекции. В оптимизированных протоколах достигалась трансфекция на уровне 63.73 ± 2.36 и 73.9 ± 1.6 % в клетках Huh-7 и Vero соответственно, в то время как максимальный уровень трансфекции с помощью jetPEI™ составлял 14.2 ± 0.69 и 28 ± 1.11 % для тех же клеток. Охлаждение клеток после трансфекции не улучшало эффективность электротрансфекции клеток Huh-7. В обеих клеточных линиях электропорация позволила достичь более высокого уровня трансфекции по сравнению с использованием химических реагентов. Представленный протокол может быть пригодным для большинства экспериментальных манипуляций, которые требуют высокого уровня трансфекции исследуемых клеточных линий.
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| fulltext |
19ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
A. HASHEMI 1, F. ROOHVAND 1, 2, M.H. GHAHREMANI 3,
M.R. AGHASADEGHI 1, R. VAHABPOUR 1, F. MOTEVALI 1, 2,
A. MEMARNEJADIAN 1
1 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran
2 NRGB, Pasteur institute of Iran, Pasteur Ave., Tehran
3 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran
University of Medical sciences
E-mail: rfarzin@pasteur.ac.ir
OPTIMIZATION OF TRANSFECTION
METHODS FOR Huh-7 AND VERO
CELLS: A COMPARATIVE STUDY
Availability of an efficient transfection protocol is the
first determinant in success of gene transferring studies in
mammalian cells which is accomplished experimentally
for every single cell type. Herein, we provide data of a
comparative study on optimization of transfection con-
dition by electroporation and chemical methods for
Huh-7 and Vero cells. Different cell confluencies,
DNA/reagent ratios and total transfection volumes were
optimized for two chemical reagents including jetPEI™
and Lipofectamine™ 2000. Besides, the effects of electric
field strength and pulse length were investigated to
improve electroporation efficiency. Transfection of cells
by pEGFP-N1 vector and tracking the expression of GFP
by FACS and Fluorescence Microscopy analysis were the
employed methods to evaluate transfection efficiencies.
Optimized electroporation protocols yielded 63.73 ±
± 2.36 and 73.9 ± 1.6 % of transfection in Huh-7 and
Vero cells respectively, while maximum achieved level of
transfection by jetPEI™ was respectively 14.2 ± 0.69
and 28 ± 1.11 % for the same cells. Post transfectional
chilling of the cells did not improve electrotransfection
efficiency of Huh-7 cells. Compared to chemical based
reagents, electroporation showed the superior levels of
transfection in both cell lines. The presented protocols
should satisfy most of the experimental applications
requiring high transfection efficiencies of these two cell
lines.
Introduction. The primary stage of every suc-
cessful gene expression study for investigations
such as functional genomics, gene therapy and
gene regulation or pathway analysis requires an
efficient and optimized method of transfection
of nucleic acids (usually in the form of plasmid
DNA) into specified mammalian cells [1, 2]. Al-
though currently several biological (virus medi-
ated) and non-biological (including chemical and
physical) transfection methods are available but
for every individual cell type the supreme method
which should be efficient and reproducible with
minimum cell toxicity, needs to be optimized
and selected experimentally through comparative
studies [2]. Chemical transfection protocols are
the most widely used methods in research which
employ cationic polymers, calcium phosphate
and cationic lipids with almost similar function-
al mechanisms [3]. Briefly, positively charged
chemicals make complex with negatively charged
nucleic acids and the complex subsequently is
fused to the negatively charged cell membrane
followed by passing through it via endocytosis or
phagocytosis [1, 2]. Cell confluency and reagent/
DNA ratio are two main parameters that should
be optimized for the cell types under study
when chemical transfection methods are being
employed [4]. Besides, the total volume of the
transfection medium is suggested to have some
minor effects on the efficiency of transfection
when chemical methods are being used [4]. The
physical transfection methods such as gene gun
or electroporation (which is the most widely used
technique) make use of physical tools to deliver
nucleic acids into cells [1]. Although the exact
mechanism of electroporation is unknown but it
is supposed that short electrical pulse makes some
holes in the cell membrane through which nucle-
ic acids can go across [5]. Since the electrical
pulse induces a high degree of cell death, having
minimal amount of cell death as well as highest
gene transfer efficiency are provided by optimiza-
tion of the electric field strength and pulse length
for each cell type [6]. In addition, some prior
studies indicated that post-transfectional chilling
of the cells to 0 °C [6] or utilization of electro-
poration buffers containing dimethyl sulphoxide
DMSO (1.25 %) during and for 24 h after the
pulse [7] may improve transfection efficiencies.
Huh-7 is an immortal and well differentiated
hepatocyte-derived cellular carcinoma cell line
that was originally taken from a liver tumor [8].
© A. HASHEMI, F. ROOHVAND, M.H. GHAHREMANI,
M.R. AGHASADEGHI, R. VAHABPOUR, F. MOTEVALI,
A. MEMARNEJADIAN, 2012
20 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
A. Hashemi, F. Roohvand, M.H. Ghahremani et al.
These cells have application in various studies,
especially understanding of hepatic processes at
the cellular and molecular levels [9, 10]. The
Vero cell line was initiated from the kidney of
a normal adult African green monkey. These
cells are being used in different studies such as;
screening for the toxin of Escherichia coli [11],
as host cells for a wide range of viruses including
polio, arboviruses and retroviruses as well as host
cells for eukaryotic parasites [12, 13].
Although, a few comparative studies have al-
ready addressed the transfection efficiency of
several chemical reagents to the electroporation
using numerous cell types such as primary and
transformed airway epithelial cells, primary fibro-
blasts, lymphoblasts, HEK 293, primary hema-
topoietic stem cells [4], embryonic stem cells [14–
15] and primary human corneal endothelial cells
[16] but there is no report (to our knowledge)
on optimization and comparing efficiency of
electroporation to chemical transfection systems
for Huh-7 and Vero cell lines.
In the present study, we provide data of a
detailed study on optimization of transfection
condition (based on transfection of a Green
Fluorescent Protein (GFP) harboring plasmid
DNA; pGFPN1) for two chemical transfection
reagents including one cationic polymer based
reagent (jetPEI™) and one cationic lipid based
reagent (Lipofectamine™ 2000) compared to
electroporation in Huh-7 and Vero cell lines and
present an efficient transfection protocol for
these two cell types.
Material and method. Cell culturing. The hu-
man hepatoma (Huh-7) (Invitrogen, USA) and
African green monkey kidney (Vero) cell lines
(National Cell Bank of Iran, Tehran) were
grown in Dulbecco’s Modified Eagle Medium
(DMEM) (Biosera) supplemented with 10 %
heat-inactivated fetal bovine serum (Gibco,
Invitrogen, USA) and antibiotics (0.1 mg/mL
streptomycin and 100 U/mL penicillin) (Biosera).
Cells were cultured at 37 °C in a humidified
incubator supplied with 5 % CO2.
Plasmid preparation. To monitor the efficiency of
transfection, pEGFP-N1 plasmid DNA (NRGB,
Pasteur Institute of Iran, Tehran) encoding En-
hanced Green Fluorescent Protein was employed
through all experiments of this study. The vector
was purified from cultured transformed bacteria
using plasmid purification Maxi kit (QIAGEN,
USA) according to the manufacturer’s pro-
tocols. The mean A260/280 ratio of the DNA
samples isolated from both cell lines was 1.8 ±
± 0.045 (n = 3).
Transfection methods. Two commercially
available transfection reagents including jetPEI™
(Polyplus, France) and Lipofectamine™ 2000
(Invitrogen, USA) were used for transfection
of cell lines by pEGFP-N1 vector based on the
manufacturer’s protocols. Each experiment was
performed three times.
After washing two times in ice-cold phosphate-
buffered saline (PBS), cells were resuspended at
1.1 · 108/ml in RPMI 1640 + 10 mM HEPES.
5 g of DNA was mixed with 0.09 ml of Huh-7
or Vero cell suspensions, transferred to a 4-mm
gapped cuvette and immediately pulsed using a
Gene Pulser Xcell (BioRad, Germany). The cells
were subsequently diluted into 10 ml prewarmed
complete growth media (DMEM-15% FBS) and
plated in 75 cm2 flask and incubated at 37 °C,
5 % CO2 for 48 hours [4]. For all studies, un-
transfected cells (mock) were included as nega-
tive (reference) control. Each experiment was
performed three times.
Evaluation of transfection efficiency. To determine
the proportion of fluorescent cells (i.e.: transfected
cells), the cells were sorted by a Partec PAS
instrument (Germany) and samples were evaluated
by FlowMax software (Tree Star Inc., Ashland,
OR). Briefly, twenty-four hours after transfection
via chemical based reagents or 48 h after transfec-
tion by electroporation, the cells were harvested by
trypsinization. After centrifugation, the cells were
resuspended at 1 · 106 cells/ml in DMEM medium.
To identify the GFP positive cells, untransfected
cells (mock) were used as the GFP negative control
to set the gates. Subsequently the gated region was
analyzed for green fluorescence (488–508 nm)
on a Partec PAS instrument supplied with a 6 W
argon laser tuned to 488 nm at 100 mW output
power. The percentage of the total cells displaying
GFP emission in the gated region was expressed
as transfection efficiency.
GFP fluorescent images of transfected cells
were acquired using an inverted fluorescence mi-
croscope (Olympus BX51, London, UK) with a
10× lens and digital images were collected using
a digital camera.
21ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
Optimization of transfection methods for Huh-7 and Vero cells: a comparative study
Results. Optimization of transfection efficiency via
chemical based reagents. To obtain the optimized cell
confluency for the highest transfection efficiency 40,
50, 60, 70 and 80 · 103 Huh-7 cells were seeded
per well (in 24-well plates) twenty four hours before
transfection. Different reagent/DNA ratios (based
on the manufacturers’ recommendation) were also
considered to screen the highest efficiency. As shown
in Fig. 1, a, the highest transfection efficiency using
Lipofectamine™ 2000 was obtained when 60 · 103
cells were seeded and the employed transfection re-
agent/DNA ratio was 4 l/0.8 g (results for other
cell confluencies are not shown). In this optimized
condition, the highest transfection efficiency was
12.2 % for Huh-7 cells (Fig. 1, b).
Different cell confluencies for Huh-7 cells
(40, 50, 60, 70 and 80 · 103) and Vero cells (10,
20, 30, 40, 50, 60 and 80 · 103 cells) were seeded
per well (in 24-well plates) twenty four hours be-
fore transfection. Different reagent/DNA ratios
(based on the manufacturers’ recommendation)
were also considered to screen the highest effi-
ciency. As shown in Fig. 1, c, e the highest trans-
fection efficiency using jetPEI™ was obtained
when 60·103 and 20·103cells were seeded in case
of Huh-7 and Vero cells respectively while the
employed transfection reagent/DNA ratio was
4 l/2 g for both cell lines (results for other cell
confluencies are not shown). In this optimized
condition, the highest transfection efficiencies
were 14.2 and 28 % for Huh-7 and Vero cells,
respectively (Fig. 1, d, f).
We also investigated if the total volume of me-
dium per well during chemical transfection would
affect the efficiencies for Huh-7 and Vero cells
as suggested by manufacturers (www.polyplus-
transfection.com). To this end the transfection
efficiencies among wells containing 500, 250,
150 l media during transfection were compared.
Interestingly when results showed no significant
differences in case of Lipofectamine™ 2000, in
case of JetPEI™ the highest transfection efficien-
cies for Huh-7 and Vero cells were obtained in
wells containing 500 and 150 l of total medium
respectively with almost 10 % variation of effi-
ciencies within the range of the measured vol-
umes (500, 250, 150 l; data not shown). These
results showed that the effect of total volume on
transfection efficiency may depend on both the
type of reagent and the cell line under study.
Optimization of transfection efficiency via Elec-
troporation. According to prior studies, applica-
tion of the square wave and low field strengths
(500–2000 V/cm) together with long time con-
stants (10–30 ms) provided the highest transfec-
tion efficiencies in case of mammalian cell types
[6]. Besides, low-resistance buffers like PBS or
culture medium (itself) are shown to be the op-
timal media for efficient electrotransfection of
these cell types [6]. Accordingly, to obtain the
condition in which cell death and electropora-
tion efficiency are approximately equilibrated,
we employed square wave form and evaluated
different low field strengths (400, 425, 450, 625,
750 V/cm corresponding to: 160, 170, 180, 250,
300 volts (for a 4-mm gapped cuvette), respec-
tively. as well as two different long pulse lengths
(20 and 40 ms) for both cell lines (Fig. 2, a). Re-
sults indicated that the optimal electroporation
condition for Huh-7 cells (electric field strength
and pulse length of 425 V/cm and 40 ms respec-
tively) led to 63.73 ± 2.36 % transfection effi-
ciency while the optimal condition for Vero cells
(625 V/cm and 20 ms) resulted in 73.9 ± 1.6 %
transfection efficiency (Fig. 2, b, c). Furthermore
the cell viability 24 h after plasmid delivery was
similar and more than 50 % for both cell lines
(data not shown).
To investigate the temperature effect on the
electroporation efficiency of Huh-7 cells, the
cuvette containing the electroporated cells was
incubated on ice for 5 min before transferring
to the growth medium. After 48 h, green fluo-
rescence was detected through flow cytometry
and compared with those cells transferred to
the medium immediately after pulsing. Interest-
ingly, our results demonstrated that chilling the
Huh-7 cells to 0 °C post-transfectionally led to
a significant decrease in cell viability by 40 %
(data not shown). Moreover, to evaluate the ef-
fect of DMSO on electroporation efficiency, a
final concentration of 1.25 % was included into
electroporation buffer during and for 24 h after
the application of the pulse. Results showed a
decrease in electroporation efficiency and cell
survival of Huh-7 cells by 60 % and 50 % respec-
tively (data not shown).
Discussion. Huh-7 and Vero cells are two im-
mortalized cell systems in two different mam-
malian species (human and monkey respectively)
22 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
A. Hashemi, F. Roohvand, M.H. Ghahremani et al.
Fig. 1. Optimization of chemically based transfection conditions in Huh-7 and Vero cells and Flow cytometry
plots of the transfected cells: a – different transfection reagent/DNA ratios using pEGFP plasmid shown for
Lipofectamine™ 2000 in Huh-7 cells also jetPEI™ in Huh-7 and Vero cells. Data represent the mean ± S.D.
(n = 3); b – the highest efficiency obtained when 60·103 cells were seeded per well and ratio was 4 l (Lipofectamine™
2000)/0.8 g (DNA) and 4 l (jetPEI™)/2 g (DNA) in Huh-7 cells. Also the highest efficiency obtained when
20·103 cells were seeded per well and ratio was 4 l (jetPEI™)/2 g (DNA) in Vero cells. Transfection efficiencies
were expressed as the ratio of the GFP-positive cells to total cells in the gated region
23ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
Optimization of transfection methods for Huh-7 and Vero cells: a comparative study
that are particularly important for characteriza-
tion of hepatitis viruses molecular mechanisms
(Huh-7) [8, 10, 17] or as host cells for a wide
range of viruses and eukaryotic parasites (Vero
cells) [11–13]. Although Huh-7 and Vero cell
lines have been employed in number of prior
studies and were transfected via different trans-
fection reagents or electroporation in separate set
of experiments but, none of the previous reports
provided an optimized transfection protocol for
these important cell types based on a compara-
tive study [18–22]. This is specially important
for Huh-7 which is generally known as a dif-
ficult to transfect cell line [23] and when high
rates of transfection efficiencies are determinant
for the aim of the study. Previously the elegant
study of Uchida and co-workers [24] based on
a comparative analysis for the transfection effi-
ciency and cytotoxicity of six non-viral chemical
reagents (Lipofectin, Lipofectamine plus, Super-
Fect, Effectene, DMRIE-C and DOTAP) in-
tended for a wide range of human cells including
Huh-7 was reported. Results of this prior study
indicated efficient transfection of Huh-7 cells by
«Lipofectamine plus» and «Effectene» compared
to other reagents (with some strong cytotoxicity
for «Effectene»). However there is no report to
compare the efficiency of transfection between
chemical methods and electroporation in Huh-7
and Vero cells and to provide an optimized and
Fig. 2. Optimization of electroporation condition on trans-
fection efficiencies of Huh-7 and Vero cells: a – the
square-wave electroporations. Varied voltage with a cons-
tant pulse length of 20 ms and 40 ms in Huh-7 and Vero cells.
Data represent the mean ± S.D. (n = 3). Values represent
the EGFP expression percentages; b – Flow cytometry plots
of electroporated cells. The highest transfection efficiency
obtained in electric field strength and pulse length of
(425 V/cm–40 ms) in Huh-7 and (625 V/cm–20 ms) in Vero
cells; c – Fluorescence microscopy images of both cell lines
electroporated in the optimum conditi on
24 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
A. Hashemi, F. Roohvand, M.H. Ghahremani et al.
practical protocol of transfection for these cell
types.
In the present study we compared two com-
mercially available transfection reagents includ-
ing «Lipofectamine™ 2000» (as a cationic lipo-
some) and «jetPEI™» (as a cationic polymer).
Optimization of the parameters in both cell lines
resulted in the transfection efficiencies of 12.2 ±
± 0.6 % in case of «Lipofectamine™ 2000» for
Huh-7 cells and 14.2 ± 0.69 and 28 ± 1.11 %
in case of «jetPEI™» for Huh-7 and Vero cells,
respectively. Although these transfection efficien-
cies are comparable with the results reported by
Uchida et al. [24] but the slightly lower trans-
fection efficiency of Huh-7 cells using «Lipo-
fectamine™ 2000» (12.2 ± 0.6 %) in our study
compared to «Lipofectamine plus» used by
Uchida et al. might be relevant to the addition of
the DNA-compacting agent to cationic lipids in
«Lipofectamine plus» composition [24]. In agree-
ment to this suggestion, addition of DNA com-
pacting peptides derived from human sequence
to cationic lipid agents were previously shown to
enhance the gene transfer efficiencies in vitro and
in vivo [25].
Endocytosis is considered as the preferred
route of cell entry of nonviral complexes and car-
gos via chemical transfection methods [26]. The
efficiency of cargo transfer by endocytic pathway
itself (and therefore the transfection efficiency of
chemical methods) is highly dependent on the
cell line type and specific characteristics of the
chemical reagent (polyplex or lipoplex) used as
carrier [27, 28]. Different transfection efficien-
cies obtained in our study for Huh-7 and Vero
cells (14.2 ± 0.69 % versus 28 ± 1.11 % respec-
tively) in spite of employing the same reagent
(jetPEI™ as a polyplex reagent) clearly indicated
the effect of the cell type in utilizing different en-
docytic pathways. Indeed, previous findings sug-
gested that the same chemical reagent (polyplex)
may be internalized by different cell lines through
various endocytic pathways leading to different
transfection efficiencies. Of note, it is suggested
that in Hela and 293 cells the caveolae-dependent
endocytic pathway leads to effective transfection
by polyplex reagent [29] while clathrin-mediated
endocytosis is the main endocytic pathway in
Huh-7 and COS-7 cells when the same chemical
reagent (polyplex) is employed for transfection
[30]. Therefore, our results for the observed dif-
ferences in the transfection efficiencies between
Vero and Huh-7 cells in case of polyplex reagent
may be related to the different fates of this re-
agent through endocytic pathways in these two
cell lines.
We also improved electroporation protocols
for gene delivery into these two cell lines by
optimizing the electric field strength and pulse
length. In accordance with previous reports the
square wave form provides higher mammalian
transfection efficiencies and is widely used for
in vitro and in vivo applications [31]. There-
fore, square wave pulsing was employed in our
study. In addition Reports using electroporation
to transform mammalian cells either employed
high field strengths and short pulse length or us-
ing lower field strengths and longer pulse length
[31]. However, later studies showed that higher
levels of expression could be obtained by the lat-
ter [6]. Our results showed that high efficiencies
as well low cell death were obtained with low
field strengths and long time constants condition
in both cell lines.
Effect of cold treatment on electrotransfection
efficiency of Huh-7 cell was also addressed in
our study. Few prior reports have indicated that
post-transfectional chilling of the cells to 0 °C
increase the transfection efficiency of some cell
types (apparently due to keeping the membrane
pores open for longer time which subsequently
provides more time for DNA to enter the cell) [6]
when on the contrary immediate transferring of
electroporated cells to pre-warmed medium have
been suggested for other cell types to increase cell
survival (probably via helping the pores created to
be resealed) [6]. Our results in accordance with
the later report demonstrated that post-transfec-
tionally chilling of the Huh-7 cells increased cell
death by 40 %. We also assessed the potential
role of dimethyl sulphoxide (DMSO) on elec-
trotransfection efficiency of Huh-7 cell in our
study. DMSO has been reported to enhance the
transfection efficiencies of DNA introduced by
polycations including polybren and DEAE [32,
33] as well as calcium phosphate method [34]. It
is suggested that DMSO enhances the uptake of
DNA by augmenting cell membrane permeabili-
zation and the osmotic shock [34]. However, the
concentration of DMSO for efficient transfection
25ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
Optimization of transfection methods for Huh-7 and Vero cells: a comparative study
is critical and should be determined experimen-
tally for each cell line. In fact optimal concentra-
tion of DMSO for one cell line may be toxic for
another one. This fact was previously reported for
chemical transfection of human epidermal kera-
tinocytes and muine NIH-3T3 cells by polybrene
reagent in which the optimal concentration of
DMSO for highest transfection efficiencies of the
cells were 27 and 15 % respectively [35]. Accord-
ing to a prior study, improvement in electrotrans-
fection efficiency and cell viability in four differ-
ent cell lines (HL60, TR146, COS-7 and L132)
using electroporation buffer containing DMSO
(1.25 %) during and for 24 h after the pulse was
reported [7]. Accordingly in our study Huh-7
cells were electroporated in buffer (media) con-
taining 1.25 % DMSO when other parameters
were kept in optimized condition. However this
modification decreased the electroporation ef-
ficiency and cell survival of Huh-7 cells by 60
and 50 % respectively (data not shown). This
result was not surprising and may indicate the
toxic effect of 1.25 % DMSO on Huh-7 cells.
Further experiments are required to access the
specific concentration of DMSO for Huh-7 cells
in which cell permeability increase is balanced
with cell viability decrease. In addition, concen-
tration of plasmid DNA might be an important
parameter for electrotransfection efficiencies that
should be determined experimentally for each
cell type. Application of 1–10 g plasmid was
suggested in different electrotransfection studies
[6, 36]. In our study 5 g of plasmid DNA (as a
mean concentration and starting point) was used
throughout all experiments. Although, it may be
possible to further increase the transfection ef-
ficiencies obtained in this study by optimizing
DMSO and plasmid DNA concentrations, but
the achieved efficiencies (63.73 ± 2.36 and
73.9 ± 1.6 % for Huh-7 and Vero cells respec-
tively) are good enough to satisfy most of the
experimental applications requiring high trans-
fection efficiencies of these two cell types.
Finally, in complete agreement with a recent
report by Maurisse et al. [4] which indicated the
superiority of electroporation/nucleofection to
chemical methods for DNA delivery into primary
and transformed mammalian cells from different
lineage, our results also showed that electrotrans-
fection was the suprime method for Huh-7 and
Vero cells. The mechanism for this superiority of
electroporation compared to chemical methods
should lie on direct delivery of the plasmid DNA
into the cytosol and passing though the endocytic
pathways as previously suggested [37].
In summary, to our best of knowledge, we
provided data of the first comparative study on
optimization of transfection condition by electro-
poration and chemical methods for Huh-7 and
Vero cells. These data indicated the superiority
of electroporation to chemical methods for DNA
delivery into Huh-7 and Vero cells. According
to our results, application of 60·103 Huh-7 cells
(confluency) and transfection reagent/DNA ra-
tios of 4 l/0.8 g and 4 l/2 g in a total vol-
ume of 500 l provided transfection efficiencies
of 12.2 % ± 0.6 and 14.2 % ± 0.69 in case of
«Lipofectamine™ 2000» and «jetPEI™» reagents
respectively, while application of the later reagent
provided 28 % efficiency using 20·103 Vero cells
and transfection reagent/DNA ratio of 4 l/2 g
in a total volume of 150 l. Alternatively, electro-
poration of both cells at a density of 1.1·108/ml
in RPMI+10 mM HEPES buffer in the pres-
ence of 5 g of plasmid DNA and application
of electric field strength and pulse length of
«425 V/cm and 40 ms for Huh-7» cells and
«625 V/cm–20 ms for Vero cells» resulted to
transfection efficiencies of 63.73 ± 2.36 and
73.9 ± 1.6 % for these two cell lines respective-
ly. The presented protocols should satisfy most
of the experimental applications requiring high
transfection efficiencies of these two cell types.
A.H received a fellowship from graduate school
of Pasteur Institute to pursue this study in the Ph.D.
program. Portions of this study were supported by
a grant (ACIP-AP20, 2010) from RIP of Pasteur
Institute Paris and Pasteur Institute of Iran.
A. Hashemia, F. Roohvand,
M.H. Ghahremanic, M.R. Aghasadeghia,
R. Vahabpoura, F. Motevali, A. Memarnejadiana
ÎÏÒÈÌÈÇÀÖÈß ÌÅÒÎÄÎÂ
ÒÐÀÍÑÔÅÊÖÈÈ ÊËÅÒÎÊ Huh-7 È VERO:
ÑÐÀÂÍÈÒÅËÜÍÛÉ ÀÍÀËÈÇ
Íàëè÷èå ýôôåêòèâíîãî ïðîòîêîëà òðàíñôåêöèè
ÿâëÿåòñÿ ïåðâûì óñëîâèåì óñïåøíûõ èññëåäîâà-
íèé ïî ïåðåíîñó ãåíîâ â êëåòêè ìëåêîïèòàþùèõ,
÷òî äîñòèãàåòñÿ ýêñïåðèìåíòàëüíî äëÿ êàæäîãî
êîíêðåòíîãî òèïà êëåòîê. Çäåñü ìû ïðèâîäèì
26 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 6
A. Hashemi, F. Roohvand, M.H. Ghahremani et al.
äàííûå ñðàâíèòåëüíîãî èññëåäîâàíèÿ ïî îïòèìè-
çàöèè óñëîâèé òðàíñôåêöèè êëåòîê Huh-7 è Vero
ñ ïîìîùüþ ýëåêòðîïîðàöèè è õèìè÷åñêèìè ìå-
òîäàìè. Äëÿ äâóõ õèìè÷åñêèõ ñîåäèíåíèé, jetPEI™
è Lipofectamine™ 2000, áûëè îïòèìèçèðîâàíû
ñî÷åòàíèÿ ðàçëè÷íûõ êëåòîê, ñîîòíîøåíèÿ ÄÍÊ/
ðåàãåíò è îáùèå îáúåìû òðàíñôåêöèè. Êðîìå òîãî,
äëÿ óëó÷øåíèÿ ýôôåêòèâíîñòè ýëåêòðîïîðàöèè
áûëî èçó÷åíî âëèÿíèå ñèëû ýëåêòðè÷åñêîãî ïîëÿ
è äëèíû èìïóëüñà. Òðàíñôåêöèÿ êëåòîê ñ ïîìî-
ùüþ âåêòîðà pEGFP-N1, îïðåäåëåíèå ýêñïðåñ-
ñèè GFP ñ ïîìîùüþ FACS è ôëþîðåñöåíòíàÿ
ìèêðîñêîïèÿ áûëè èñïîëüçîâàíû äëÿ îöåíêè ýô-
ôåêòèâíîñòè òðàíñôåêöèè. Â îïòèìèçèðîâàííûõ
ïðîòîêîëàõ äîñòèãàëàñü òðàíñôåêöèÿ íà óðîâíå
63.73 ± 2.36 è 73.9 ± 1.6 % â êëåòêàõ Huh-7 è
Vero ñîîòâåòñòâåííî, â òî âðåìÿ êàê ìàêñè-
ìàëüíûé óðîâåíü òðàíñôåêöèè ñ ïîìîùüþ jet-
PEI™ ñîñòàâëÿë 14.2 ± 0.69 è 28 ± 1.11 % äëÿ
òåõ æå êëåòîê. Îõëàæäåíèå êëåòîê ïîñëå òðàíñ-
ôåêöèè íå óëó÷øàëî ýôôåêòèâíîñòü ýëåêòðîòðàíñ-
ôåêöèè êëåòîê Huh-7.  îáåèõ êëåòî÷íûõ ëè-
íèÿõ ýëåêòðîïîðàöèÿ ïîçâîëèëà äîñòè÷ü áîëåå
âûñîêîãî óðîâíÿ òðàíñôåêöèè ïî ñðàâíåíèþ ñ
èñïîëüçîâàíèåì õèìè÷åñêèõ ðåàãåíòîâ. Ïðåäñòàâ-
ëåííûé ïðîòîêîë ìîæåò áûòü ïðèãîäíûì äëÿ
áîëüøèíñòâà ýêñïåðèìåíòàëüíûõ ìàíèïóëÿöèé,
êîòîðûå òðåáóþò âûñîêîãî óðîâíÿ òðàíñôåêöèè
èññëåäóåìûõ êëåòî÷íûõ ëèíèé.
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Received 26.11.11
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/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
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| id | nasplib_isofts_kiev_ua-123456789-126499 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0564-3783 |
| language | English |
| last_indexed | 2025-12-07T19:03:50Z |
| publishDate | 2012 |
| publisher | Інститут клітинної біології та генетичної інженерії НАН України |
| record_format | dspace |
| spelling | Hashemi, A. Roohvand, F. Ghahremani, M.H. Aghasadeghi, M.R. Vahabpour, R. Motevali, F. Memarnejadian, A. 2017-11-25T14:05:11Z 2017-11-25T14:05:11Z 2012 Optimization of transfection methods for Huh7 and Vero cells: a comparative study / A. Hashemi, F. Roohvand, M.H. Ghahremani, M.R. Aghasadeghi, R. Vahabpour, F. Motevali, A. Memarnejadian // Цитология и генетика. — 2012. — Т. 46, № 6. — С. 19-27. — Бібліогр.: 37 назв. — англ. 0564-3783 DOI: 10.3103/S0095452712060035 https://nasplib.isofts.kiev.ua/handle/123456789/126499 Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± ± 2.36 and 73.9 ± 1.6 % of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was respectively 14.2 ± 0.69 and 28 ± 1.11 % for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines. Наличие эффективного протокола трансфекции является первым условием успешных исследований по переносу генов в клетки млекопитающих, что достигается экспериментально для каждого конкретного типа клеток. Здесь мы приводим данные сравнительного исследования по оптимизации условий трансфекции клеток Huh-7 и Vero с помощью электропорации и химическими методами. Для двух химических соединений, jetPEI™ и Lipofectamine™ 2000, были оптимизированы сочетания различных клеток, соотношения ДНК/реагент и общие объемы трансфекции. Кроме того, для улучшения эффективности электропорации было изучено влияние силы электрического поля и длины импульса. Трансфекция клеток с помощью вектора pEGFP-N1, определение экспрессии GFP с помощью FACS и флюоресцентная микроскопия были использованы для оценки эффективности трансфекции. В оптимизированных протоколах достигалась трансфекция на уровне 63.73 ± 2.36 и 73.9 ± 1.6 % в клетках Huh-7 и Vero соответственно, в то время как максимальный уровень трансфекции с помощью jetPEI™ составлял 14.2 ± 0.69 и 28 ± 1.11 % для тех же клеток. Охлаждение клеток после трансфекции не улучшало эффективность электротрансфекции клеток Huh-7. В обеих клеточных линиях электропорация позволила достичь более высокого уровня трансфекции по сравнению с использованием химических реагентов. Представленный протокол может быть пригодным для большинства экспериментальных манипуляций, которые требуют высокого уровня трансфекции исследуемых клеточных линий. A.H received a fellowship from graduate school of Pasteur Institute to pursue this study in the Ph.D. program. Portions of this study were supported by a grant (ACIP-AP20, 2010) from RIP of Pasteur Institute Paris and Pasteur Institute of Iran. en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Оригинальные работы Optimization of transfection methods for Huh7 and Vero cells: a comparative study Оптимизация методов трансфекции клеток huh-7 и Vero: сравнительный анализ Article published earlier |
| spellingShingle | Optimization of transfection methods for Huh7 and Vero cells: a comparative study Hashemi, A. Roohvand, F. Ghahremani, M.H. Aghasadeghi, M.R. Vahabpour, R. Motevali, F. Memarnejadian, A. Оригинальные работы |
| title | Optimization of transfection methods for Huh7 and Vero cells: a comparative study |
| title_alt | Оптимизация методов трансфекции клеток huh-7 и Vero: сравнительный анализ |
| title_full | Optimization of transfection methods for Huh7 and Vero cells: a comparative study |
| title_fullStr | Optimization of transfection methods for Huh7 and Vero cells: a comparative study |
| title_full_unstemmed | Optimization of transfection methods for Huh7 and Vero cells: a comparative study |
| title_short | Optimization of transfection methods for Huh7 and Vero cells: a comparative study |
| title_sort | optimization of transfection methods for huh7 and vero cells: a comparative study |
| topic | Оригинальные работы |
| topic_facet | Оригинальные работы |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/126499 |
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