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099-46

Biologia 65/5: 919—924, 2010

Section Zoology
DOI: 10.2478/s11756-010-0095-6

Biological and morphological characterization of human neonatal

fibroblast cell culture B-HNF-1

Vanda Repiská1, Ivan Varga2,3,4, Ivan Lehocký5, Daniel Böhmer1, Milan Blaško1,
Štefan Polák2, Marián Adamkov6 & Ľuboš Danišovič1,4*
1
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava,
Sasinkova 4, SK-81108 Bratislava, Slovakia; e-mail: lubos.danisovic@fmed.uniba.sk
2
Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, SK-81108
Bratislava, Slovakia
3
Institute of Histology and Embryology, Faculty of Medical Specialty Studies, Slovak Medical University, Limbová 12,
SK-83303 Bratislava, Slovakia
4
Institute of Laboratory Medicine, St. Elizabeth University of Health and Social Sciences, Palackého 1, SK-81000 Bratislava,
Slovakia
5
Department of Biology, Institute of Forensic Science of Police Corps, Sklabinská 1, SK-81272 Bratislava, Slovakia
6
Institute of Histology and Embryology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Malá
Hora 4, SK-03754 Martin, Slovakia

Abstract: In the present study, human neonatal fibroblasts were isolated from a two-month-old human male. The purpose
of the present investigation was the analysis of the morphology (light and transmission electron microscopy), karyotype and
growth characteristics of the human neonatal fibroblast cell culture B-HNF-1. Moreover, STR typing and mitochondrial
DNA amplification and sequencing was also performed. Analysis of chromosomes count showed that B-HNF-1 cell culture is
diploid and has normal male karyotype 46, XY, which was stable during cultivation. The transmission electron microscopy
demonstrated the ultra-structure of the B-HNF-1 cells; they have typical morphological features of proteosynthesis-active
cells. Large number of fibroblasts bearing different shapes and surface characteristics adhered to the substrate with microvilli
and filopodia. Our in vitro expanded fibroblasts have a large and irregular nucleus with prevalence of euchromatin. One
to three nucleoli are present in each nucleus. The cytoplasm contains a richly developed rough endoplasmic reticulum
and prominent Golgi apparatus cisterns. The result of the fragment analysis is a DNA profile defined as multiplex of STR
markers and sex determining system. Sequencing analysis of hypervariable segments of control region of mitochondrial DNA
showed haplotype that belongs to haplogroup W. In this study, we complexly characterized a new cell culture of human
neonatal fibroblasts B-HNF-1 from different points of view. This culture should be used for further biomedical experiments.
Key words: neonatal fibroblasts; cell culture; morphological analysis; STR typing; mitochondrial DNA analysis

Introduction cur. Another important advantage of in vitro system

Recently, a large amount of chemicals or materials that
come into close contact with the human body are pro-
duced. For that reason it is necessary to perform battery
of tests for cytotoxicity, genotoxicity and mutagenicity.
Classical toxicological assays are usually performed on
animal models that represent an enormous time, per-
sonnel and economic load. Moreover, some organiza-
tions point out the non-ethical and cruel treatment of
experimental animals (Gad 2006; Hayes 2007). Since
the second half of the eighties of the last century in vivo
testing has been increasingly replaced by in vitro sys-
tems as the first step of biohazard testing. Their usage
leads to the reduction of experimental animals as well as
to non-negligible economic effect. In many cases, based
on their predictive results, in vivo testing does not oc-
is the possibility to define strict experimental condi-
tions with the elimination of external factor influence
(Davila et al. 1998). In addition, in vitro system pro-
vides more information about the mechanisms of ac-
tion of tested compounds, which is difficult to obtain
from animal model due to its complexity (Barber et
al. 2009). For that reason, primary isolated cells, cell
cultures and strictly defined cell lines of animal or hu-
man origin belong to most used in vitro models in the
biohazard testing (Zucco et al. 2004; Vojtaššák et al.
2006).
Fibroblasts are spindle-shaped cells found in the
majority of tissues and organs of the human body as-
sociated with extracellular matrix (ECM) molecules.
When activated, fibroblasts exhibit an abundant en-
doplasmic reticulum and prominent Golgi apparatus

* Corresponding author

c 2010 Institute of Zoology, Slovak Academy of Sciences
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920
associated with the synthesis and secretion of ECM
molecules including collagens, proteoglycans and fi-
bronectin as well as proteases capable of degrading the
ECM (McAnulty 2007). It has been estimated that each
cell can synthesise approximately 3.5 million procolla-
gen molecules per day (McAnulty et al. 1991). Fibrob-
lasts also play an important role in regulating tissue
interstitial fluid volume and pressure (Wiig et al. 2003).
The purpose of the present investigation was the
analysis of the morphology, karyotype and growth char-
acteristics of the human neonatal fibroblast cell culture
B-HNF-1. Moreover, STR (short tandem repeat) typing
and mitochondrial DNA amplification and sequencing
was also performed.
Material and methods
Sampling procedure
Human neonatal fibroblasts were isolated from biopsy spec-
imen of right musculus quadriceps femoris of a two-month-
old human male. Sampling procedure was indicated due to
cytogenetical analysis and was performed by a physician un-
der aseptic conditions according to the Helsinki declaration
after the subject’s parents’ informed consent. A sample of
tissue was immediately transported to the laboratory of cell
culture in sterile physiologic saline with gentamicin (Lék,
Slovenia) in final concentration of 200 µg ml−1 .
Isolation and cell culture
The obtained biopsy specimen was carefully rinsed with
phosphate buffered solution (PBS; Oxoid, GB) and sec-
tioned into small fragments by sterile scalpel. The frag-
ments were washed twice with PBS. Subsequently, they
were transferred into a plastic 40 mm Petri dish (TPP,
Switzerland) with Eagle’s minimal essential medium (E-
MEM, PAA, Austria) supplemented with 10% fetal calf
serum (FCS, PAA, Austria), gentamicin (Lék, Slovenia) in
final concentration of 160 µg ml−1 and cultured at 37 ◦C
in a humidified atmosphere containing 5% CO2 . After 72
hours, some fragments adhered and migration of fibroblasts
started. Non-adherent tissues were removed by changing the
culture medium. During subsequent cultivation, the medium
was refreshed twice a week. After eight days, when the cul-
ture became almost confluent, fibroblasts were detached by
0.25% trypsin (Gibco, USA) and sub-cultured.
In the fourth passage, cultivation was terminated. Cells
were trypsinized and the obtained suspension was tested for
viability by trypan blue exclusion test as well as for my-
copalsma contamination by PCR. Viability was 97 % and
results for mycopalsma were negative. After that, cells were
frozen according to a standard method (Freshney 2005) and
stored in liquid nitrogen. Fibroblasts from the fourth pas-
sage were also used for morphological and karyotype analy-
sis as well as for STR typing and mtDNA sequencing.
Growth curve
The evaluation of the growth characteristics of B-HNF-1
cells was performed by generating a growth curve prepared
according to a standard method published by Mather &
Roberts (1998). Briefly, the cell suspension in concentration
of 1 × 105 cells per milliliter was seeded into Petri dishes (40
mm). During the next seven days of cultivation, three dishes
were monitored on a daily basis for density. Growth curves
were plotted and population doubling time was calculated.
V. Repiská et al.
Karyotype analysis
For karyotype analysis, cultures of exponentially growing
cells were treated with demecolcine (Sigma-Aldrich, Ger-
many) in concentration of 0.05 µg ml−1 . After three hours
of incubation, the karyological slides were prepared accord-
ing to conventional method (Freshney 2005). The slides were
air-dried and stained with 2% Giemsa solution for ten min-
utes. Chromosomes were analysed in three series of 100
metaphase cells.
Morphological analysis
Fibroblasts appointed for light microscopy observation were
cultured on the sterile slides under the same conditions as
mentioned above. Before analysis, they were gently washed
with PBS, fixed with metanol (Centralchem, Slovakia) for
ten minutes and stained with hematoxylin and eosin (Merck,
Germany). Cells were then analyzed under light microscope
Nikon (Nikon Corporation, Japan) with/without Nomarski
interference contrast.
Samples for transmission electron microscopy (TEM)
were fixed in 2.5% glutaraldehyde (Serva, Germany), pH 7.2,
at 4 ◦C for four hours. After fixation, samples were rinsed
in phosphate buffered saline (PBS, Oxoid, GB) and post-
fixed in 2% osmium tetra oxide (Serva, Germany) for two
hours, then rinsed in distilled water and dehydrated in a
graduated series of ethanol (Centralchem, Slovakia). Sub-
sequently, the samples were embedded in durcupan (Serva,
Germany) and cut into semi-thin sections. Ultra-thin sec-
tions were mounted on 200 mesh copper grids, then double
stained using uranyl acetate and lead citrate (Serva, Ger-
many) and examined using a transmission electron micro-
scope Morgagni 268D.
STR typing
DNA was isolated from the cell pellet of culture line by di-
gestion with Proteinase K and subsequent extraction with
Phenol/chloroform method. The amount of DNA was de-
termined by agarose gel electrophoresis in comparison to a
lambda DNA standard.
STR analysis was conducted using the multiplex-
PCR-based Identifiler kit (Applied Biosystems, USA). Loci
analyzed include CSF1PO, D2S1338, D3S1358, D5S818,
D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433,
D21S11, FGA, TH01, TPOX, vWA and amelogenin.
Electrophoreogram data were collected on an ABI 3130
Genetic Analyzer (Applied Biosystems, USA) and analyzed
using GeneMapper ID Software Version 3.1 – Human iden-
tification analysis (Applied Biosystems, USA).
Mitochondrial DNA amplification and sequencing
DNA was isolated from the cell line by a standard method
mentioned above. PCR amplification was performed using
primers H15975 and L16420 for HV1 region and H8 and
L429 for HV2 region. The amplification products of the
hypervariable regions were precipitated in absolute ethanol
and purified by washing in 70% ethanol.
Sequencing was performed using primers H15975 and
L16420 for HV1 region and H8 and L429 for HV2 region.
Sequencing reactions were performed using the BigDye

Terminator v3.1 Cycle Sequencing kit (Applied Biosystems,
USA). The sequencing products were precipitated in solu-
tion containing sodium acetate, absolute ethanol and dis-
tilled water for each sample and purified from residual ter-
minators by washing in 70% ethanol. DNA analysis was
run on Applied Biosystems 3130 Genetic Analyzer (Applied
Biosystems, USA).
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Characterization of cell culture B-HNF-1 921

Sequencing of HV1 and HV2 regions was performed

using DNA Sequencing Analysis Software version 5.2 (Ap-
plied Biosystems, CA, USA) and sequences were aligned us-
ing ABI PRISM
SeqScape
Software version 2.5 (Applied
Biosystems, CA, USA). Quality control: According to ISFG
recommendations. The double evaluation of the profiles as-
certained the quality of the results.

Results and discussion

Fibroblasts are the major components of the connective

tissue. The fibroblast synthesizes and continuously se-
cretes mature proteoglycans and glycoproteins and the
precursor molecules of various types of collagens and
elastin. Many in vitro models used fibroblast, for exam-
ple for modelling of wound healing (Moulin et al. 1996), Fig. 1. Human neonatal fibroblast cell B-HNF-1 culture (× 100).
remodelling of the bronchial wall in asthma (Michalik
et al. 2009), specific alterations in myocardial fibrob-
lasts biology after myocardial infarction (Squires et al.
2005), attachment behaviour of periodontal ligament fi-
broblasts to dentin surface (Al-Nazhan 2004) or to de-
scribe the effect of laser irradiation on cultured human
periodontal fibroblasts (Chen et al. 2005). Moreover,
cell cultures of fibroblasts belong to the most used bi-
ological models in the evaluation of cytotoxicity, geno-
toxicity and mutagenicity of chemical compounds and
materials under in vitro conditions (Ata & Yavuzyilmaz
2009; Unyayar et al. 2006; Vojtaššák et al. 2003).
In the present study, we have performed a compre-
hensive analysis of biological and morphological prop-
erties of human neonatal fibroblast culture B-HNF-1 as
well as STR typing and mitochondrial DNA amplifica-
tion and sequencing.
The cell culture was established by a standard
method of primary explants, which was originally devel-
oped by Harrison (1907). This technique was modified
and simplified several times and recently it has been
widely used for the initiation of primary cultures of ad- Fig. 2. Growth curve of the B-HNF-1 cells.
herent cells (Li et al. 2009; Wang et al. 2003; Kinugawa
et al. 1998).
After the initiation of the cell culture, some tis- 96 hours and the population doubling time was about
sue fragments adhered and the migration of fibroblasts 24 hours. Results obtained by other authors were quite
as well as epithelial cells was observed after 72 hours. diverse and they were dependent on the species origin
During the following five days, the cells reached con- and age of the tissue donor (Li et al. 2009; Wu et al.
fluence and were trypsinized to obtain monocellular 2008; Veelken et al. 1994).
suspension for further sub-cultivation. In case of dif- Every test on bacterial or mycopalsma infection
ferent toleration to trypsin during following passages, prior to freezing was negative. The viability was 97%.
epithelial cells disappeared. Purified fibroblast culture These results indicated that B-HNF-1 cell line was
was obtained after second passage. They are of a typical healthy and ready for storage in liquid nitrogen and
long spindle shape (Fig. 1). Moreover, after passage, the for subsequent utilization.
cell growth accelerated until next confluence. Contact Analysis of chromosomes count (Fig. 3) showed
inhibition was observed if they were not sub-cultured that B-HNF-1 cell culture is diploid; the modal num-
promptly. A similar course of fibroblast cultivation was ber of chromosomes was 46 and has normal male kary-
described by many authors (Li et al. 2009; Wu et al. otype 46, XY, which was stable during cultivation. We
2008; Vojtaššák et al. 2006). did not record any chromosomal aberration, neither in
Fig. 2 shows the growth curve of the B-HNF-1 structure nor in number.
cells. The lag time was almost undetectable; the fibrob- Fig. 4 illustrates the normal morphological features
lasts adhered very quickly after cell seeding. Cells then of fibroblast in light microscopy level, with Nomarski in-
started to proliferate and their number increased log- terference contrast. Each spindle-shaped fibroblast has
arithmically. They reached the stationary phase after a large nucleus with one to four nucleoli and many long
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922 V. Repiská et al.

Fig. 3. Karyotype analysis of B-HNF-1 cell culture: normal male karyotype 46, XY.

Fig. 5. Transmission electron microscopic observation of human

Fig. 4. Light electron microscopic observation of human neona- neonatal fibroblasts B-HNF-1. Each cell have large, pale nucleus
tal fibroblast cell culture B-HNF-1 with Nomarski interference (1), in the cytoplasm abundant dilated cisterns of rough endo-
contrast. Each nucleus contains 1 to 4 nucleoli (× 400). plasmatic reticulum (2). The cells adhered to the substrate with
microvilli and filopodia (3). (× 8900).

and branched processes. The transmission electron mi-

croscopy demonstrated the ultra-structure of the hu-
man neonatal fibroblasts B-HNF-1; we can see the
typical morphological features of proteosynthesis-active
cells (Figs 5–8). Large number of fibroblasts bearing dif-
ferent shapes and surface characteristics adhered to the
substrate with microvilli and filopodia. The filopodia
and lamellipodia of in vitro cultivated fibroblasts were
also described by Al-Nazhan (2004) with scanning elec-
tron microscopy. Our in vitro expanded fibroblasts have
a large and irregular nucleus with prevalence of euchro-
matin. One to four nucleoli are present in each nucleus.
The cytoplasm contains a richly developed rough endo-
plasmic reticulum and prominent Golgi apparatus cis-
terns. In contrast to Chen et al. (2005), we did not find
collagen fibrils around the fibroblasts. However, cited
authors use a 28-day-old fibroblast culture for the TEM Fig. 6. TEM observation of human neonatal fibroblasts B-HNF-1.
We can see typical features of proteosynthetic active cell: large,
analysis; we observed only a seven-days-old cell culture. pale nucleus (1), markedly nucleolus (2), in the cytoplasm abun-
The result of the fragment analysis is a DNA profile dant cisterns of rough endoplasmatic reticulum (3). (× 7100).
defined as multiplex of STR markers and sex determin-
Licensed to Andressa Thomazzoni - andressathomazzoni123@gmail.com - 046.377.099-46
Characterization of cell culture B-HNF-1
Fig. 7. TEM observation of human neonatal fibroblasts B-HNF-
1. Large, pale nucleus with two nucleoli (1), in the cytoplasm
abundant cisterns of rough endoplasmatic reticulum (2). The cells
adhered to the substrate with microvilli and filopodia (3). (×
7100)
Fig. 8. TEM observation: detail of human neonatal fibroblasts
B-HNF-1. Pale nucleus (1), richly developed rough endoplasmic
reticulum (2) and prominent Golgi apparatus (3). (× 36000).
ing system. Biological material of cell line belongs to the
male individual. This type of identification is essential
for the individual identification of biological material of
unknown origin. The probability of occurrence of deter-
mined DNA profile in our population is 5.58 × 10−20 ,
which means that one out of 1.79 × 1019 people is a
holder of this DNA profile.
Sequencing analysis of hypervariable segments of
control region of mitochondrial DNA plays an impor-
tant role – the result is a haplotype mtDNA. Al-
though haplotype presents group identification, it in-
forms about the origin of biological material – phyloge-
netic parameter. Determined haplotype belongs to hap-
logroup W (Line 1: 16223T, 16292T; 73G, 189G, 194T,
195C, 204C, 207A, 263G, 315.1C). This haplogroup oc-
curs in West Eurasia and its frequency in our popula-
tion was calculated by comparison with the database
of mitochondrial DNA hypervariable regions 1 and 2
sequences of individuals from Slovakia. Frequency of
haplogroup W is 0.27% (Lehocký et al. 2008).
923
In this study, we complexly characterized a new
cell culture of human neonatal fibroblasts B-HNF-1
from different points of view: from morphological (light
and transmission electron microscopy), growth poten-
tial (growth curve) as well as molecular biology and
genetics (karyotype, STR typing and mitochondrial
DNA analysis). This culture should be used for further
biomedical experiments.
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Received January 8, 2010
Accepted February 10, 2010