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 ¨ hmer1, Milan Blaško1, Vanda Repiská1, Ivan Varga2,3,4, Ivan Lehocký5, Daniel Bo 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:
[email protected] 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 Recently, a large amount of chemicals or materials that come into close contact with the human body are produced. 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, personnel and economic load. Moreover, some organizations 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 systems 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* Corresponding author
c 2010 Institute of Zoology, Slovak Academy of Sciences
cur. Another important advantage of in vitro system is the possibility to define strict experimental conditions with the elimination of external factor influence (Davila et al. 1998). In addition, in vitro system provides more information about the mechanisms of action 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 human 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 associated with extracellular matrix (ECM) molecules. When activated, fibroblasts exhibit an abundant endoplasmic reticulum and prominent Golgi apparatus
920 associated with the synthesis and secretion of ECM molecules including collagens, proteoglycans and fibronectin as well as proteases capable of degrading the ECM (McAnulty 2007). It has been estimated that each cell can synthesise approximately 3.5 million procollagen molecules per day (McAnulty et al. 1991). Fibroblasts 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 characteristics 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 specimen of right musculus quadriceps femoris of a two-monthold human male. Sampling procedure was indicated due to cytogenetical analysis and was performed by a physician under 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 sectioned into small fragments by sterile scalpel. The fragments were washed twice with PBS. Subsequently, they were transferred into a plastic 40 mm Petri dish (TPP, Switzerland) with Eagle’s minimal essential medium (EMEM, 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 culture 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 mycopalsma 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 passage were also used for morphological and karyotype analysis 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, Germany) in concentration of 0.05 µg ml−1 . After three hours of incubation, the karyological slides were prepared according to conventional method (Freshney 2005). The slides were air-dried and stained with 2% Giemsa solution for ten minutes. 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 postfixed 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). Subsequently, the samples were embedded in durcupan (Serva, Germany) and cut into semi-thin sections. Ultra-thin sections were mounted on 200 mesh copper grids, then double stained using uranyl acetate and lead citrate (Serva, Germany) and examined using a transmission electron microscope Morgagni 268D. STR typing DNA was isolated from the cell pellet of culture line by digestion with Proteinase K and subsequent extraction with Phenol/chloroform method. The amount of DNA was determined by agarose gel electrophoresis in comparison to a lambda DNA standard. STR analysis was conducted using the multiplexPCR-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 identification 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 solution containing sodium acetate, absolute ethanol and distilled water for each sample and purified from residual terminators by washing in 70% ethanol. DNA analysis was run on Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).
Characterization of cell culture B-HNF-1
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Sequencing of HV1 and HV2 regions was performed using DNA Sequencing Analysis Software version 5.2 (Applied Biosystems, CA, USA) and sequences were aligned using ABI PRISM SeqScape Software version 2.5 (Applied Biosystems, CA, USA). Quality control: According to ISFG recommendations. The double evaluation of the profiles ascertained the quality of the results.
Results and discussion Fibroblasts are the major components of the connective tissue. The fibroblast synthesizes and continuously secretes mature proteoglycans and glycoproteins and the precursor molecules of various types of collagens and elastin. Many in vitro models used fibroblast, for example for modelling of wound healing (Moulin et al. 1996), remodelling of the bronchial wall in asthma (Michalik et al. 2009), specific alterations in myocardial fibroblasts biology after myocardial infarction (Squires et al. 2005), attachment behaviour of periodontal ligament fibroblasts to dentin surface (Al-Nazhan 2004) or to describe the effect of laser irradiation on cultured human periodontal fibroblasts (Chen et al. 2005). Moreover, cell cultures of fibroblasts belong to the most used biological models in the evaluation of cytotoxicity, genotoxicity 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 comprehensive analysis of biological and morphological properties of human neonatal fibroblast culture B-HNF-1 as well as STR typing and mitochondrial DNA amplification and sequencing. The cell culture was established by a standard method of primary explants, which was originally developed 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 adherent cells (Li et al. 2009; Wang et al. 2003; Kinugawa et al. 1998). After the initiation of the cell culture, some tissue fragments adhered and the migration of fibroblasts as well as epithelial cells was observed after 72 hours. During the following five days, the cells reached confluence and were trypsinized to obtain monocellular suspension for further sub-cultivation. In case of different toleration to trypsin during following passages, epithelial cells disappeared. Purified fibroblast culture was obtained after second passage. They are of a typical long spindle shape (Fig. 1). Moreover, after passage, the cell growth accelerated until next confluence. Contact inhibition was observed if they were not sub-cultured promptly. A similar course of fibroblast cultivation was described by many authors (Li et al. 2009; Wu et al. 2008; Vojtaššák et al. 2006). Fig. 2 shows the growth curve of the B-HNF-1 cells. The lag time was almost undetectable; the fibroblasts adhered very quickly after cell seeding. Cells then started to proliferate and their number increased logarithmically. They reached the stationary phase after
Fig. 1. Human neonatal fibroblast cell B-HNF-1 culture (× 100).
Fig. 2. Growth curve of the B-HNF-1 cells.
96 hours and the population doubling time was about 24 hours. Results obtained by other authors were quite diverse and they were dependent on the species origin and age of the tissue donor (Li et al. 2009; Wu et al. 2008; Veelken et al. 1994). Every test on bacterial or mycopalsma infection prior to freezing was negative. The viability was 97%. These results indicated that B-HNF-1 cell line was healthy and ready for storage in liquid nitrogen and for subsequent utilization. Analysis of chromosomes count (Fig. 3) showed that B-HNF-1 cell culture is diploid; the modal number of chromosomes was 46 and has normal male karyotype 46, XY, which was stable during cultivation. We did not record any chromosomal aberration, neither in structure nor in number. Fig. 4 illustrates the normal morphological features of fibroblast in light microscopy level, with Nomarski interference contrast. Each spindle-shaped fibroblast has a large nucleus with one to four nucleoli and many long
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Fig. 3. Karyotype analysis of B-HNF-1 cell culture: normal male karyotype 46, XY.
Fig. 4. Light electron microscopic observation of human neonatal fibroblast cell culture B-HNF-1 with Nomarski interference contrast. Each nucleus contains 1 to 4 nucleoli (× 400).
and branched processes. The transmission electron microscopy demonstrated the ultra-structure of the human neonatal fibroblasts B-HNF-1; we can see the typical morphological features of proteosynthesis-active cells (Figs 5–8). Large number of fibroblasts bearing different 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 electron microscopy. Our in vitro expanded fibroblasts have a large and irregular nucleus with prevalence of euchromatin. One to four nucleoli are present in each nucleus. The cytoplasm contains a richly developed rough endoplasmic reticulum and prominent Golgi apparatus cisterns. 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 analysis; we observed only a seven-days-old cell culture. The result of the fragment analysis is a DNA profile defined as multiplex of STR markers and sex determin-
Fig. 5. Transmission electron microscopic observation of human neonatal fibroblasts B-HNF-1. Each cell have large, pale nucleus (1), in the cytoplasm abundant dilated cisterns of rough endoplasmatic reticulum (2). The cells adhered to the substrate with microvilli and filopodia (3). (× 8900).
Fig. 6. TEM observation of human neonatal fibroblasts B-HNF-1. We can see typical features of proteosynthetic active cell: large, pale nucleus (1), markedly nucleolus (2), in the cytoplasm abundant cisterns of rough endoplasmatic reticulum (3). (× 7100).
Characterization of cell culture B-HNF-1
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 potential (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. References
Fig. 7. TEM observation of human neonatal fibroblasts B-HNF1. 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 determined 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 important role – the result is a haplotype mtDNA. Although haplotype presents group identification, it informs about the origin of biological material – phylogenetic parameter. Determined haplotype belongs to haplogroup W (Line 1: 16223T, 16292T; 73G, 189G, 194T, 195C, 204C, 207A, 263G, 315.1C). This haplogroup occurs in West Eurasia and its frequency in our population 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).
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