Development and Cytogenetic Characterization of a Continuous Bovine Kidney Cell Line (IRKHBK) and Ev

Development and Cytogenetic Characterization of a Continuous Bovine Kidney Cell Line (IRKHBK) and Evaluation its Susceptibility to some Viruses Archives of Razi Institute, Vol. 70, No. 3 (2015) 1 63-169 Copyright © 2015 by Razi Vaccine & Serum Research Institute INTRODUCTION Viruses are obligate intracellular parasites that h ijack the host’s cellular machinery during their replicat ion cycle. Cultured cells, specific pathogen free eggs and laboratory animals may be used for virus isolation. Although this approach is often slow and requires considerable technical expertise, it has been regar ded for decades as the “gold standard” for the laborato ry diagnosis of viral disease (Leland & Ginocchio 2007 ). Fast replication in cell culture and high productio n yields are the key success factors for the broader adoption of cell culture technology for virus isola tion in most laboratories (Freshney 2005). Propagation of viruses in cell cultures was first described as ear ly as 1913 for vaccinia virus, and in the 1930s for both smallpox virus and yellow fever virus (Storch 2000, Lednicky & Wyatt 2012). Primary cell culture is use ful Original Article Development and Cytogenetic Characterization of a Continuous Bovine Kidney Cell Line (IRKHBK) and Evaluation its Susceptibility to some Viruses Masoudi ∗ ∗∗ ∗ , S. Department of Research and Production of Poultry Viral Vaccine , Razi vaccine and Serum Research Institute, Karaj, I ran Received 19 May 2015; accepted 29 August 2015 Author for correspondence. Email: [email protected] c.ir ABSTRACT In this syudy a continuous bovine kidney cell line derived from a primary bovine kidney cells was established for the first time in Iran. The cells w ere originating from two-day-old normal male calf o f Holstein breed. The cell cultures were continuously passaged following complete proliferation of prima ry cells. The specific properties or characteristics o f the cell were defined using cytogenetic and tumor igenicity analysis. An increasing in cell proliferation was o bserved at 30 th passage. Subsequently chromosomes analysis was shown the first chromosomal adhesion. In karyotyping a decrease in number of the cell ′ s chromosomes (n=59) was detected compared to the nor mal bovine cells chromosome count (2n=30). The cell obtained unlimited proliferation capacity from 70th passage and was identified as infinite cells at passage 90th. The continuous cell line named Iran Razi Khedmati Bovine Kidney (IRKHBK) and was deposited in National Cell Bank of Iran (NCBI), Pas teur Institute. Susceptibility of the IRKHBK cell line for isolation and replication processes of bovine herpesvirus-1 (BHV-1) and bovine virus diarrhea-mucosal disease (BVD-MD) were also evaluated. The results showed that this c ell is more susceptible to the viruses compared to primary bovine kidney cells. According to our results, IRKHBK cell is recommended for routine assays of viruses as a substitution for pri mary bovine kidney cells. Keywords: Cell line, IRKHBK , Karyotype, Chromosome Masoudi et al / Archives of Razi Institute, Vol. 70 , No. 3 (2015) 163-169 164 for virological methods purpose but preparation of primary cells from various organs of animals has several disadvantages; they must be prepared at reg ular intervals from a reliable supply of animal tissue w hich is costly and time consuming. A primary cell cultur e may be composed of mixtures of cell types and canno t be characterized (Pye 1989, Leland & Ginocchio 2007). Moreover there is possibility of the primary and secondary contamination of the cells to latent viru ses because it cannot be readily tested for freedom fro m contamination prior to use (Leland & Ginocchio 2007 ) . By the early 1970s, diagnostic virology expanded dramatically, largely because of the availability o f highly of purified reagents and commercially prepar ed cell lines. Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. The cell lines do not have the above mentioned disadvantages and can be stored in liquid nitrogen until used (Pye 1989, Lednicky & Wyatt 2012). The role of virus isolation is most signific ant in the diagnosis of new or unexpected infection, and i n yielding infectious virus for further study. Mode clinical virology relies on rapid virus detection f or timely infection control and antiviral therapy (Ogi lvie 2001, Storch 2000). Thus a continuous cell line, which is sensitive for virus replication with high prolif eration capacity, is required. Here the development and cytogenetically characterization of a continuous ce ll line from a normal bovine kidney were described. MATERIALS AND METHODS Cell source. The standard methods were essentially followed in preparing primary bovine kidney (BK) cells for culture (Freshney 2005). A 2-day-old healthy ma le calf of Holstein breed was selected. The kidneys we re removed aseptically, minced, washed with PBS two times and then dispersed 0.25% trypsin in PBS, pH 7 .2 for 15 minutes at room temperature using a magnetic stirrer. The dispersed cells were centrifuged at 15 00 rpm at 4 ° C for 10 minutes. The supeatant was discarded and the cells were suspended to a concentration of 10 4 cells per ml in the Earle's solution supplemented with 10% new bo calf serum, %0.4 per cent lactalbumin hydrolysate, and %0.08 per cent ye ast extract (YLE) medium, 100 units penicillin, 0.1 mg streptomycin and 100 units mycostatin per ml. The medium was adjusted to pH 7.0 with 1% saturated NaHCO 3 . Sterile bottles were seeded with 100 ml of the cell suspension per bottle. Cells were grown at 37ºC for 5-7 days at which time they were usually conflu ent and ready for passaging. Cell passaging. The confluent culture of the primary cell culture is selected for passaging. The cell pa ssaging involved standard procedures based on trypsin-verse ne solution, and fresh culture were initiated using sp lit ratio (of 1:2-1:6) (Freshney 2005). Briefly, the me dium was discarded, and the culture was rinsed with 3 ml trypsin-versene for 1-2 minutes to aid the cell lib eration process, following which, the bottle was stored at room temperature for about 5 minutes. After complete cel l liberation, 200 ml fresh YLE medium containing 10% serum was added to each bottle and the bottles were vigorously shaken until a homogeneous suspension wa s obtained and the cell suspension was divided to 2 – 6 Roux bottles. The culture was incubated at 37 ° C for 5- 7 days until they were ready for next passage. The morphological analyses of the cell line were carrie d out using microphotographs of the cultures, taken with an inverted microscope (Zeiss, Jena, Germany). Cytogenetic. Cytogenetic analysis was carried out every ten passages of the cell line that was in an exponential growth phase, with the standard procedu re (McConnell et al 1990). The cells were arrested in metaphase with 0.0016% colchicines, harvested with 0.05% trypsin, treated with hypotonic solution (0.0 75 KCl) for about 20 minutes at 37ºC, and fixed with 3:1 methanol/ acetic acid. Slides were prepared and submitted to standard Giemsa staining (G banding) (Lima et al 2004, Freshney 2005, Chauffaille et al 2003) and abnormalities was described. Cryopreservation. The cell line which was named IRKHBK cryopreserved according to the protocol used by others with some modification (Lima et al 2004, Masoudi et al / Archives of Razi Institute, Vol. 70 , No. 3 (2015) 163-169 165 Chauffaille et al 2003, Freshney 2005, Simione 2009). The cells were removed in a trypsin-EDTA solution and cryopreserved in 10% dimethylsulfoxide (DMSO), 50% stoker medium, 20% fetal calf serum, 10% 0.5 M sucrose, 10% bovine albumin, aliquot in vials and t he temperature is then gradually reduced to -20 ºC for 0.5 h, -70 ºC for 1 h, and the cells are then immersed in liquid N2 or stored at -70 ° C. For thawing, the cells were rapidly thawed in 37 ° C water after which they are placed in a rehydrating solution (10% fetal cal f serum, and 25% stoker solution). After this procedu re, the material is washed once or twice and then transferred to tissue culture bottles, where it is incubated and maintained at 37 ° C. Tumorigenicity test. Cells of monolayer cultures were trypsinized and counted in a haemocytometer (Freshney 2005). Then the cells suspended in Stoker medium with 2% bovine serum. Ten million cells were injected subcutaneously in the sold of mouse. Susceptibility test. Susceptibility of different passages of IRKHBK cell line to viruses was examined by isolation of bovine herpesvirus-1 (BHV-1) and Bovine viral diarrhea Virus (BVDV) . RESULTS Development of the cell line. Cultured primary bovine kidney ( BK ) cells were immortalized in vitro by spontaneous transformation; a biological phenomenon leads to produce the Iran Razi Khedmati Bovine Kidney (IRKHBK) cell line. The BK cells were mixture of fibroblast and epithelial like cells morphologicall y. As shown in figure 1 at the 70 th passage the cells were established and a uniform population of cells in as pect of morphology and growth rate did all the succeedin g passages. The cells were featured epithelial-like c ells with cell cycle approximately 24 hours long, with maximum confluence at 36 hours. Cytogenetic analysis. The cell incubated at 37 ° C up to 6 days. Chromosome analyses were performed on 2 nd , 20 th , 30 th , 60 th , 94 th , 155 th and 200 th passages. Karyological examination of the cell line revealed that the ploidy of the IRKHBK cell line evolved from diploid at 20 th passage to aneuploid at the 30th passage (Figure 2). The Karyotype data are detailed in Tabl e 1. Analysis of the different IRKHBK cell passages indicates the cell features at 2 nd and 20 th passages was normal and no differences observed between their karyotype data and primary cell culture. The first change in cell karyotype was found at 30 th passage in which two chromosomes joined together from the centromer site. The translocation leads to reductio n in number of chromosomes (n=59) compared to the diploid normal bovine somatic cells (n=60) (Figure 2). By continued passaging number of cell chromosome was decreased to 55, 48, 47 and 46 at 60 th , 94 th , 155 th and 200 th passages respectively (Figures 3, 4, 5, 6). Tumorigenicity test. Tumorigenicity is defined as the ability of viable cultured cells to give rise t o progressively growing tumor nodules, showing viable and mitotically active cells. In case, 20 adult mic e were inoculated with the cell line and observed twice a week for appearance of tumors. After 6 months no tumors were developed at the inoculation site. Susceptibility test. The results show that the IRKHBK cell line was highly sensitive for isolation of BHV-1 and BVD MD compared with primary bovine kidney cell. The viruses were replicate easily in IRKHBK cell (Table 2) and the marked cytophatic effect due to the virus replication was observed at shorter time than primary cell culture (Figure 7). DISCUSSION In order to reduce the time required for the virus isolation and development of viral vaccines, host c ell lines should be easily available and safe in labora tories. Because of some disadvantages associated with manufacture in primary cells, unlimited life span continuous cell lines have been developed. The cell s have reduced host complexity lead to reduction in v irus strain diversity and vaccine production yields. (Lednicky & Wyatt 2012, Matsuura et al 2011, Leland et al 2007, Ogilvie 2001, Pye 1989). In this study a bovine cell line, IRKHBK , was developed during extended in vitro passaging. Cytogenetic analysis forms Masoudi et al / Archives of Razi Institute, Vol. 70 , No. 3 (2015) 163-169 166 Table1. Characteristics of IRKHBK cell line at 2-200 passages levels. The numbers of Chromosomes in 50 or more cells from each passage level were counted. Passage level P 2 P 15 P 30 P40 P 50 P 55 P 94 P 15 5 p 200 Total cells 51 50 52 51 56 50 50 50 50 Normal (diploid) 46(92%) 45(90%) 44(86%) 13(26 %) - - - - - polyploidy 2 (4%) 3(6%) 3 (6 % ) 4 (8%) 3 (6 %) 4 ( 8 %) 4 4 (8 %) 1 (2 %) 3 (6 %) Hypodiploidy 1 (2%) 2 (4%) 1 (2%) - - - - - - Hyperdiploids 1 (2%) - - - - - - - - Break or Gap 1 (2%) - 2 (4%) 2 (4%) - - 1 (2%) - - Structural aberration % - - 2 (4%) 32 (64%) 56(100% ) 50(100%) 50(100%) 50(100%) 50(100%) Table 2. CPE effect of viruses on Primary BK cell and IRKHBK cell line Viruses Primary BK cell IRKHBK cell line BHV-1 3-4 * 1-2 PI3 4-5 2-3 RPV 5-6 3-4 BVD-MD 6-7 4-5 PPR 7-8 5-6 * Days after inoculation of cell line with viruses th at CPE seen. Figure 1. (a) Primary bovine kidney cells, 100X; (b) Establi shed IRKHBK cell in 70 th passage 100X. A B A B A X Y Figure 2. In this photograph the chromosomes have been stain ed with Giemsa (G-banding) and arranged in a standard fashion. This standard arran gement of an individual chromosome is known as a karyotype. (a) Karyotype of normal bovine somatic c ells; (b) Karyotype of IRKHBK at 30 th passage. Joining of two chromosomes from centromeric site ar e shown with flash (n =59). Masoudi et al / Archives of Razi Institute, Vol. 70 , No. 3 (2015) 163-169 167 Figure 3. Karyotype of IRKHBK cell line at 60 th passage. Numbers of chromosome of the cell is 55 (n=55). Translocated chromosomes are shown with flash. Figure 4. Karyotype of IRKHBK at 94 th passage. Numbers of chromosome of the cell is 48 (n=48). Figure 5. Karyotype of IRKHBK at 155 th passage. Numbers of chromosome of the cell is 47 (n=47). Figure 6. Karyotype of IRKHBK at 200 th passage. Numbers of chromosome of the cell is 46 (n=46). b an essential part of characterizing and identifying cell lines. The analysis is performed on cell cultures t o perform identity checks by verifying species of ori gin or the retention of key chromosome rearrangements i n cell lines. Karyotypic changes occurred in parallel with the phenotypic changes as shown in figures 1 and 2 and immortalization occurred between passages 70 and 90 . The cell was successfully passaged and until the 15 th passage did not show any changes neither in growth rate nor in its morphology. At this passage the growth rate of the cell and als o the transferable cell population rate began to decrease . We had an increase in cell growth rate from the 18 th passage but it is decreased severely again and we h ad to collect the cells of 2-4 bottle in one 2 oz till 30 th passage. The first chromosomal change was observed (Figure 2) following gradually increase in growth r ate of the cells at this passage and although the prima ry BK culture was a mixture of fibroblast and epithelial like a Figure 7. (a) IBR virus-infected IRKHBK Continuous cell line, 100X. (b) BVD - MD virus - infected IRKHBK Continuous cell line. b Masoudi et al / Archives of Razi Institute, Vol. 70 , No. 3 (2015) 163-169 168 Figure 8. IRKHBK Continuous cell line Foot and Mouth Disease virus Infected after 72 hours ( Rafiei et al 2014). cells, the epithelial cells began to predominant fr om 40 th passage, and at the 60 th passage the fibroblast-like cells were eliminated. The growth rate of epithelial-like cells was increa sed in the next passages until establishment of a uniform population of cells in aspect of morphology and gro wth rate. From the 70 th passage the cells were obtained an unlimited growth capacity in which at passage 90 th identified as infinite cell. At 30 th passage two chromosomes joined together revealed the first chan ge was occurred in the cell karyotype. The number of chromosomes in somatic cells is constant and termed the diploid number or 2n (Simione 2009). Despite th e normal bovine somatic diploid cell (2n=30) (Mircham si et al 1978) number of IRKHBK cell chromosome at 30th passage decreased indicating the ploidy of the cell was switch to aneuploid. Decrease in chromosome number was continued up to the last passage (200 th passage). The IRKHBK cell is easy cultured and has a satisfactory growth rate with high confluences (36 hr). The continuous cell line supports the virus’s repli cation and is even more sensitive for parasite proliferati on. Several studies on impact of IRKHBK cell line for virus isolation indicated that the examined viruses inclu de BHV-1 , equine herpes virus-1, human herpes virus-1, foot and mouth disease virus (Figure 8), and herpes simplex virus type-1 have been well isolated (Pishraft Sabet et al 2003, Rafiei et al 2014, Rafiei et al 2015, Shirvani et al a 2011, Shirvani et al b 2011, Soleiman Jahi et al 2004, Taghipour-Bazargani 2014). Mahmodzadeh and the co-workers (2008) have studied the proliferation of Pneumocystis carinii on IRKHBK cell line and compared its growth rate with Vero an d MRC-5 cell lines. They conclude that the difference between IRKHBK and two other cell lines was significant (p = 0.023) and IRKHBK cell line is suitable for parasite proliferation assay. The IRKHBK continuous cell line also known as Razi Bovine Kidney (RBK) was submitted to National Cell Bank of Iran (NCBI) under C541 code. Ethics I hereby declare all ethical standards have been respected in preparation of the submitted article. Conflict of Interest The authors declare that they have no conflict of i nterest. Grant Support This work was supported by Razi Vaccine & Serum Research Institute of Iran. Acknowledgments The author wishes to thank Dr. K Khedmati for generously providing materials and helpful discussi ons. The author thanks Dr. S. Shahsavandi for a critical reading of the manuscript and Dr. M Lotfi, Dr. A Mirjalili, Mr. F Kamali, Mr. R Sarmast, and Mr. A Bakhtiary for their assistance. References Chauffaille, M. F., Pinheiro, R. F., Stefano, J. T. , and Kerbauy, J. (2003). Karyotype of cryopreserved bone marrow cells. Brazilian Joual of Medical and Biological Research 36: 845-850. Freshney, R. (2005). Culture of animal cells : A Manual of Basic Technique . 5th ed. New York, Wiley- LISS Publications Ltd. Jeon, K. L., and Hwang, K. K. (2014). Immortalizati on of bovine primary epithelial cells to isolate foot-and -mouth disease virus. Joual of Preventive Veterinary Medicine 38: 15-25. Khedmati, K., Kargar Moaakhar, R., and Masoudi, SH. (1998). Determination of sensitivity of IRKHBK to animal

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