Establishment and Characterisation of a Canine Osteoblastic Cell Line

Normal canine osteoblast cultures are important in experimental studies for comparisons with osteosarcomas and their responses to test therapies. Although canine osteosarcoma cells are available commercially, normal canine osteoblastic cultures are scarce. Osteoblasts are the main cells present in bones that are responsible for the synthesis of the organic matrix during bone formation, regeneration, healing and remodelling. The purpose of this study was to establish and characterise a canine osteoblast cell line, named COBS. Cells were harvested from a bone callus of a 10-month-old German Spitz female dog. Cells were cultivated in a complete medium specific for canine osteoblast growth. Cells underwent seven cycles of culturing to establish a cell lineage. COBS cells were characterised using cytogenetic analysis and immunofluorescence with osteopontin, alkaline phosphatase, and SPARC (secreted protein, acidic and rich in cysteine; also known as osteonectin). Results confirmed that the cells had 78 chromosomes and expressed the osteoblast biomarkers. COBS cells are now available and can be used as controls in anticancer research.


Introduction
Osteoblasts account for 4-6% of the total resident cells in the bone and are commonly known for their bone building function.
Osteoblasts, thus, exert a crucial role in the achievement and maintenance of a correct bone mass, which is accomplished through a close cross-talk with other bone cells, i.e. osteoclasts and osteocytes Capulli et al. [1]. Osteoblasts are derived from mesenchymal stem cells Grigoriadis et al. [2], specialised mesenchymal cells present in bones, that are able to synthesise the dense, crosslinked collagen and specialised proteins, including osteocalcin and osteopontin, which comprise the organic matrix of bone Stein et al. [3]. Osteoblast cultures are becoming more and more important for several purposes, like, for example, bone tissue and bioengineering research Czekanska et al. [4]. Commercial canine osteoblast lineages are scarce, and they are especially important when a normal cell is necessary as controls for studies involving osteosarcoma cells. The aims of this study were, therefore, to establish and characterise a canine osteoblastic cell line for use in experimental studies.

Material and Methods
The study was performed in accordance with the protocols Osteoblasts were collected from a bone callus formed after a tibia fracture in a 10month-old female German Spitz dog. Upon

Abstract
Normal canine osteoblast cultures are important in experimental studies for comparisons with osteosarcomas and their responses to test therapies. Although canine osteosarcoma cells are available commercially, normal canine osteoblastic cultures are scarce. Osteoblasts are the main cells present in bones that are responsible for the synthesis of the organic matrix during bone formation, regeneration, healing and remodelling. The purpose of this study was to establish and characterise a canine osteoblast cell line, named COBS. Cells were harvested from a bone callus of a 10-month-old German Spitz female dog. Cells were cultivated in a complete medium specific for canine osteoblast growth. Cells underwent seven cycles of culturing to establish a cell lineage. COBS cells were characterised using cytogenetic analysis and immunofluorescence with osteopontin, alkaline phosphatase, and SPARC (secreted protein, acidic and rich in cysteine; also known as osteonectin). Results confirmed that the cells had 78 chromosomes and expressed the osteoblast biomarkers. COBS cells are now available and can be used as controls in anticancer research. anaesthesia, the bone callus was scrapped and the osseous material was immediately collected and cultured in a complete medium, specific for canine osteoblast growth (canine osteoblast growth medium, code: CN417500, Sigma). Cells were recovered and recultured at least seven times to establish an osteoblast cell line. Prior to freezing, the osteoblasts were initially washed with PBS and then trypsinised for 10 min in a CO 2 incubator at 37°C.
After cell detachment, confirmed via microscope analysis, the cells were collected and canine osteoblast growth medium was added with 10% FBS for trypsin inactivation. The cell suspension was centrifuged for 10 min at 1200 rpm. After centrifugation, the supernatant was discarded and the cells were resuspended in canine osteoblast growth medium. An aliquot was automatically counted in an automatic cell counter (Countess™, Life Technologies), and the concentration was adjusted to 106 cells/mL with freeze medium (10% DMSO + 90% suspension of cells in FBS). The suspension, already prepared with DMSO, was then aliquoted into properly labelled cryotubes (Corning®), and these were stored in a freezing tank (Nalgene® Mr. Frosty®) and placed in a freezer at -80°C. After freezing, the cryotubes were quickly removed from the freezer and placed in a drum containing liquid nitrogen until use.
Defrosting was performed whenever necessary, by removing the cryotubes from the nitrogen drum and placing them in a water bath at 37°C. Once the cell suspensions were thawed, canine osteoblast growth medium was added with FBS and then centrifuged for 10 min at 1200 rpm. After centrifugation, the cells were resuspended   Pasteur pipettes, the supernatant was carefully discarded and the pellet was resuspended by adjusting the vial volume to 5 mL with hypotonic solution (0.075M KCl) at a temperature of 37°C. The tube was incubated in a water bath at 37°C for 10 min, then removed and placed at room temperature. Eight drops of ice-cold fixative (75% methanol and 25% acetic acid) were added and the mixture was allowed to homogenise for another 10 min at 37°C. After the hypotonisation period, the material was centrifuged at 1500 rpm for 5 min. For material fixation, the supernatant was discarded and the vial volume was adjusted to 5 mL with ice-cold fixative. The contents were homogenised and centrifuged again at 1500 rpm for 5 min.
The supernatant was discarded with the aid of the Pasteur pipette and the pellet was resuspended with the frozen fixative, adjusting the volume to 5 mL. This procedure was performed two additional times to remove cellular debris from the preparation. Finally, the cell pellet was resuspended in 1 mL of frozen fixative and stored in a freezer at -20°C until the preparation of the metaphase slides, the staining, and the analyses. After all steps of the material processing were performed, and a cytogenetic pellet was obtained, the dilution of the material was adjusted with the fixative so that the material was not saturated. The slide used was first cleaned with absolute alcohol and gauze, and then dipped in distilled water and held at an angle so that a drop with the cytogenetic material (dripped with the aid of a Pasteur pipette) could flow across the slide. After drying the material (24 h at room temperature, 25°C), the prepared slides were subjected to staining techniques. The slides were incubated for 30 sec in a trypsin solution diluted 1:250 with Dulbecco's buffer.
The digestion reaction was then blocked with a rapid dip of the slides in Dulbecco's buffer solution plus 5% foetal bovine serum.
The slides were stained with Giemsa (1:30) for 10 min and washed with distilled water. After drying, the slides were analysed. Images

Discussion and Conclusions
Ahs been established canine osteoblastic cell line, called COBS, from a bone callus scraping from a tibia fracture of a 10-monthold female Spitz-German dog. We observed that the normal COBS cell line that was established in our laboratory has somatic cells with 78 chromosomes (2n = 78), which is consistent with the normal number of chromosomes of C. lupus familiaris. In this cell line, we observed the expression of bone markers such as alkaline phosphatase, osteopontin, and SPARC. These results confirmed that the COBS cell line is comprised of isolated osteoblasts, which allows their use as controls in antineoplastic research.