M E T H O D S I N M O L E C U L A R M E D I C I N E TM Bone Research Protocols Edited by Miep H. Helfrich, PhD Stuart H. Ralston, MD Bone Research Protocols Edited by Miep H. Helfrich, PhD Stuart H. Ralston, MD Human Osteoblast Culture 3 3 From: Methods in Molecular Medicine, Vol. 80: Bone Research Protocols Edited by: M. H. Helfrich and S. H. Ralston © Humana Press Inc., Totowa, NJ 1 Human Osteoblast Culture James A. Gallagher 1. Introduction Osteoblasts are the cells responsible for the formation of bone; they synthesize almost all of the constituents of the bone matrix and direct its subsequent mineralization. Once a phase of active bone formation is com- pleted the osteoblasts do not become senescent but instead redifferentiate into one of two other cell types: osteocytes and bone lining cells, both of which play a major role in the regulation of calcium homeostasis and bone remodeling. Researchers have endeavored to culture osteoblasts from human bone for several reasons: 1. To investigate the biochemistry and physiology of bone formation. 2. To investigate the molecular and cellular basis of human bone disease. 3. To investigate the role of cells of the osteoblastic lineage in regulating bone resorption. 4. To screen for potential therapeutic agents. 5. To develop and test new biomaterials. 6. To use cell therapy in tissue engineering and bone transplantation. The structure of bone tissue, the heterogeneity of cell types, the cross- linked extracellular matrix, and the mineral phase combine to make bone a difficult tissue from which to extract cells. Consequently, early attempts to culture osteoblasts avoided human tissue and instead relied on enzy- matic digestion of poorly mineralized fetal or neonatal tissue from experimental animals. The first attempt to isolate cells from adult human bone, using demineralization and collagenase digestion, was reported by Bard and co-workers (1). The cultured cells were low in alkaline phos- phatase and collagen synthesis, which were then regarded as the best 01/Gall/1-18/F1 2/26/03, 10:44 AM3 4 Gallagher markers of the osteoblastic phenotype. Although the cells remained viable for up to 2 wk they did not proliferate, and it was concluded that osteocytes were the predominant cell type present. Mills et al. used the alternative approach of explant culture and were successful in culturing cell populations that included parathyroid hormone (PTH) responsive and alkaline phosphatase positive cells (2). The first successful attempts to isolate large numbers of cells that expressed an osteoblastic phenotype from human bone were undertaken in Graham Russell’s laboratory at the University of Sheffield in the early 1980s. The defining characteristics of these studies were (1) the use of explant cultures, which avoided the need for digestion of the tissue and (2) the availability of an appropriate phenotypic marker. Successful culture of any cell type can be achieved only if there is a specific marker of the phenotype that can be used to confirm the identity of the cells in vitro. In this case, the marker was the then recently discovered bone gla protein as measured by a radioimmunoassay developed by Jim Poser (3,4). Nearly 20 yr later, bone gla protein, now known as osteocalcin, undoubtedly remains the most specific marker of the osteoblas- tic phenotype. Although this culture system has been extensively modified by several groups of researchers (see Note 1), the vast majority of published reports on isolation of human osteoblasts still essentially use this simple but highly repro- ducible explant technique. This technique and its modifications have been described, compared, and reviewed elsewhere (5,6). The aim of this chapter is to describe the basic methodology that is used in the author’s laboratory. This is shown schematically in Fig 1. The nomenclature used by various research groups to describe the isolated cells includes “human bone cells,” “human osteoblasts in vitro,” “human osteoblastic cells,” and “HOBS.” We have pre- ferred the conservative term “human bone derived cells” (HBDCs), and this is used throughout this chapter. HBDCs have been widely used to investigate the biology of the human osteoblast, and their use has facilitated several major developments in our understanding of the hormonal regulation of human bone remodeling. These cells have also been used to investigate the cellular and molecular pathology of bone disease. The major milestones in the culture of human osteoblasts are summarized in Table 1. Figure 2 shows the increase in the application of human osteoblast cultures since the initial reports in 1984. Human bone cell culture is now becoming an important tool in tissue engi- neering to test the biocompatibility and osteogenicity of novel biomaterials and also for autologous transplantation of osteoblastic populations expanded in vitro. 01/Gall/1-18/F1 2/26/03, 10:44 AM4 Human Osteoblast Culture 5 2. Materials 2.1. Tissue-Culture Media and Supplements 1. Phosphate-buffered saline (PBS) without calcium and magnesium, pH 7.4 (Invitrogen). 2. Dulbecco’s modification of minimum essential medium (DMEM) (Invitrogen) supplemented to a final concentration of 10% with fetal calf serum (FCS), 2 mM L-glutamine, 50 U/mL of penicillin, 50 µg/mL streptomycin. Freshly prepared 50 µg/mL of L-ascorbic acid should be added to cultures in which matrix synthesis or mineralization is being investigated (see Note 2). 3. Serum-free DMEM (SFM). 4. FCS (see Note 3). 5. Tissue culture flasks (75 cm 2 ) or Petri dishes (100-mm diameter) (see Note 4). 2.2. Preparation of Explants 1. Bone rongeurs from any surgical instrument supplier. 2. Solid stainless steel scalpels with integral handles (BDH Merck). Fig. 1. Technique used to isolate cells expressing osteoblastic characteristics (HBDCs) from explanted cancellous bone. E1, explant 1; E2, explant 2. 01/Gall/1-18/F1 2/26/03, 10:44 AM5 6 Gallagher 2.3. Passaging and Secondary Culture 1. Trypsin–EDTA solution: 0.05% Trypsin and 0.02% EDTA in Ca 2+ - and Mg 2+ - free Hanks’ balanced salt solution, pH 7.4 (Invitrogen). 2. 0.4% Trypan blue in 0.85% NaC1 (Sigma Aldrich). 3. 70-µm “Cell Strainer” (Becton Dickinson). 4. Neubauer hemocytometer (BDH Merck). 5. Collagenase (Sigma type VII from Clostridium histolyticum). 6. DNase I (Sigma Aldrich). Table 1 Phenotypic Milestones in the Culture and Characterization of Osteoblastic Cells (HBCDs) from Human Bone Isolation of viable cells from human bone Bard et al., 1972 (1) Introduction of explant culture Mills et al., 1979 (2) Production of osteocalcin Gallagher et al., 1984 (3) Beresford et al., 1984a (4) High alkaline phosphatase activity Gallagher et al., 1984 (3) Beresford et al., 1984 (4) Gehron-Robey and Termine 1985 (7) Auf’molk et al., 1985 (8) Responsiveness to PTH Beresford et al., 1984 (4) MacDonald et al., 1984 (9) Gehron-Robey and Termine 1985 (7) Auf’mkolk et al., 1985 (8) MacDonald et al., 1986 (10) Synthesis of type I but not type III collagen Beresford et al., 1986 (11) Synthesis of other bone matrix proteins Gehron-Robey and Termine 1985 (7) Fedarko et al., 1992 (12) Response to cytokines Beresford et al., 1984 (13) Gowen et al., 1985 (14) Response to oestrogen Vaishnav et al., 1984 (15) Eriksen et al 1988 (16) Expression of purinoceptors Schoefl et al., 1992 (17) Bowler et al., 1995 (18) Production of nitric oxide Ralston et al., 1994 (19) Investigation of specific pathologies Marie et al., 1988 (20) Walsh et al., 1995 (21) Formation of mineralised nodules Beresford et al., 1993 (22) Formation of bone in vitro and in vivo Gundle et al., 1995 (23) 01/Gall/1-18/F1 2/26/03, 10:44 AM6 Human Osteoblast Culture 7 2.4. Phenotypic Characterization 1. 1,25-Dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] (Leo Pharmaceuticals or Sigma Aldrich). 2. Menadione (vitamin K3) (Sigma Aldrich). 3. Alkaline phosphatase assay kit (Sigma Aldrich). 4. Staining Kit 86-R for alkaline phosphatase (Sigma Aldrich). 5. Osteocalcin radioimmunassay (IDS Ltd., Boldon, UK) (see Note 5). 6. Polymerase chain reaction (PCR) primers and reagents for a panel of osteoblastic markers including osteocalcin (IDS Ltd., Boldon, UK). 2.5. In Vitro Mineralization 1. Dexamethasone (Sigma Aldrich). 2. Hematoxylin (BDH Merck). 3. L-Ascorbic acid (see Note 2). 4. Inorganic phosphate solution: Mix 500 mM solutions of Na 2 HPO 4 and NaH 2 PO 4 in a 4:1 (v/v) ratio. Sterile filter and store at 4°C prior to use. 2.6. Cryopreservation of Cells 1. Dimethyl sulfoxide (DMSO) (Sigma Aldrich). 2. Cryoampules. 3. Cell freezing container. Fig. 2. Graph showing the increase in the application of human osteoblast cultures since the initial reports in 1984. 01/Gall/1-18/F1 2/26/03, 10:44 AM7 8 Gallagher 3. Methods 3.1. Establishing Primary Explant Cultures A scheme outlining the culture technique is shown in Fig. 1. 1. Transfer tissue, removed at surgery or biopsy, into a sterile container with PBS or serum-free medium (SFM) for transport to the laboratory with minimal delay, preferably on the same day (see Note 6). An excellent source is the upper femur of patients undergoing total hip replacement surgery for osteoarthritis. Cancel- lous bone that would otherwise be discarded is removed from this site prior to the insertion of the femoral prosthesis. The tissue obtained is remote from the hip joint itself, and thus from the site of pathology, and is free of contaminating soft tissue (see Note 7). 2. Remove soft connective tissue from the outer surfaces of the bone by scraping with a sterile scalpel blade. 3 Rinse the tissue in sterile PBS and transfer to a sterile Petri dish containing a small volume of PBS (5–20 mL, depending on the size of the specimen). If the bone sample is a femoral head, remove cancellous bone directly from the open end using sterile bone rongeurs or a solid stainless steel blade with integral handle. Disposable scalpel blades may shatter during this process. With some bone samples (e.g., rib), it may be necessary to gain access to the cancellous bone by breaking through the cortex with the aid of the sterile surgical bone rongeurs. 4. Transfer the cancellous bone fragments to a clean Petri dish containing 2–3 mL of PBS and dice into pieces 3–5 mm in diameter. This can be achieved in two stages using a scalpel blade first, and then fine scissors. 5. Decant the PBS and transfer the bone chips to a sterile 30-mL “universal con- tainer” with 15–20 mL of PBS. 6. Vortex-mix the tube vigorously three times for 10 sec and then leave to stand for 30 sec to allow the bone fragments to settle. Carefully decant off the supernatant containing hematopoietic tissue and dislodged cells, add an additional 15–20 mL of PBS, and vortex-mix the bone fragments as before. Repeat this process a mini- mum of three times, or until no remaining hematopoietic marrow is visible and the bone fragments have assumed a white, ivory-like appearance. 7. Culture the washed bone fragments as explants at a density of 0.2–0.6 g of tissue/ 100-mm diameter Petri dish or 75-cm 2 flask (see Note 4) in 10 mL of medium at 37° in a humidified atmosphere of 95% air, 5% CO 2 . 8. Leave the cultures undisturbed for 7 d, after which time replace the medium with an equal volume of fresh medium taking care not to dislodge the explants. 9. Check for outgrowth of cells at 7–10 d (see Note 8). 10. Replace the medium at 14 d and twice weekly thereafter until the desired cell density has been attained. 3.2. Passaging Cells and Establishing Secondary Cultures 1. Remove and discard the spent medium. 2. Gently wash the cell layers three times with 10 mL of PBS without Ca 2+ and Mg 2+ . 01/Gall/1-18/F1 2/26/03, 10:44 AM8 Human Osteoblast Culture 9 3. To each flask add 5 mL of freshly thawed trypsin–EDTA solution at room tem- perature (20°C) and incubate for 5 min at room temperature with gentle rocking every 30 sec to ensure that the entire surface area of the flask and explants is exposed to the trypsin–EDTA solution. 4. Remove and discard all but 2 mL of the trypsin–EDTA solution, and then incu- bate the cells for an additional 5 min at 37°C. 5. Remove the flasks from the incubator and examine under the microscope. Look for the presence of rounded, highly refractile cell bodies floating in the trypsin– EDTA solution. If none, or only a few, are visible tap the base of the flask sharply on the bench top in an effort to dislodge the cells. If this is without effect, incu- bate the cells for a further 5 min at 37°C. 6. When most of the cells have become detached from the culture substratum, trans- fer to a “universal container” with 5 mL of DMEM with 10% FCS to inhibit tryptic activity. 7. Wash the flask two to three times with 10 mL of SFM and pool the washings with the original cell isolate. 8. Centrifuge at 250g for 5 min to pellet the cells. 9. Remove and discard the supernatant, invert the tube, and allow the medium to drain briefly. 10. Resuspend the cell in 2 mL of SFM. If the cells are clumping see Note 9. If required, the cell suspension can be filtered through a 70-µm “Cell Strainer” (Becton Dickinson) to remove any bone spicules or remaining cell aggregates. For convenience and ease of handling the filters have been designed to fit into the neck of a 50-mL polypropylene tube. Wash the filter with 2–3 mL of SFM and add the filtrate to the cells. 11. Take 20 µL of the mixed cell suspension and dilute to 80 µL with SFM. Add5 µL of trypan blue solution, mix, and leave for 1 min before counting viable (round and refractile) and nonviable (blue) cells in a Neubauer Hemocytometer. Using this procedure, typically 1–1.5 × 10 6 cells are harvested per 75-cm 2 flask, of which ≥75% are viable. 12. Plate the harvested cells at a cell density suitable for the intended analysis. We routinely subculture at 5 × 10 3 –10 4 cells/cm 2 and achieve plating efficiencies measured after 24 h of ≥70% (see Note 10). 3.3. Phenotypic Characterization The phenotypic characterization of HBDCs is described in detail in ref. 5. The simplest phenotypic marker to investigate is the enzyme alkaline phos- phatase, a widely accepted marker of early osteogenic differentiation. Alkaline phosphatase can be measured by simple enzyme assay or by histochemical staining. Basal activity is initially low, but increases with increasing cell den- sity. Treatment with 1,25-(OH) 2 D 3 increases alkaline phosphatase activity. The most specific phenotypic marker is osteocalcin. This is a protein of Mr 5800 containing residues of the vitamin K-dependent amino acid γ-carboxyglutamic 01/Gall/1-18/F1 2/26/03, 10:44 AM9 10 Gallagher acid. In humans its synthesis is restricted to mature cells of the osteoblast lin- eage. It is an excellent late stage markers for cells of this series despite the fact that its precise function in bone has yet to be established. Osteocalcin can be measured by one of the many commercially available kits. 1,25-(OH) 2 D 3 increases the production of osteocalcin in cultures of HBDCs, but not fibro- blasts obtained from the same donors. More recently, researchers have adopted the use of reverse transcription (RT)-PCR to look at the expression of osteo- blastic markers in HBDCs. PCR primers for a panel of osteoblastic markers including osteocalcin are shown in Table 2. 3.4. Phenotypic Stability in Culture As a matter of routine we perform all of our studies on cells at first passage. Other investigators have studied the effects of repeated subculture on the phe- notypic stability of HBDCs and found that they lose their osteoblast-like char- acteristics. In practical terms this presents real difficulties, as it is often desirable to obtain large numbers of HBDCs from a single donor. As an alter- native to repeated subculture, trabecular explants can be replated at the end of primary culture into a new flask (see Fig. 1). Using this technique, it is possible to obtain additional cell populations that continue to express osteoblast-like characteristics, including the ability to mineralize their extracellular matrix, and maintain their cytokine expression profile (6). Presumably, these cultures are seeded by cells that are situated close to the bone surfaces, and that retain Table 2 PCR Primers for a Panel of Osteoblastic Markers Osteoblastic phenotype marker Primer pairs T m (°C) Product size (bp) Osteocalcin 5'-ccc tca cac tcc tcg ccc tat-3' 5'-tca gcc aac tcg tca cag tcc -3' 65 246 PTH receptor 5'-agg aac aga tct tcc tgc tgc a-3' 5'-tgc atg tgg atg tag ttg cgc gt-3' 55 571 Alkaline 5'-aag agc ttc aaa ccg aga tac aag-3' phosphatase 5'-ccg agg ttg gcc ccg at-3' 68 715 CBFA1 5'-ccc cac gac aac cgc acc-3' 5'-cac tcc ggc cca caa atc tc-3' 60 388 Osteoprotegerin 5'-ggg cgc tac ctt gag ata gag tt-3' 5'-gag tga cag ttt tgg gaa agt gg-3' 60 760 RANKL 5'-act att aat gcc acc gac atc-3' 5'-aaa aac tgg ggc tca atc ta-3' 54 462 01/Gall/1-18/F1 2/26/03, 10:44 AM10 Human Osteoblast Culture 11 the capacity for extensive proliferation and differentiation. The continued sur- vival of these cells may be related to the gradual release over time in culture of the cytokines and growth factors that are known to be present in the extracellu- lar bone matrix, many of which are known to be produced by mature cells of the osteoblast lineage. The addition of 25 µM L-ascorbic acid (50 µg/mL) (see Note 2) to HBDCs in secondary culture (E1P1) produces a sustained increase in the deposition of matrix due to an increase in the synthesis of collagen and noncollagenous protein and bone sialoprotein and osteocalcin. 3.5. Passaging Cells Cultured in the Continuous Presence of Ascorbate Because of their synthesis and secretion of an extensive collagen-rich extra- cellular matrix, HBDCs cultured in the continuous presence of ascorbate can- not be subcultured using trypsin–EDTA alone. They can, however, be subcultured if first treated with purified collagenase. The basic procedure is as follows: 1. Rinse the cell layers twice with SFM (10 mL/75-cm 2 flask). 2. Incubate the cells for 2 h at 37°C in 10 mL of SFM containing 25 U/mL of puri- fied collagenase (Sigma type VII) and 2 mM additional calcium (1:500 dilution of a filter-sterilized stock solution of 1 M CaCl 2 ). 3. Gently agitate the flask for 10–15 sec every 30 min. 4. Terminate the collagenase digestion by discarding the medium (check that there is no evidence of cell detachment at this stage). 5. Gently rinse the cell layer twice with 10 mL of Ca 2+ - and Mg 2+ -free PBS. To each flask add 5 mL of freshly thawed trypsin–EDTA solution, pH 7.4, at room temperature (20°C). 6. Typically this procedure yields ~3.5–4 × 10 6 cells/75-cm 2 flask after 28 d in pri- mary culture. Cell viability is generally ≥90%. 3.6. Setting Up Mineralizing HBDC Cultures The function of the mature osteoblast is to form bone. Despite the over- whelming evidence that cultures of HBDCs contain cells of the osteoblast lin- eage, initial attempts to demonstrate the presence of osteogenic (i.e., bone forming) cells proved unsuccessful. Subsequently, several authors reported that culture of HBDCs in the presence of ascorbate and millimolar concentrations of the organic phosphate ester β-glycerol phosphate (β-GP) led to the forma- tion of mineralized structures resembling the nodules that form in cultures of fetal or embryonic animal bone derived cells (reviewed in ref. 22). These have been extensively characterized and shown by a variety of morphological, bio- chemical, and immunochemical criteria to resemble embryonic/woven bone formed in vivo. An alternative to the use of β-GP is to provide levels of inor- 01/Gall/1-18/F1 2/26/03, 10:44 AM11 [...]... relationship to the expression of FGF receptors 1-4 Bone 27, 185–195 01/Gall/1-18/F1 18 2/26/03, 10:44 AM Bone Cells from Calvariae and Long Bones 19 2 Osteoblast Isolation from Murine Calvariae and Long Bones Astrid Bakker and Jenneke Klein-Nulend 1 Introduction When conducting in vitro research on bone, a choice has to be made between using bone organ or bone cell cultures When one decides to use the... range of bone- specific gene expression characteristic of primary bone cells This means that in certain experiments, the use of primary bone cells is preferred to the use of cell lines Peck and co-workers initiated the use of primary bone cell cultures in 1964 (1) They isolated cells from frontal and parietal bones of fetal and neonatal rat calvariae by collagenase digestion of the uncalcified bone matrix... 1,25-(OH)2D3] treatment than bone cells obtained from adult bone (10) Because vitamin D3 stimulates immature bone cell differentiation, this supports the notion that neonatal cell cultures contain more immature, rapidly growing cells than cultures from adult bone Thus, for in vitro studies investigating the cellular behavior of adult bone, it seems advisable to use cells from adult bone fragments to reproduce... derived cells in vitro: a model of bone formation? Am J Med Genet 45, 163–178 23 Gundle, R G., Joyner, C J., and Triffitt, J T (1995) Human bone tissue formation in diffusion chamber culture in vivo by bone derived cells and marrow stromal cells Bone 16, 597 24 Wergedal, J E and Baylink, D J (1984) Characterisation of cells isolated and cultured from human trabecular bone Proc Soc Exp Biol Med 176, 60–69... used as a tool to improve our knowledge of bone biology (7–9) This chapter describes the isolation of primary mouse bone cells from adult mouse calvariae and long bones, as well as the process of isolation of bone cells from neonatal mouse calvariae Owing to their difference in origin and method of isolation, it is to be expected that each of the primary bone cell cultures described will have its own... W., and Russell, R G G (1984) Production of osteocalcin by human bone cells in vitro Effects of 1,25(OH)2D3, parathyroid hormone and glucocorticoids Metab Bone Dis Rel Res 5, 229–234 5 Gallagher, J A., Gundle, R., and Beresford, J N (1996) Isolation and culture of bone forming cells (osteoblasts) from human bone, in Human Cell Culture Protocols (Jones, G E., ed.), Humana Press Totowa, NJ 6 Gundle, R.,... for a sterile no 11 scalpel Remove the muscles from the long bones in the limb (femur; tibia and fibula; or humerus, radius, and ulna), and scrape the bone with a scalpel until it is clean (see Note 2) Excise the long bone and place it in a Petri dish with PBS When all the long bones have been removed, cut off the epiphyses Flush out the bone marrow with PBS, using a 5-mL syringe and a 27-gauge needle... to inhibit collagenase activity Rinse the bone pieces three times with cCM 11 Transfer the bone pieces to 25 cm2 flasks, containing 5 mL of cCM, at a density of approx 20–30 fragments per flask 12 Change the medium three times per week Adult mouse bone cells will start to migrate from the bone chips after 3–5 d On average the cell monolayer growing from the bone fragments will reach confluency after... AM Mineralizing Fibroblast-Colony-Forming Assays 37 5 Methods of harvesting bone marrow cells: The centrifugation method is optimized for use with rat bones; however, with very minor modifications it can be adapted for use with mouse bones and with human bone chips Another method of harvesting is to flush the cells from the bone using a syringe but the variability of the yield will be greatly increased... the order of £10,000 (approx $15,000) or more References 1 Mundy, G R (1995) No bones about fluoride Nat Med 1, 1130–1131 2 Friedenstein, A J (1990) Osteogenic stem cells in the bone marrow Bone Miner Res 7, 243–272 3 Maniatopoulos, C., Sodek, J., and Melcher, A H (1988) Bone formation in vitro by stromal cells obtained form bone marrow of young rats Cell Tissue Res 254, 317–330 4 Nefussi, J-R., Boy-Lefevre, . H O D S I N M O L E C U L A R M E D I C I N E TM Bone Research Protocols Edited by Miep H. Helfrich, PhD Stuart H. Ralston, MD Bone Research Protocols Edited by Miep H. Helfrich, PhD Stuart H receptors 1-4. Bone 27, 185–195. 01/Gall/1-18/F1 2/26/03, 10:44 AM18 Bone Cells from Calvariae and Long Bones 19 19 From: Methods in Molecular Medicine, Vol. 80: Bone Research Protocols Edited. Calvariae and Long Bones Astrid Bakker and Jenneke Klein-Nulend 1. Introduction When conducting in vitro research on bone, a choice has to be made between using bone organ or bone cell cultures.