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The aim of this paper is to present several popular methods for in vitro culture of osteocytes and osteocyte cell lines. Osteocytes are located extremely suitably within the calcified bone matrix to sense mechanical signals, and are equipped with a multitude of molecular features that allow mechanosensing. However, osteocytes are more than specialized mechanosensing cells. Several signaling molecules are preferentially produced by osteocytes, and osteocytes hold a tight reign over osteoblast and osteoclast formation and activity, but also have a role as endocrine cell, communicating with muscles or organs as remote as the kidneys. In order to facilitate further research into this fascinating cell type, three protocols will be provided in this paper. The first protocol will be on the culture of mouse (early) osteocyte cell lines, the second on the isolation and culture of primary mouse bone cells, and the third on the culture of fully embedded human osteocytes within their own three-dimensional bone matrix.
Bone research has long focused on the formation and function of osteoblasts and osteoclasts, the cells that form and resorb bone. This while osteocytes were regarded as rather inactive cells, entombed within the calcified matrix, unable to move. One of the pioneers in osteocyte research was Peter Nijweide, who was the first to isolate osteocytes from the calvariae of chickens,1 using an antibody to what was later discovered to be the avian variant of Phex.2 The protocol for the isolation of these osteocytes is not dissimilar to other techniques allowing the isolation of osteoblasts and osteocyte-like cells from mouse and human bone.3,4 Since the development of that first protocol, osteocytes were discovered to have a key role in the mechanical adaptation of bone to mechanical loading, and to be dynamic cells that produce a vast multitude in signaling molecules.5,6,7 One of these signaling molecules produced, more or less exclusively by osteocytes, sclerostin, may inhibit bone mass accrual by osteoblasts, and sclerostin antibodies are currently being investigated as a potential osteoporosis therapy.8 Osteocytes, rather than osteoblasts, are the main source of Receptor activator of nuclear factor kappa-B ligand (RANKL) in adults, determining the extent of osteoclast formation and activity.9,10 Osteocytes also have a role in phosphate homeostasis, by communicating with the kidney, making bone a truly endocrine organ.11 In order to facilitate future research in this cell, having such an important role in regulation of bone metabolism, we provide three protocols for the culture of osteocytes, either as cell line, primary cell or as cells in their original three-dimensional matrix.
In the first part of this paper, we provide a detailed protocol for culturing MLO-Y4 and MLO-A5 cells, which are currently still one of the most frequently used osteocyte cell lines. Immortal clonal cells such as MLO-A5 and the MLO-Y4 cells are routinely used as osteocyte models, which represent different stages of osteoblast to osteocyte differentiation. MLO-A5 cells are derived from osteocalcin promoter-driven T-antigen transgenic mice and have higher expression of ALP and osteocalcin compared to primary osteoblasts and MLO-Y4 osteocytes.12 They mineralize spontaneously in culture, even in the absence of phosphate supplementation and are thought to represent post-osteoblast/pre-osteocyte cells that mineralize the osteoid matrix they are embedded in.12 MLO-Y4 cells are a more mature osteocyte cell model compared with MLO-Y5 cells, derived from the same transgenic mice used for the generation of MLO-A5 cells, although they likely still represent a relatively early osteocyte. Even though these cells represent early osteocytes, they already display the stellate morphology, with dendritic processes typical of osteocytes embedded in matrix. MLO-Y4 cells also possess the ability to respond to mechanical stimulation by releasing Prostaglandin E2,13 ATP14 and nitric oxide,15 integral to osteocytes' orchestration of adaptive bone remodeling. This makes them very useful for studying factors that alter the response of osteocytes to mechanical stimuli. MLO-Y4 cells have relatively high expression of osteocalcin and connexin-43 with low collagen type 1, periostin and alkaline phosphatase activity compared with primary osteoblasts and clonal cells.12,16 However, both MLO-A5 and MLO-Y4 cells have their limitations, such as the lack of sclerostin expression and low DMP-1 expression by MLO-Y4 cells. This makes MLO-Y4 cells less suitable for studying signal molecule production by mature osteocytes. Alternative cell lines that have been used to study sclerostin expression include the SaOS2 osteosarcoma cell line and the osteoblast-like UMR-106 cells, which are described elsewhere.17,18 IDG-SW319 and Ocy45420 are osteocyte cell lines that express relatively high levels of SOST/sclerostin as well as FGF23, both key regulators of bone homeostasis, and could therefore be used to study osteocyte signaling toward other cell types. Readers are encouraged to pick the osteocyte cell line that best suits their research question.
The second part of this paper describes the isolation and culture of primary osteocytes from mice. This protocol allows the study of osteocytes with deletions of specific genes, by isolation of osteocytes from knockout mice. As with all osteocyte culture models, care should be taken to monitor the osteocyte-like phenotype of these cells, as osteocytes in vivo are no longer able to divide, and will be quickly overgrown by other cell types, limiting the useful life span of the culture. In addition, osteocytes are embedded in calcified bone matrix in vivo, determining their three-dimensional shape, and thereby likely affecting their ability to sense mechanical signals. It remains to be shown to what extent osteocytes cultured in two dimensions on tissue culture plastic retain their complete osteocyte-like phenotype. Therefore, several protocols have been developed lately for the culture of osteocytes or osteocyte-like cells in three dimensions in a collagen matrix; for example, the co-culture of osteoblasts on top of MLO-Y4 osteocytes cultured in three-dimensional collagen gels.21 Although these cultures likely give a better approximation of the natural environment of the osteocyte, these cultures still require the use of cell lines or isolated cells, with all their limitations. In addition, gels do not exactly mimic the physical, anatomical, and chemical make-up of the matrix. Therefore, the third part of this protocol describes a method to culture human osteocytes embedded in their native matrix. Human bone tissue is cut in to small pieces and treated with collagenase to remove lining cells and bone marrow cells. These denuded bone pieces, containing osteocytes in their native matrix, can be cultured for up to 7 days.
Altogether, these protocols represent current popular osteocyte culture methods, although new methods that more thoroughly represent the bone niche are continuously being developed.
The following steps should be performed in aseptic conditions.
Experiments with MLO-Y4 cells can be set-up in collagen-coated multiwell plates at a seeding density of 8 × 103 cells per cm2. Outcomes of cell survival and dendricity could be assessed by counting cell numbers, mean dendrites per cell and mean dendritic length from crystal violet stained cultures.
Start with coating the tissue culture plastic with collagen type I, as described in ‘Coating tissue culture plastic with collagen' for protocol I. All dissection equipment should be sterilized in 70% ethanol prior to use. The collagenase and EDTA solutions should be warmed to 37°C before use.
To improve the yield, the digested bone pieces can be homogenized to release osteocytes remaining within the bone pieces after digestion by using the following protocol:
The cells isolated from digest 9 and the bone particles are alkaline phosphatase negative, E11 antigen positive, and display a dendritic morphology as described in ref. 27.
To further increase the cell yield, the cells from digests 7 and 8 may also be harvested in addition to digest 9. These digests contain many osteocytes, but will be more heterogeneous than digest 9, which contains primarily osteocytes. Cell fractions after EDTA or collagenase digest should be preferably collected into serum-containing culture medium and kept on ice prior to seeding.
If osteoblasts are also required for cultures, these can be collected from digests 5 and 6. Earlier digests contain a relatively large amount of fibroblasts.
If the bones from more than two mice are being used, the volumes of collagenase and EDTA solutions can be scaled up accordingly.
Note that even digest 8 and 9 still contain other cells than osteocytes (for example, cells from the hematopoietic lineage, osteoblasts and fibroblasts). Fibroblasts can quickly overgrow the osteocyte cultures, and primary osteocytes lose their osteocyte-like phenotype in two dimensional culture, limiting the life span of these osteocyte-rich cultures. Cells obtained are best used within days. In addition, the presence of cell types other than osteocytes can affect the results obtained,28 and care should be taken when drawing conclusions about osteocyte properties when experiments are performed using primary cell cultures.
The authors of this section prefer to use trabecular bone samples obtained from the anterior iliac crest, since this bone can be obtained from healthy people as surgical waste material when originally collected for example, sinus floor elevation surgery. Trabecular bone can also be obtained in large quantities during total hip or knee replacement surgery for osteoarthritis, but one should keep in mind that this bone might differ in cell consistency from healthy bone.
Detection of sclerostin will confirm the presence of osteocytes in the bone pieces.
The basis for many protocols for the isolation of osteoblasts or osteocyte-like cells from bone is formed by the avian osteocyte isolation protocol, which has been described in detail the following publication: Semeins CM et al.29 The isolation of primary mouse osteoblasts has also been described in detail elsewhere.27 For those of you interested in subjecting cultured osteocytes to a precisely controlled mechanical stimulation, several methods for mechanical stimulation of two-dimensionally cultured skeletal cells have been described in detail in the following publication: Huesa and Bakker.30
We thank Matt Prideaux for his help in realizing the protocol on isolation of mouse primary osteocytes.
The authors declare no conflict of interest.