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187
Miep H. Helfrich and Stuart H. Ralston (eds.), Bone Research
Protocols, Methods in Molecular Biology, vol. 816,DOI
10.1007/978-1-61779-415-5_13, Springer Science+Business Media, LLC
2012
Chapter 13
RANKL-Mediated Osteoclast Formation from Murine RAW 264.7
cells
Patricia Collin-Osdoby and Philip Osdoby
Abstract
Extensive research efforts over the years have provided us with
great insights into how bone-resorbing osteoclasts (OCs) develop
and function and, based on such work, valuable antiresorptive
therapies have been developed to help combat the excessive bone
loss that occurs in numerous skeletal disorders. The RAW 264.7
murine cell line has proven to be an important tool for in vitro
studies of OC formation and function, having particular advantages
over the use of OCs generated from primary bone marrow cell
populations or directly isolated from murine bones. These include
their ready access and availability, simple culture for this pure
macrophage/pre-OC population, sensitive and rapid development into
highly bone-resorptive OCs expressing hallmark OC characteristics
following their RANKL stimulation, abundance of RAW cell-derived
OCs that can be generated to provide large amounts of study
material, relative ease of transfection for genetic and regulatory
manipulation, and close correlation in characteristics, gene
expres-sion, signaling, and developmental or functional processes
between RAW cell-derived OCs and OCs either directly isolated from
murine bones or formed in vitro from primary bone marrow precursor
cells. Here, we describe methods for the culture and RANKL-mediated
differentiation of RAW cells into bone-resorptive OCs as well as
procedures for their enrichment, characterization, and general use
in diverse analytical assays.
Key words: Osteoclast , Osteoclast development , Bone resorption
, RANKL , Mouse macrophage , RAW 264.7 cells
Osteoclasts (OCs) are cells uniquely responsible for dissolving
the organic and inorganic components of bone during bone
develop-ment and remodeling throughout life. They originate from
hematopoietic precursors of the monocyte/macrophage lineage present
in the bone marrow and peripheral circulation, and their numbers
and/or activity frequently increase in a wide array of
1. Introduction
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188 P. Collin-Osdoby and P. Osdoby
clinical disorders associated with excessive bone loss ( 1 ) .
For many years, investigations into how OCs develop and function
were hampered by considerable diffi culties associated with
isolating and culturing these normally rare cells. Whereas cell
lines have fre-quently provided an invaluable research tool and are
widely used to decipher mechanisms involved in osteoblast (OB)
differentiation and bone formation, no immortalized cell lines for
mature OCs exist and the few pre-OC cell lines that were reported
either did not undergo full OC differentiation ( 2, 3 ) or involved
coculture systems and cells that were not readily available to all
researchers ( 4 6 ) . To further compound the problem, it was diffi
cult to reli-ably generate bone-resorptive OCs expressing mature OC
charac-teristics from primary bone marrow or circulating precursor
cells in vitro. This all changed with the breakthrough discovery of
the key OC differentiation signal, receptor activator of nuclear
factor B ligand (RANKL), that triggers the full development and
activa-tion of OCs both in vitro and in vivo ( 7 9 ) . During OB
develop-ment or in response to specifi c hormonal or local signals,
RANKL becomes highly expressed on the surface of OB/stromal cells
and interacts with a receptor, RANK, upregulated by macrophage
colony-stimulating factor (M-CSF) on the surface of pre-OCs to
stimulate their fusion, differentiation, and resorptive function.
Many researchers now routinely form OCs in vitro through the
exogenous addition of soluble recombinant RANKL (in combina-tion
with M-CSF to stimulate pre-OC proliferation, survival, and RANK
expression) to cultures of primary bone marrow cells or peripheral
blood monocytes derived from various species (e.g., human, mouse,
rat, rabbit, or chicken, as discussed in other chapters in this
volume). However, such procedures still require the isola-tion of
primary precursor populations, and in suffi cient numbers, to
provide enough in vitro generated OCs for experimentation or
characterization.
In addition to primary cells, at least one pre-OC cell line,
murine macrophage RAW 264.7 cells, responds to RANKL stim-ulation
in vitro to generate bone resorbing multinucleated OCs (RAW-OCs)
with the hallmark characteristics expected for fully differentiated
OCs ( 10 12 ) . RAW cells have been extensively employed in
macrophage studies for >30 years and were origi-nally
established from the ascites of a tumor induced in a male mouse by
intraperitoneal injection of Abelson leukemia virus (although RAW
cells do not secrete detectable virus particles) ( 13 ) . RAW cells
express the c-fms receptor for M-CSF ( 14 ) as well as M-CSF,
perhaps explaining why they also express high levels of RANK ( 10 )
and do not require M-CSF as a permissive factor in their
RANKL-induced formation into RAW-OCs. RAW cells are often used in
studies of OC differentiation and function, in parallel or as a
prelude to studies with OCs formed from primary cells. There are
many advantages of this system over the
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18913 RANKL-Mediated Osteoclast Formation from Murine
generation of OCs from primary cell populations, including the
following: (1) ready access (making it unnecessary to schedule
experiments around when primary cells may become available) and
widespread availability of this cell line to most researchers, (2)
easy culture and homogeneous nature of the pre-OC popula-tion
(devoid of OBs, stromal, lymphocytes, or other cell types), (3)
sensitive and very rapid RANKL-mediated formation of RAW-OCs
(within days), (4) very large number of RAW-OCs that can be
generated (and, thus, RNA or protein for study), (5) high bone pit
resorptive capability and expression of OC characteristics
exhibited by RAW-OCs, (6) relative ease of transfection for genetic
and regulatory manipulation, and (7) close correlation in
characteristics, gene expression, signaling, and developmental or
functional processes between RAW-OCs, OCs formed from primary
precursor cells in vitro, and isolated in vivo formed OCs. In this
chapter, we describe methods for the culture and RANKL-mediated
differentiation of RAW cells into bone-resorptive RAW-OCs, the
preparation of RAW-OC enriched populations by serum density
gradient fractionation, and the culture and characteriza-tion of
RAW-OCs. Such in vitro generated OCs can be analyzed using
biochemical, immunological, physiological, molecular, functional,
or other assays according to commonly employed procedures; see also
various other chapters on osteoclasts in this volume.
All media and solutions are prepared with glass distilled
water.
1. Culture medium: mix 90 ml of sterile Dulbeccos modifi ed
Eagle medium (DMEM) supplemented with 4 mM L -glutamine, 1.5 g/l
sodium bicarbonate, 4.5 g/l glucose, and 1.0 mM sodium pyruvate
with 10 ml of fetal bovine serum (FBS, Invitrogen-Gibco) and 1 ml
of a 100 stock of antibiotic/anti-mycotic (a/a, Invitrogen-Gibco);
store at 4C and prewarm to 37C for use with cells.
2. Phosphate buffered saline, pH 7.2 (PBS). 3. RANKL (Enzo Life
Sciences, PeproTech, EMD4Biosciences,
R&D Systems, or homemade): reconstitute and store as a
con-centrated stock solution (typically 100 g/ml in PBS) in
ali-quots (~1050 l) at 80C as recommended by the manufacturer,
briefl y thaw and dilute into culture medium (to 35 ng/ml fi nal
concentration for murine recombinant soluble RANKL) immediately
before use with RAW cells, and refreeze remaining RANKL (and aim to
thaw individual vials no more than three times to retain optimal
bioactivity).
2. Materials
2.1. Tissue Culture Medium, Solutions, and Supplies
-
190 P. Collin-Osdoby and P. Osdoby
4. Mosconas high bicarbonate (MHB): add 8 g of NaCl, 0.2 g of
KCl, 50 mg of NaH 2 PO 4 , 1.0 g of NaHCO 3 , 2 g of dextrose, 10
ml of a/a, and 990 ml of water; check pH is 7.2 and sterile-fi
lter.
5. Hanks balanced salt solution (HBSS, Invitrogen-Gibco), pH
7.2.
6. Collagenase (Type 3): prepare a 3% stock (3 g in 100 ml)
solu-tion in HBSS; store in aliquots (0.51.0 ml) at 20C.
7. Trypsin: 1% stock (1 g in 100 ml) solution in HBSS; store in
aliquots (1.0 ml) at 20C.
8. Collagenasetrypsin digestion solution: briefl y thaw and add
71 l of 3% collagenase solution and 141 l of 1% trypsin solu-tion
to 3 ml of MHB (per dish) immediately before use with cells.
9. Protease (EC 3.4.24.31, Sigma P-8811): 0.1% (100 mg in 100
ml) stock solution in PBS; store at 4C for up to several months or
in aliquots (0.5 ml) at 20C for long-term storage.
10. EDTA: 2% (2 g in 100 ml) stock solution (using EDTA sodium
salt) in PBS; store at 4C.
11. ProteaseEDTA digestion solution: briefl y thaw and add 50 l
of 0.1% protease solution and 50 l of 2% EDTA solution to 5 ml of
PBS (per dish) immediately before use with cells.
12. Supplies: sterile bottles, fl asks, and tissue culture
dishes; rubber cell scrapers (Fisher); hemocytometer.
13. Devitalised bone or dentine slices, prepared as described
(see Section 3.1 ). Ivory is obtained through donation from a local
zoo or, in the USA, the Federal Department of Fish and Wildlife
Services (or similar Department in other countries). Bovine
cortical bone is obtained from a local slaughterhouse.
1. Segments of ivory and bovine cortical bone are thoroughly
cleaned and washed (multiple HBSS and 70% ethanol rinses), sliced
into small chunks and then reduced to rectangular 0.4-mm thick
sheets using a low-speed Isomet saw (Buehler, Lake Bluff, IL).
2. The sheets are rinsed three times with 70% ethanol, incubated
in 70% ethanol overnight, and then washed for several hours in HBSS
before circular disks are cut using a 5-mm paper punch.
3. Methods
3.1. Preparation of Devitalized Bone or Dentine Slices
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19113 RANKL-Mediated Osteoclast Formation from Murine
3. The disks are soaked repeatedly in 70% ethanol in sterile
50-ml tubes (alcohol changes can be gently poured off because the
disks tend to stick to the side of the tube), and stored in 70%
ethanol at 20C.
4. For experimental use, the required number of disks are
removed from the tube using alcohol-presoaked tweezers (to maintain
sterility) in a tissue culture hood, transferred to a new sterile
50-ml polypropylene tube, rinsed extensively by inversion and mild
shaking at least three times with ~40 ml sterile HBSS per wash, and
the disks transferred using sterile tweezers into culture wells or
dishes containing sterile HBSS for 324 h of preincubation in a
tissue culture incubator prior to the plating of cells. HBSS is
removed only immediately before the disks are used so that they do
not dry prior to RAW cell or RAW-OC seeding.
RAW 264.7 cells are obtained from the ATCC or similar cell
repos-itory. They represent a murine macrophage cell line that has
the capability to be grown indefi nitely as an OC precursor
population or can be differentiated by treatment with RANKL into
multinu-cleated bone resorptive OCs expressing the hallmark
characteristics of in vivo formed OCs (see Subheading 3.3 ).
All work should be performed in a sterile hood using sterile
solutions and supplies.
1. If starting from a frozen (liquid nitrogen) vial of RAW
cells, quickly (
-
192 P. Collin-Osdoby and P. Osdoby
with additional medium as needed to yield 8 ml per 100-mm dish
or 0.5 ml (or 1.0) per well of a 24-well dish, and then place the
cells into a tissue culture incubator.
7. To grow the RAW cells for an extended period of time, refeed
the cultures every 23 days and subculture when they reach confl
uency as in steps 4 6 .
This method is based on the published procedure of Hsu et al. (
10 ) .
1. Culture RAW cells to confl uency (see Subheading 3.2 , steps
1 3 ).
2. Subculture confl uent RAW cells into 24-well dishes as
described in Subheading 3.1 , steps 4 6 (see Notes 3 6 ). If the
cells are to be used for cytochemical or immunological staining,
replate the RAW cell suspension into 24-well dishes that contain a
sterile glass coverslip in each well. If bone resorption is to be
evaluated in parallel with OC development in the RAW cell cultures,
replate the RAW cell suspension into 24-well dishes that contain 24
small disks of bone or ivory (see Subheading 3.1 ) per well (with
or without a glass coverslip under the disks).
3. Immediately add soluble recombinant RANKL to the dishes at a
fi nal concentration of 35 ng/ml to initiate OC development (day 0)
and increase the volume in the wells to 0.5 ml (or 1.0) with
additional culture medium (see Note 7).
4. Culture to day 3. Briefl y examine the cells under a
microscope for evidence that RAW cells are beginning to fuse into
multi-nucleated RAW-OCs. Refeed the developing RAW-OC cell cultures
with 0.5 ml (or 1.0) of fresh medium containing 35 ng/ml RANKL.
5. Culture until day 5 or 6 when many multinucleated RAW-OCs
have formed but have not completely covered the dish (see Note 8).
The day 5 or 6 RAW-OC populations may be imme-diately fi xed and
used for cytochemical or immunological stain-ing, harvested for
biochemical or molecular studies, or analyzed for bone resorption
(see other Chapters in this volume). For greater bone resorption,
such cultures may be incubated until days 79. Alternatively,
RAW-OCs can be purifi ed further by serum gradient density
fractionation (see Subheading 3.4 ).
Because not all RAW cells fuse into multinucleated RAW-OCs by
day 5 or 6, those that have can be purifi ed from the remaining
mononuclear cells using serum density gradient fractionation (see
Note 9). This procedure is a modifi cation of the one we routinely
use to purify in vitro formed OCs or OC-like cells from chick or
human origin ( 15 ) . Directions are provided for RAW-OCs formed on
100-mm tissue culture dishes. All steps are conducted at room
3.3. RAW-OC Formation ( See Note 4 )
3.4. Serum Gradient Purifi cation of RAW-OC
-
19313 RANKL-Mediated Osteoclast Formation from Murine
temperature, unless otherwise noted, and are performed in a
sterile hood using sterile solutions and supplies.
1. Remove the spent culture medium from two 100-mm dishes of day
5 or 6 RANKL-generated RAW-OCs.
2. Gently add 10 ml of MHB to each 100-mm dish to wash the
cells. Remove and discard the washes.
3. Repeat step 2 to wash the RAW-OCs twice more with MHB. 4. Add
10 ml of MHB to each dish and place them into a tissue
culture incubator at 37C for 15 min. 5. Remove and discard the
MHB solution from each dish. 6. Add 5 ml of freshly prepared
collagenasetrypsin digestion
solution to each dish and incubate at 37C for 5 min. 7. Remove
the dishes from the incubator and shake the plates
gently by hand back and forth (e.g., slide the dish on a fl at
surface) for ~30 s to detach and loosen the interaction of cells
with extracellular matrix produced by the cells.
8. Completely remove the collagenasetrypsin solution contain-ing
the released matrix material from each dish and discard (see Note
10).
9. Gently wash the adherent cells on each dish by releasing 10
ml of PBS slowly against the side wall of the dish. Completely
remove and discard the washes.
10. Repeat step 9 to wash the cells on each dish with PBS twice
more.
11. Add 5 ml of proteaseEDTA digestion solution to each dish.
Incubate at 37C for 1015 min (see Note 11).
12. Loosen the adherent cells on each dish by fl ushing the
proteaseEDTA incubation solution with a pipette gently over the
sur-face of the cell layer to free the cells (see Note 12).
13. Transfer the cell suspensions from two 100-mm dishes into
one 50-ml sterile centrifuge tube containing 1.0 ml FBS (to inhibit
further protease action).
14. Centrifuge the cells at 100 g for 5 min. 15. Remove and
discard the supernatant. Gently resuspend the cell
pellet in 15 ml of MHB by repeatedly drawing up and releasing
from a pipette (not too vigorously, see Note 12).
16. Prepare 16 ml of 70% FBS in MHB (11.2 ml FBS plus 4.8 ml of
MHB) in a 50-ml centrifuge tube, and 16 ml of 40% FBS in MHB (6.4
ml FBS plus 9.6 ml MHB) in another 50-ml tube.
17. Prepare an FBS gradient in a 50-ml round-bottom centrifuge
tube. To do this, carefully dispense 15 ml of the 70% FBS-MHB
solution (from step 16) into the bottom of the tube. Very slowly
overlay this with 15 ml of the 40% FBS-MHB solution (from
-
194 P. Collin-Osdoby and P. Osdoby
step 16), using a pipette held at a 45 angle against the side of
the tube just above the 70% FBS layer and slowly releasing the 40%
FBS solution in a thin stream so as not to deform the sur-face of
the 70% FBS layer.
18. Let the tube stand undisturbed for 30 min (at room
tempera-ture) to allow the larger multinucleated RAW-OCs to settle
under normal gravity and penetrate the FBS layers (see Note
13).
19. Carefully take off the top 17 ml which contains mononuclear
cells, and transfer it into a 50-ml tube.
20. Then, remove the 16 ml middle layer, which contains
primarily mononuclear cells and some small multinucleated RAW-OCs,
and transfer this into another 50-ml tube.
21. The bottom 12 ml fraction contains predominantly large
multinucleated RAW-OCs.
22. Centrifuge the purifi ed RAW-OC bottom fraction (and the
other fractions if they are also to be cultured and/or analyzed) at
100 g for 5 min.
23. Gently resuspend the RAW-OC pellet in culture medium, count
an aliquot in a hemocytometer, and plate 1,0004,000 cells per well
of a 24-well dish. Typically, the purifi ed RAW-OCs from two 100-mm
dishes can be cultured in 210 wells of a 24-well dish (with 0.51.0
ml medium per well) for 624 h (see Note 14). The top and middle
fractions from the serum gradient fractionation are typically
cultured in 2040 wells and 1530 wells of a 24-well dish,
respectively. Alternatively, RAW-OCs (and the top and middle
fractions, if desired) may be used immediately for analysis (see
Subheading 3.2 , step 5).
Serum gradient fractionation routinely provides 4,00010,000
purifi ed RAW-OCs from one 100-mm dish (this depends on the effi
ciency of ones technique and, more importantly, on the exact stage
of RAW-OC used to purify the cells; see Notes 6, 8, 13, and 14). In
unfractionated RANKL-generated RAW-OC cultures, multinucleated
(more than three nuclei) RAW-OCs typically rep-resent ~1% on a per
cell basis and 25% on a per nuclear basis of the total cell
population (Fig. 1 , left panel). By contrast, serum gradi-ent
purifi ed RAW-OCs (with more than three nuclei) typically comprise
6090% on a per cell basis and 96% on a per nuclear basis of the
total cell population in the bottom serum fraction (Fig. 1 , lower
right panel). On average, RAW-OCs in this bottom serum fraction
contain 1530 nuclei per cell.
Standard protocols can be used to evaluate the morphological
(light, scanning electron microscopy), ultrastructural
(transmission elec-tron microscopy), histochemical (general or
enzymatic activity stains), or immunocytochemical staining (e.g.,
for OC developmen-tal markers) characteristics of RAW cells
representing pre-OCs and
3.5. Phenotypic and Functional Characterization of RAW-OCs
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19513 RANKL-Mediated Osteoclast Formation from Murine
in vitro RANKL-formed RAW-OCs (see Chapter 9 , this volume ).
Whereas untreated RAW cells do not stain for tartrate resistant
acid phosphatase (TRAP) activity, a key marker and enzyme involved
in OC bone resorption, RANKL-differentiated RAW cell cultures
develop both TRAP+ mononuclear and multinucleated cells (Fig. 2a, c
). The RAW-OCs formed by cell fusion contain multiple nuclei
clustered together, and the cells may appear either spread out or
partially elongated when cultured on plastic (Fig. 2a ). RAW-OCs
cultured on bone or ivory (either during RANKL development or
following replating of the differentiated cells) frequently display
a more compact and highly motile elongated shape with numerous
pseudopodial extensions (Fig. 2c ). Resorption pits formed by
RAW-OCs are typifi ed by clusters of multilobulated excavation
cavities or long resorption tracks (which may also be
multilobulated) adjacent to or underlying RAW-OCs actively engaged
in bone resorption (Fig. 2c ). Molecular, immunological, and/or
biochemical analyses have shown that RAW-OCs express the key
hallmark properties of OCs including TRAP, calcitonin receptor,
cathepsin K, matrix
Fig. 1. RANKL-mediated RAW-OC formation and serum gradient
purifi cation. ( Left ) RAW cells were cultured with 35 ng/ml
murine recombinant RANKL for 6 days and then subjected to serum
gradient fractionation. A well cultured in parallel was fi xed and
stained for TRAP activity to show the proportion of mononuclear and
multinucleated TRAP+ cells that arise by day 6 in
RANKL-differentiated RAW cell cultures. The cells were viewed using
a light microscope and images were captured with a computer-linked
digital camera. (Reduced from original magnifi cation, 100.) (
Right ) The top , middle , and bottom fractions from the serum
gradient fractionation were replated and cultured on plastic for
several hours, after which the cells were fi xed and stained for
TRAP activity. ( Upper right ) The top fraction consists entirely
of mononuclear cells, some of which are TRAP+ (in contrast to
untreated RAW cells which are all TRAP-, not shown). (Reduced from
original magnifi ca-tion, 200.) ( Middle right ) The middle
fraction primarily contains mononuclear cells, a portion of which
are TRAP+, and some small multinucleated RAW-OC. (Reduced from
original magnifi cation, 200.) ( Lower right ) The bottom fraction
con-sists primarily of large multinucleated RAW-OC, although a few
mononuclear cells may still be present. (Reduced from original
magnifi cation, 100).
-
196 P. Collin-Osdoby and P. Osdoby
metalloproteinase-9, integrin v 3, and c-src (refs. 10, 16 , our
unpublished data ). Both the phenotypic and functional
characteris-tics of RANKL-differentiated RAW-OCs resemble those of
in vivo formed isolated murine OCs or RANKL-differentiated OCs
(MA-OC) formed from murine bone marrow cells in the presence of
M-CSF (Fig. 2b, d ). Thus, like RAW-OCs, TRAP+ MA-OCs exhibit a
well-spread morphology on plastic (Fig. 2b ) and a more compact,
motile phenotype on bone or ivory (Fig. 2d ). Multilobulated
resorp-tion pits and tracks formed by MA-OCs (Fig. 2c ) also
resemble those formed by RAW-OCs, with well defi ned margins and
deep resorption lacunae (Fig. 2d ). Resorption pit formation by
RAW-
Fig. 2. RANKL-mediated RAW-OC or MA-OC formation and bone pit
resorption. ( a , c ) RAW cells were cultured with 35 ng/ml murine
recombinant RANKL for 6 days on plastic ( a ) or ivory ( c ), and
then fi xed and stained for TRAP activity. Note the well spread
morphology of RAW-OCs on plastic ( a ) compared with the more
compact and motile phenotype of such cells actively engaged in bone
resorption on ivory ( c ). Abundant resorption pits and tracks were
evident that were frequently composed of connecting excavation
cavities. These represent periods of RAW-OC attachment and pit
formation, followed by RAW-OC movement to an adjacent area of ivory
for further resorption. ( a ) and ( b ) reduced from original
magnifi cation, 200). ( b , d ) Murine bone marrow cells were
isolated and cultured at 5.6 10 5 cells per well of a 24-well dish
(1.9 cm 2 per well) with 25 ng/ml of murine M-CSF and 35 ng/ml of
murine RANKL for 6 days on plastic ( c ) or ivory ( d ), after
which the cells (MA-OCs) were fi xed and stained for TRAP activity.
Like RAW-OCs, the TRAP+ MA-OCs were well spread on plastic ( b )
and more compact on ivory ( d ). Resorption pits and tracks formed
by MA-OCs ( d ) were indistinguishable from those formed by RAW-OCs
( b ). ( b ) and ( d ) reduced from original magnifi cation, 100
and 200, respectively).
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19713 RANKL-Mediated Osteoclast Formation from Murine
OCs, in the presence or absence of modulators, can be quantifi
ed as for other OCs (see Chapters 8 12 , this volume). In addition
to these phenotypic and functional analyses, RAW-OCs provide
abun-dant material (protein, RNA, etc.) for investigations of gene
or pro-tein expression or microarray profi ling, receptors and
signal transduction pathways, production of various factors
(cytokines, chemokines, growth factors, and arachidonic acid
metabolites), release of other substances (free radicals and
enzymatic activities), cell and matrix interactions, and diverse
regulatory mechanisms (particularly since RAW cells are more easily
transfected than pri-mary bone marrow cells) (see Note 15).
1. Some DMEM formulations may produce a visible dark
precipi-tate that causes rapid cell death of RAW cells during
culture. In such cases, we fi nd it best to obtain new DMEM lacking
Fe(NO 3 ) 3 along with a separate stock (1,000) of Fe(NO 3 ) 3 that
is stored in aliquots (1 ml) at 20 C. This iron stock is only
thawed and added to DMEM at the time that complete medium is
prepared (containing FBS and a/a) for current use. Typically, this
complete medium is usable for 10 days to 2 weeks for cell refeeding
before evidence of precipitation occurs, at which time any
remaining medium should be discarded. Some inves-tigators have
reported that RAW cells also grow and form OCs well in -MEM medium,
so this may be an alternative to DMEM if problems are encountered
with the latter.
2. We fi nd that a rubber-tipped cell scraper works best because
it completely contacts the surface of the tissue culture fl ask (or
dish) and causes the least cell damage.
3. In general, RAW cells should be subcultured at a ratio of
1:31:6.
4. If more cells will be needed than are provided by a
reasonable number of T25 fl asks, the confl uent RAW cells can be
subcul-tured into T75 fl asks (at a 1:31:6 ratio) and then grown to
confl uency.
5. The number of RAW cell passages affects RANKL-mediated OC
formation. In our hands, RAW-OCs seem to better form after passage
4 and will no longer form in response to RANKL stimulation once
they have undergone 1820 passages from the time that they were
received from the ATCC repository. The reason for this is not fully
clear, although other researchers have similarly noted that not all
RAW 264.7 cell lines (or passages?) will form OCs after RANKL
treatment, and subclones of RAW 264.7 cells can be derived that are
more or less effi cient at
4. Notes
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198 P. Collin-Osdoby and P. Osdoby
RANKL-mediated OC formation ( 17, 18 ) . It is also possible
that particular lots of FBS may differentially infl uence
RANKL-mediated RAW-OC formation. Therefore, different lots and
sources of FBS may be tested if there are diffi culties
encoun-tered in trying to form RAW-OCs, although RAW-OCs have been
formed even in serum-deprived conditions ( 19 ) .
6. The density of RAW cells plated affects the rate and yield of
RAW-OC development, as well as the subsequent analysis of RAW-OCs
formed. Too low a cell density (100500 cells/cm 2 ) delays RAW-OC
formation and decreases the fi nal yield. For most purposes (e.g.,
testing the effects of various agents on RAW-OC development), the
plating density for RAW cells should be in the range of 10 3 3 10 4
/cm 2 to facilitate count-ing or characterization of the RAW-OCs
formed and still gen-erate a suffi cient number for analysis. If
RAW-OCs are to be purifi ed by serum density gradient
fractionation, the initial plating density of RAW cells should be
considerably higher (1.5 10 5 /cm 2 ) so that enough RAW-OCs are
obtained fol-lowing their purifi cation (see Subheadings 3.1 and
3.2 ). However, too high a density of RAW cells (4.57.5 10 5 /cm 2
) inhibits RAW-OC formation.
7. In our experience, the potency of recombinant soluble RANKL
for inducing OC formation is dependent upon its source (not only
for RAW cells, but also for human monocyte, mouse bone marrow, or
chicken bone marrow preparations). Thus, differ-ent commercial
RANKL preparations vary signifi cantly in the dose required,
kinetics of OC formation, and fi nal yield of bone pit resorptive
OCs obtained. This is not strictly due to species compatibility
issues because human or murine recombinant RANKL are similarly effi
cient for inducing OCs from murine RAW or bone marrow-derived
cells, chicken bone marrow cells, or human peripheral blood
monocytes (although all but RAW cells require M-CSF costimulation).
Although we have successfully used various commercial RANKL
preparations, we typically prepare soluble recombinant mouse RANKL
in our own lab that exhibits high osteoclastogenic activity with
murine RAW or bone marrow cells, chicken bone marrow cells, or
human monocytes. At 35 ng/ml, this mouse RANKL induces
multinucleated TRAP+ cell formation that is fi rst apparent on days
34 of RAW cell culture. Lower RANKL concentrations delay the
kinetics and fi nal yield (and size) of RAW-OC forma-tion. Others
have used recombinant RANKL preparations in the range of 20100
ng/ml (depending upon its source and bioactivity) to form
multinucleated TRAP+ cells that usu-ally fi rst appear on days 34
of RAW cell culture ( 11, 18, 20, 21 ) . However, certain
recombinant mouse RANKL prepara-tions appear to require an
additional anti-RANKL antibody
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19913 RANKL-Mediated Osteoclast Formation from Murine
cross-linking step to induce osteoclastogenesis ( 16 ) .
Therefore, it is recommended that pilot studies be performed with
each new source and preparation of recombinant RANKL to ascertain
an appropriate dose to achieve the level of RAW-OC formation needed
(see also the discussion in Chapter 7 , this volume).
8. In our model of RANKL-mediated RAW-OC development, TRAP+
cells fi rst become apparent on day 2 of culture, and
multinucleated TRAP+ cells appear on days 34 and nearly reach a
peak on days 56 of culture. It is important to either use the cells
or subject them to serum gradient purifi cation at this point (and
not wait another day until the full peak of RAW-OC formation has
occurred) because if the cells become overconfl uent and overfused,
they die very rapidly (
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200 P. Collin-Osdoby and P. Osdoby
that few viable RAW-OCs will be recovered following the serum
gradient purifi cation.
14. Even under controlled conditions of RANKL-mediated RAW-OC
formation as discussed in this chapter, once such cells have formed
they tend to apoptose very rapidly and the cells can be lost within
24 h if allowed to develop too long (see also discussion in Chapter
8 , this volume). Short survival after formation in culture is
specifi cally a problem with mouse osteo-clasts and not seen with
for example human osteoclasts. Addition of 10 ng/ml IL-1 to promote
RAW-OC survival on plastic only slows the apoptotic process
slightly, and a recent report indicates that it preferentially
activates larger over smaller RAW-OCs ( 22 ) . Therefore, we
typically use RAW-OCs formed in tissue culture dishes by days 56
for analysis within 624 h (e.g., staining, RNA or protein
extraction, etc.). If RAW-OCs have been formed on bone or ivory,
resorption pits are usually evident by day 4 and maximal by day 6
or 7; modu-lators can be added at appropriate times to observe
stimulatory or inhibitory effects on resorption. When RAW-OCs are
puri-fi ed by serum gradient fractionation and replated onto tissue
culture dishes, their viability is usually extended for an
addi-tional 24 h. Alternatively, purifi ed RAW-OCs can be replated
onto bone or ivory (~800 cells per well of a 48-well dish
con-taining one piece of ivory or bone) and cultured with 35 ng/ml
RANKL and 10 ng/ml IL-1 , in the presence or absence of other
modulators, for 56 days to ascertain effects primarily on preformed
RAW-OCs (although some additional RAW-OC development also occurs
during this time period since the 70% serum purifi ed fraction
still contains some mononuclear cells). Purifi ed RAW-OCs typically
do not exhibit pit formation within the fi rst 24 h after replating
onto bone or ivory.
15. Although many of the phenotypic and functional
characteris-tics of RAW-OCs match those of RANKL-differentiated
pri-mary murine bone marrow-derived OCs or isolated in vivo formed
murine OCs, this cannot automatically be assumed to be true for any
particular property being evaluated. The most obvious difference is
the requirement for M-CSF in RANKL-stimulated OC formation from
bone marrow cells (which have relatively low RANK prior to M-CSF
exposure) but not for transformed RAW cells (which already make
M-CSF and express high RANK levels). In addition,
apoptosis/survival pathways (including ERK) may differ between
primary bone marrow cells and transformed RAW cells, and various
other differences have been noted. Therefore, it is important to
consider that the specifi c attribute under study in the RAW-OC
cell system may not necessarily refl ect that of normal murine OC
formation or function. However, because RAW cells are
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20113 RANKL-Mediated Osteoclast Formation from Murine
easier to obtain and culture than primary bone marrow cells,
represent a pure population of pre-OCs (defi cient in osteo-blasts,
stromal cells, lymphocytes, etc.), are more readily trans-fected,
and provide abundant material for study, they provide a highly
valuable resource for rapidly and effi ciently screening and
determining mechanisms underlying OC-related processes. Therefore,
we recommend that RAW cell studies are subse-quently followed by at
least a limited number of experiments using primary murine OCs
(directly isolated and/or RANKL-generated in vitro) to confi rm
that these processes are likewise observed in normal murine OCs and
are not unique to trans-formed RAW cells or RAW-OCs.
Acknowledgments
We are greatly indebted to the Drs. Xuefeng Yu and Hong Zheng
for their advice and many valuable contributions to an earlier
version of this chapter. This work was supported by NIH Grants
AR32927, AG15435, and AR32087 to P.O.
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Chapter 13: RANKL-Mediated Osteoclast Formation from Murine RAW
264.7 cells1. Introduction2. Materials2.1. Tissue Culture Medium,
Solutions, and Supplies
3. Methods3.1. Preparation of Devitalized Bone or Dentine
Slices3.2. RAW 264.7 Cell Culture3.3. RAW-OC Formation ( See Note
4)3.4. Serum Gradient Purification of RAW-OC3.5. Phenotypic and
Functional Characterization of RAW-OCs
4. NotesReferences