Chondrocyte Culture and Assay UNIT 12.

Chondrocytes are the cells solely responsible for the synthesis and maintenance of
cartilage. On the basis of comparisons of morphology and content of the matrix, three
types of cartilage have been identified: elastic cartilage, fibrocartilage and hyaline
cartilage (von der Mark, 1986). Elastic cartilage contains elastin and is found in the ear
and epiglottis. Fibrocartilage consists predominantly of type I collagen; it is found, for
example, in the vertebral nucleus pulposus and joint meniscus and is a transitional form
between hyaline cartilage and dense connective tissue. Hyaline cartilage in mammals is
uniquely characterized by a high content of collagen II and of aggrecan, a large,
cartilage-specific proteoglycan species. It serves as a developmental precursor to bone
and is present in adult mammals as nasal cartilage and intercostal cartilage. As skeletal
development progresses and cartilagineous precursors are calcified to form bone, the
endings of bones that are within articulated (moving) joints remain covered with hyaline
cartilage. Proximal to the bone ending is growth plate cartilage, and at the surface,
articular cartilage, both of which are subtypes of hyaline cartilage. The function of
articular cartilage is to ensure the almost-frictionless movement of bone surfaces within
vertebrate joints. Osteoarthritis, a disease which is especially prevalent in the elderly, is
characterized by defects in or deterioration of articular cartilage. Thus, articular cartilage
is the focus of many pharmacological studies. Although the following protocols are
applicable to all types of hyaline cartilage, they are most commonly used for work with
articular cartilage, which in this unit is referred to as simply “cartilage”.
The structural integrity of cartilage derives from the fibrous network formed by the
cartilage-specific protein, collagen II, which is immersed in a gel formed by the very large
proteoglycan species aggregan, and by water, which binds so avidly to it that cartilage is
∼70% water by total weight. Free movement of joint surfaces is allowed by the resulting,
unique extracellular matrix. Remarkably, the architecture of cartilage is dictated solely
by a single cell type, chondrocytes, which make up <10% of the total volume of human
articular cartilage. Chondrocytes synthesize and secrete the extracellular matrix that
constitutes cartilage, which contains a cartilage-specific collagen (collagen II; 70% of
cartilage dry weight), a very large proteoglycan (aggregan; 20% of dry weight), and, in
lesser amounts, several smaller proteoglycan and collagen species that modulate the
architecture of cartilage. This cell type also secretes numerous additional factors crucial
to the maintenance and remodeling of cartilage, notably metalloproteinases, cytokines,
and growth factors. A fundamental consideration for tissue culture is that the full
repertoire of chondrocyte actions is maintained only when they are in 3-dimensional
matrices that allow them to remain spherical (von der Mark, 1986).
This unit presents a method of isolating and culturing chondrocytes in monolayer from
articular cartilage (see Basic Protocol 1) and two alternative culture systems: chondrocyte
culture in alginate (see Alternate Protocol 1) and cartilage chip culture, also termed
“explant culture” (see Alternate Protocol 2). Chondrocyte monolayer culture conforms to
conventional cell culture procedures and facilitates the experimental manipulation of
chondrocytes. Alternate Protocol 1 and Alternate Protocol 2 each provide for 3-dimen-
sional culture of chondrocytes, allowing them to retain their characteristic phenotype and
range of actions. Dissection of cartilage from joints is described (see Support Protocol
1). Incorporation of 35S into chondrocytes or cartilage (see Basic Protocol 2) is a widely
used assay of proteoglycan synthesis, a basic measure of cartilage function. An alternative,
staining-based assay, amenable to higher-throughput pharmacological studies, is also
presented (see Alternate Protocol 3). Either of these assays is suitable for the evaluation
of drug or cytokine effects on proteoglycan synthesis, which provides a measure of the
In Vitro Cellular
Assays
Contributed by Jeffrey Liebman and Ronald L. Goldberg 12.2.1
Current Protocols in Pharmacology (2001) 12.2.1-12.2.18
Copyright © 2001 by John Wiley & Sons, Inc. Supplement 12
 extent to which chondrocytes are actively synthesizing components of cartilage. Finally,
an assay of DNA content is provided to allow normalization of proteoglycan synthesis
relative to cell number (see Support Protocol 2).
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO2
incubator unless otherwise noted.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.
NOTE: Perform all chondrocyte isolation and culture procedures under standard cell
culture conditions (UNIT 12.1). Medium should always contain both antibiotic and antifun-
gal chemicals.
Unless otherwise indicated, perform all procedures including centrifugation and assays
at room temperature. Follow Universal Precautions whenever human tissue material is
used (Office of Health and Safety, Center for Disease Control web page, 1999).

BASIC CHONDROCYTE MONOLAYER CULTURE

PROTOCOL 1 Shavings are excised from articular cartilage after dissection (see Support Protocol 1),
and are then digested successively with protease and collagenase to release chondrocytes.
The chondrocytes are pelleted by centrifugation, resuspended, and cultured as monolayers
according to conventional cell culture techniques.
Before initiating tissue culture work, ensure that all materials have been rendered sterile.
If not already sterilized by the manufacturer, materials can be sterilized by autoclaving,
or in the case of liquids by passing through a 0.2-µm filter, except as indicated.
Materials
Pronase E, type XIV (Sigma)
DMEM/antibiotic-antifungal solution with FBS as indicated (see recipe)
Cartilage shavings (see Support Protocol 1)
150 to 1250 U/mg high clostripain bacterial collagenase (Worthington
Biochemical)
10 mg/ml ascorbic acid, sterile (optional)
0.8-, 0.45-, and 0.22-µm disposable filter units
150-mm sterile, plastic petri dishes
Sterile disposable scalpels, no. 21, no. 15, and no. 10
100-ml spinner flask with side arms
Impeller assembly for spinner flasks, stainless steel (Bellco)
Magnetic stirrer
20-µm nylon filter membrane (Spectra-Mesh, Spectrum Laboratories)
Autoclaved glass funnel
50-ml centrifuge tubes, sterile
Clinical centrifuge
Culture vessels (see Table 12.2.1)

Dissociate chondrocytes from cartilage

1. Dissolve 1 g of pronase E in 100 ml DMEM/5% FBS/antibiotic-antifungal solution
and pass successively through 0.8-, 0.45-, and 0.22-µm filter units.
Filtration may be slow.
Use pronase E in DMEM solution within 1 hr after preparing. Pronase E is believed to
digest proteins that would otherwise impede digestion cartilage by bacterial collagenase.
Chondrocyte
Culture and Assay

12.2.2
Supplement 12 Current Protocols in Pharmacology
 Table 12.2.1 Recommended Chondrocyte Monolayer
Seeding Densities

Vessel Vol. medium (ml) Cells (× 106)

96-well plate 0.1 0.05
24-well plate 0.5 0.2
6-well plate 2.4 1
25-cm2 flask 6 2.5
100-mm petri dish 14 5.7
75-cm2 flask 19 7.5

2. Transfer the cartilage shavings (see Support Protocol 1) to a 150-mm plastic petri
dish containing 30 ml DMEM/antibiotic-antifungal solution.
3. Using two sterile stainless steel disposable scalpels (no. 21 or equivalent), mince the
cartilage shavings into small chips (i.e., 0.5- to 1-mm across) and transfer to a 100-ml
spinner flask equipped with side arms.
4. Add the filtered pronase E in DMEM solution from step 1 to the spinner flask, and
place the impeller assembly in the flask. Put the flask with the attached impeller on
a magnetic stirrer in a 37°C, 5% CO2 humidified cell culture incubator and digest
with stirring for 1 hr.
Do not use a magnetic stir bar in place of the impeller assembly. The cells will be damaged
as they pass between the stir bar and the bottom of the flask.
5. During the pronase E digestion, dissolve 350 mg of high clostripain bacterial
collagenase in 100 ml DMEM/5% FBS/antibiotic-antifungal solution and pass
successively through 0.8-, 0.45-, and 0.22-µm filter units.
Do not substitute mammalian collagenase for bacterial collagenase. Bacterial collagenase
digests collagen much more thoroughly.
Use the collagenase/DMEM solution within 1 hr of preparation
6. Decant the pronase E–containing medium through the side arm of the spinner flask,
taking care to retain the chips at the bottom of the flask. Wash the chips two times,
each time with 100 ml of DMEM/antibiotic-antifungal medium. Add the col-
lagenase/DMEM/5% FBS/antibiotic-antifungal solution (from step 5) to chips and
resume stirring for ∼3 hr in the 37°C, 5% CO2 humidified incubator.
Because FBS is not needed for wash steps, its omission is economical. FBS is needed during
digestion and culture steps.
The chips should remain largely intact during pronase E digestion, but should shrink and
become almost invisible during collagenase digestion. The collagenase digestion time may
need to be modified depending on the species, type of cartilage, and batch of collagenase.
7. Fold an autoclaved piece of 20-µm nylon filter membrane into an autoclaved glass
funnel. Decant the collagenase solution from step 6 through this filter into 50-ml
sterile centrifuge tubes.
The dissociated chondrocytes will pass through the filter, leaving undigested matrix behind.

Place chondrocytes in culture

8. Form a chondrocyte pellet by centrifuging for 10 min at 250 × g (1000 rpm in a
conventional clinical centrifuge), room temperature. Aspirate the supernatant (me- In Vitro Cellular
dium). Assays

12.2.3
Current Protocols in Pharmacology Supplement 12
 9. Wash the cells two times in DMEM (serum is not necessary), centrifuging after each
wash as in step 8.
10. Add ∼2 ml medium per hoof (the precise volume is not critical). Resuspend cells by
pipetting up and down, then count cells.
A single bovine calf metacarpophalangeal joint should yield ∼3 × 106 cells, depending on
how much cartilage is excised. See Support Protocol 1.
11. Plate the chondrocytes in cell culture vessel(s) as indicated in Table 12.2.1. Approxi-
mately 24 hr later (usually the following morning) add an equivalent volume of
medium. Change medium 1 day later.
Freshly isolated primary chondrocytes require up to 24 hr to attach completely. The medium
becomes conditioned during this interval, which is beneficial for the chondrocytes.
12. Optional: if needed, add ascorbic acid (10 to 50 µg/ml culture) after the first 2 days
of culture.
Unless collagen synthesis is of interest, monolayer chondrocyte cultures do not require
ascorbic acid.
13. Begin experiments (e.g., drug or cytokine studies) on monolayered bovine chondro-
cytes within 4 days of plating and complete the experiments by 10 days. Do not
subculture.
Primary monolayered bovine chondrocytes begin to assume a fibroblast phenotype after
several days in culture, and subculturing accelerates this undesirable dedifferentiation
process. Therefore, if the chondrocyte phenotype is desirable or required, subculturing
should be avoided.

ALTERNATE CHONDROCYTE CULTURE IN ALGINATE

PROTOCOL 1
This protocol differs from Basic Protocol 1 in that chondrocytes are embedded in a
3-dimensional matrix composed of alginate. The precautions indicated in Basic Protocol
1 are equally applicable to this protocol.
Additional Materials (also see Basic Protocol 1)
1.2% (w/v) alginate, sodium salt, medium viscosity (Sigma) in 0.9% sodium
chloride solution (sterilize by autoclaving)
102 mM calcium chloride
0.9% saline
DMEM/10% FBS/antibiotic-antifungal solution (see recipe for solution with 5%
FBS) containing 10 to 50 µg ascorbic acid/ml
20-G needle and syringe
6-well culture dishes
1. Perform Basic Protocol 1, steps 1 to 10. Pellet cells by centrifugation for 10 min at
250 × g (1000 rpm in a conventional clinical centrifuge), room temperature. Resus-
pend cell pellet in 1.2% alginate to 4 × 106 cells/ml of suspension.
2. While cells are being centrifuged, add 5 ml calcium chloride into each well of a 6-well
culture dish (one dish per ∼4 × 106 cells).
3. Draw the cell suspension through a 20-G hypodermic needle into a sterile disposable
syringe large enough to accommodate the volume of suspension and dispense
dropwise 25 drops into each well. Allow the beads to polymerize in this solution for
Chondrocyte ≥10 min.
Culture and Assay

12.2.4
Supplement 12 Current Protocols in Pharmacology
 The droplets coalesce individually as they enter the solution and polymerize into discrete
beads, provided that they do not come into contact with other droplets during polymeriza-
tion. It is important to move the needle around as the suspension is dispensed, thus
minimizing contact between the drops. Avoid contacting the calcium chloride solution with
the dispensing needle.
Approximately 40,000 chondrocytes per bead can be expected.
4. Gently tip the plate forward, letting the beads settle. Slowly tilt the plate back and
aspirate the calcium chloride solution from the other side of each well while retaining
the beads in the well.
5. Wash the beads by adding 5 ml of sterile saline to each well and rocking the plates
gently to rinse the beads. Aspirate the saline and repeat the wash once. Gently
resuspend the washed beads in DMEM/10% FBS/antibiotic-antifungal mix contain-
ing 10 to 50 µg ascorbic acid/ml medium. Incubate under standard cell culture
conditions (see UNIT 12.1). Allow ≥7 to 14 days for the matrix to form before beginning
experiments that include, for example, addition of drugs or cytokines.
Ascorbic acid is essential for extracellular matrix to form in alginate beads.
Chondrocytes can be maintained in alginate for weeks to months or as long as the cultures
are free of microbial contamination. At any time during culture, alginate can be dissolved
according to Basic Protocol 2 steps 2b to 4b, followed by step 6a.

CARTILAGE CHIP CULTURE ALTERNATE

PROTOCOL 2
This is a simple procedure for culturing intact cartilage. The precautions indicated in Basic
Protocol 1 are equally applicable to this protocol.
Additional Materials (also see Basic Protocol 1)
Sterile forceps
1. Obtain long shavings according to Support Protocol 1.
2. Section these shavings perpendicular to the long axis into similarly sized segments
(∼1 mm wide).
Each chip will then comprise a cross-section incorporating all layers of cartilage.
3. Use sterile forceps to place each chip individually into a single well of a 24-well dish
containing 1 ml of DMEM/10% FBS/antibiotic-antifungal solution and 10 to 50 µg
ascorbic acid per milliliter.
Ascorbic acid is essential for maintenance of intact chips.
4. Allow the chips to incubate 2 to 3 days in a 37°C, 5% CO2 humidified incubator
before beginning experiments that include, for example, addition of drugs or cytok-
ines.

DISSECTION OF BOVINE ARTICULAR CARTILAGE SUPPORT

PROTOCOL 1
The following describes a widely used method for excising articular cartilage from freshly
obtained bovine metacarpophalangeal joints. Use analogous dissection procedures for
freshly obtained rabbit shoulder, knee, and hip joints. In rabbit, shoulder is the best source,
but the shavings are much thinner. Cartilage is similarly excised from human hip and knee
specimens, which should be dissected within 2 days after donation. Frozen cartilage does
not yield viable cells and cannot be used.
In Vitro Cellular
Assays

12.2.5
Current Protocols in Pharmacology Supplement 12
 Materials
Calf hooves (from local slaughterhouse)
70% ethanol
1× phosphate-buffered saline (PBS; see recipe)
DMEM/antibiotic-antifungal solution (without serum; see recipe)
DMEM/10% FBS/antibiotic-antifungal solution (see recipe)
Sterile disposable scalpels, no. 10, no. 15, and no. 21
150-mm petri culture dishes
1. Obtain several calf hooves that also include the first joint above the hoof of ∼2- to
6-month-old calves.
This is the metacarpophalangeal joint, commonly termed the fetlock.
Calf joints yield thicker cartilage than do adult joints.
2. Wash hoof with water to remove all traces of dirt from the skin and the hoof. Wipe
with ethanol swabs or paper towels moistened with 70% ethanol.
3. Using a no. 21 disposable scalpel, make a lengthwise incision and dissect away the
skin, taking care not to pierce the joint capsule. Wash the skinned limb with water,
then 70% ethanol.

A B

C D

Figure 12.2.1 Dissection of cartilage from the bovine metacarpophalangeal joint. (A) The skin is
removed, exposing the outer joint capsule. (B) An incision through the anterior surface of the joint
capsule is made which spares the cartilage within. (C) After the joint has been opened and exposed,
Chondrocyte a scalpel is used to excise articular cartilage by shaving along the curved surface of the exposed
Culture and Assay
joint. (D) The same joint with some of the cartilage removed, revealing the underlying bone.
12.2.6
Supplement 12 Current Protocols in Pharmacology
 4. Place the skinned limb upright, anterior side forward, inside of a sterile cell culture-
type hood on top of absorbent laboratory diapers (see Fig. 12.2.1A). Flex the limb
back at the joint.
The biological safety cabinet used should preferably be one that is not also used for routine
cell culture work.
From this point in the dissection, use sterile materials only.
5. Make a horizontal incision through the anterior side of the joint capsule, taking care
not to cut into cartilage (see Figure 12.2.1B), and continue the incision around the
sides until the joint is exposed on three sides. Carefully sever the internal ligament
and open the joint fully (see Figure 12.2.1C), washing away the clear, viscous
synovial fluid with sterile 1× PBS.
If blood is visible in the metacarpophalangeal joint capsule when it is opened, the joint
should be discarded as it may be necrotic or contaminated with microorganisms.
6. Using a sterile no. 15 disposable scalpel, obtain shavings from the metacarpopha-
langeal condylar surface (see Figure 12.2.1D). Follow the curve of the joint as closely
as possible so as to obtain long, uniformly shaped shavings, ideally in one or two
long pieces per joint surface.
Long pieces are especially desirable when cartilage is to be sectioned into chips (e.g., see
Alternate Protocol 2).
If uniformity of cartilage shavings is not critical (i.e., if chips are to be enzymatically
digested to release chondrocytes for culture), material can also be excised from other
cartilage surfaces within the joint to increase the overall yield of chondrocytes.
Excessive oozing blood from the shavings indicates that the incision was deep and included
bone, which (unlike cartilage) is vascularized in younger calves.
7. Transfer the shavings into a 150-mm petri dish containing DMEM/antibiotic-anti-
fungal mixture (do not include serum) and place the dishes in a conventional cell
culture hood.
Shavings from each hoof should be placed in a separate dish to minimize the possibility of
cross-contamination.
8. Wash the shavings two times with DMEM/antibiotic-antifungal mixture and immerse
in DMEM/10% FBS/antibiotic-antifungal solution. Store in a 37°C, 5% CO2 humidi-
fied cell culture incubator.
To allow timely detection of microbial contamination, it is advisable to incubate chips for
48 hr before chondrocyte dissociation is initiated (see Basic Protocol 1). Microbial
contamination typically presents as cloudy medium (bacteria) or visible mold.

INCORPORATION OF 35S INTO CHONDROCYTES OR CARTILAGE BASIC

PROTOCOL 2
Chondrocytes add sulfate, including exogeneous 35S, to the glycosaminoglycans (GAG)
that are attached to the proteoglycan core protein. Virtually all free sulfate is used in the
sulfation of GAGs, so the amount of 35S incorporated is proportional to the rate of
proteoglycan (largely aggregan) synthesis, an indirect index of cartilage formation.

In Vitro Cellular
Assays

12.2.7
Current Protocols in Pharmacology Supplement 12
 Materials
∼1000 to 1500 Ci/mmol 35S radionuclide (5 mCi/ml) in aqueous solution (NEN)
Chondrocyte sample: 6- or 24-well dish of cultured chondrocytes (see Basic
Protocol 1), alginate-embedded chondrocytes, or cartilage chips
PBS, calcium-free and magnesium-free
0.25% trypsin, without calcium or magnesium
DMEM/10% FBS/antibiotic-antifungal solution (see recipe for 5% FBS solution)
10× pronase E solution: 100 mg of pronase E (type XIV; Sigma; alternatively
designated as “protease”) in 10 ml water (for alginate-embedded chondrocytes
only)
HPLC-grade or molecular biology-grade water
Scintillation cocktail
0.9% saline
Alginate dissolving buffer (see recipe)
DMEM/antibiotic-antifungal solution (see recipe)
100% ethanol
15-ml polypropylene centrifuge tubes
56°C incubator (nonhumidified)
Sephadex G-25 molecular sieve columns (PD-10, Pharmacia; for monolayered and
alginate-embedded chondrocytes)
96-well scintillation counting plate
Scintillation counter with plate reader
Rocking platform at 4°C
1.5-ml microcentrifuge tubes
CAUTION: Radioactive materials require special handling; radioactive waste must be
disposed of appropriately.

Assay monolayered chondrocytes

1a. Add 25 µCi (0.925 MBq) of 35S radionuclide to each well of a 6-well dish of
chondrocytes (see Basic Protocol 1). Incubate for 5 hr under standard cell culture
conditions (see UNIT 12.1).
If conditioned medium samples are needed, for example, to analyze the release of sulfated
glycosaminoglycans or other released components of cartilage, pipet the required volumes
of medium into other tubes or vessels before adding 35S.
For convenience, this step can be done at the bench (rather than in a biosafety hood),
provided that the subsequent incubation with 35S does not exceed several hours. Subsequent
steps should be performed at the bench. More prolonged incubation of samples after bench
work may increase the risk of microbial contamination.
Adjust the amount of radionuclide correspondingly if vessels containing different volumes
are used.
2a. After incubation, save the medium from one sample in a scintillation vial for later
quantification of radioactivity. Discard all other overlaying medium according to
established radiation safety procedures.
Approximately 50,000 cpm per 10 ìl should be detectable in the culture medium when fresh
35
S is used as described.
3a. Wash each well with 5 ml calcium-free, magnesium-free PBS, and then aspirate.
4a. Add 0.5 ml of 0.25% trypsin (without calcium or magnesium) to each well and let
stand for 5 min at room temperature to release cells from dish surfaces.
Chondrocyte
Culture and Assay Trypsin does not interfere with the recovery of extracellular matrix components.
12.2.8
Supplement 12 Current Protocols in Pharmacology
5a. Add ∼2 ml of DMEM/10% FBS/antibiotic-antifungal solution to each well, transfer
to 15-ml polypropylene centrifuge tubes, and centrifuge for 10 min at 250 × g (1000
rpm).
Pooling of samples from wells may be needed at this point to optimize assay sensitivity
because monolayer culture yields relatively low counts as compared with alginate or
cartilage chip culture.
6a. Remove supernatant by pipetting. Add 1 ml of 10× pronase E solution to the pellet,
vortex to resuspend, and incubate overnight at 56°C.
This step digests the remaining matrix complexes. Samples are ready for processing the
following morning or can be refrigerated for up to 2 to 3 days.
7a. Equilibrate a Sephadex G-25 (PD-10) column with 25 ml of HPLC-grade or molecu-
lar biology-grade water. Apply 0.5 ml of the lysate and 2 ml of water successively,
discarding the flowthrough. Then add 3.5 ml of water and collect the eluate.
The void volume contains 35S incorporated into macromolecules, primarily sulfated
glycosaminoglycans (S-GAG). Unincorporated 35S remains in the column and is discarded.
8a. Add 100 µl eluate and 100 µl scintillation cocktail to duplicate wells of a 96-well
scintillation counting plate, and perform scintillation counting. Normalize the counts
to the relative DNA content of the samples (see Support Protocol 2).

Assay alginate-embedded chondrocytes

1b. Follow steps 1a to 2a above.
2b. Wash the beads two times with 5 ml of 0.9% saline per well, aspirate saline, and add
1 ml of alginate dissolving buffer per well.
3b. Incubate the dishes for 15 min at 4°C on a rocking platform.
4b. Transfer the samples to 15-ml polystyrene centrifuge tubes and centrifuge for 10 min
at 250 × g (1000 rpm in a low-speed centrifuge).
5b. Follow steps 6a to 8a above.

Assay cartilage chips

1c. Follow steps 1a and 2a above, but use a 24-well dish and half the amount of radioactive
label or less.
2c. Add DMEM/antibiotic-antifungal solution to each well and incubate 30 to 45 min in
a 37°C, 5% CO2 humidified incubator.
3c. Aspirate the radioactive medium and wash two times with 1× PBS, incubating ≥15
min (preferably 30 min) between washes at room temperature.
This incubation period allows free 35S to diffuse out of the chip, thus reducing background.
4c. Wash two times with 100% ethanol, allowing 10 to 15 min incubation in 100% ethanol
each time.
The ethanol precipitates the macromolecules in the cartilage chip.
5c. Aspirate the ethanol and evaporate the residual ethanol in a ∼37° to 56°C nonhumidi-
fied incubator.
6c. Transfer the chips to pre-labeled, screw-cap 1.5-ml microcentrifuge tubes.
Do not use flip-cap tubes.
In Vitro Cellular
Assays

12.2.9
Current Protocols in Pharmacology Supplement 12
 7c. Add 1 ml of 2 mg/ml of 10× pronase E solution, vortex and incubate overnight at
56°C. Vortex again after incubation.
Make sure the chips are completely digested. No residue should be visible.
8c. To individual wells of a 96-well scintillation counting plate, add 100 µl of scintillation
cocktail, and 100 µl protease-digested samples, mix well and quantify radioactivity
using a scintillation counter. Normalize 35S incorporation relative to DNA content
(see Support Protocol 2).

BASIC DIMETHYLMETHYLENE BLUE STAINING TO DETERMINE

PROTOCOL 3 PROTEOGLYCAN CONTENT
1,9-Dimethylmethylene blue (DMB) binds to sulfated glycosaminoglycans, causing a
change from a blue to a purple color (i.e., metachromasia). Based on this principle, an
assay is described that is more rapid and economical than the 35S incorporation assay (see
Basic Protocol 2), although potentially susceptible to greater variability between wells.
This assay is suitable for monolayer chondrocyte cultures, but more difficult to apply to
alginate cultures because of alginate interference. The assay is unsuitable for chip cultures
because excessive background radioactivity is obtained in the digests.
Materials
Medium and protease digests from cartilage or chondrocyte cultures
PBS-BSA: 1× phosphate-buffered saline (see recipe) containing 1% (w/v) purified
bovine serum albumin
1 mg/ml chondroitin sulfate (CS) from shark cartilage (Sigma) with 0.005%
sodium azide as a preservative
2× 1,9-dimethylmethylene blue (DMB) working solution (see recipe)
96-well plates, flat clear bottom polystyrene, nonsterile (e.g., Costar)
Plate reader able to read absorbance between 520 to 530 nm (if this bandwidth is
not available, the bandwidth 590 to 600 nm can be used).
1. Dilute the samples (protease digests from cartilage or chondrocyte cultures; step 6a
or 7c of Basic Protocol 2) in PBS-BSA in a 96-well plate.
BSA is necessary for stabilization of the S-GAG-DMB complex in 96-well dishes.
Pipet carefully and avoid forming bubbles; they will interfere with plate readings.
2. Prepare six serial 1:1 dilutions of the original sample. Add 100 µl of each dilution to
the corresponding well.
3. Construct a standard absorbance curve by adding CS (as a standard) successively at
concentrations from 4 µg/well to 0.125 µg/well in successive 1:1 dilutions, in a final
volume of 100 µl.
4. Just before reading the plates, add 100 µl of 2× DMB working solution to each well.
5. Place the plate on a plate shaker for 5 to 30 sec.
Avoid vigorous vortexing or elevated temperatures as undesirable precipitation may occur.
6. Read absorbance at 525 nm any time between 5 and 30 min later. Use a filter between
520 and 530 nm.
If this bandwidth is not accessible, a 580 to 600 nm filter may be used, but results tend to
be more consistent at 520 to 530 nm.
Upon dye binding, proteoglycan absorbance increases at 525 nm and decreases at 590 nm.
The readable color range is from blue to purple. Disregard any pink samples, as their
absorbance values are outside the linear assay range.
Chondrocyte
Culture and Assay

12.2.10
Supplement 12 Current Protocols in Pharmacology
 7. Normalize calculated proteoglycan absorbance values to DNA content values, if
available (see Support Protocol 2).

MEASUREMENT OF DNA CONTENT SUPPORT

35 PROTOCOL 2
Values for S incorporation (see Basic Protocol 2) or 1,9-dimethylmethylene blue (DMB)
staining (see Basic Protocol 3) in monolayered or alginate-embedded chondrocytes
should be normalized for cell number. It is virtually impossible to count cells within intact
cartilage or alginate matrix, but content of DNA is generally considered to correlate with
cell number and can be used as a surrogate measure.
Materials
Hoechst dye working solution (see recipe), prepare fresh daily
Lysed or protease-digested chondrocyte samples (e.g., see Basic Protocol 1,
Alternate Protocol 1, or Alternate Protocol 2)
100 µg/ml bovine (calf) thymus DNA or other reference DNA sample (see recipe)
TNE buffer (see recipe)
96-well black cluster plate, clear bottom
Fluorescence plate reader
1. To duplicate wells of the cluster plate, add 200 µl of Hoechst dye working solution
and 50 µl of the lysed or protease-digested chondrocyte samples.
The Hoechst dye intercalates with DNA and emits detectable fluorescence, allowing
detection and quantitation of tissue DNA.
2. Prepare a standard curve using six serial dilutions (0.156 to 10 µg) of 100 µg/ml
bovine (calf) thymus DNA or other reference DNA in TNE buffer. Add each
concentration in duplicate to selected wells of a 96-well black cluster plate.
If chondrocytes are released with protease, protease solution should be used as the diluent
for the reference DNA samples.
3. Measure fluorescence using a fluorescence plate reader set at 350 to 360 nm for
excitation and 450 to 460 nm for emission.
The minimum detectable level should be ∼100 ng/ml DNA. The assay is linear up to 100
ìg/ml (see Fig. 12.2.2).

14000 1600
12000 y = 127.37x − 101.86 1400 y = 151.25x + 12.258
Fl 360/450

Fl 360/450

10000 R 2 = 0.9996 1200 R 2 = 0.9977

1000
8000
800
6000 600
4000 400
2000 200
0 0
0 25 50 75 100 0 1 2 3 4 5 6 7 8 9 10
[DNA] (µg/ml) [DNA] (µg/ml)

Figure 12.2.2 Representative standard curves for assay of DNA content. Known concentrations
of DNA were serially diluted in a 96-well plate in buffer (indicated on the horizontal axis) and Hoechst
Dye was added as described. The vertical axis of the figure indicates fluorescence as measured
with an excitation filter of 360 and emission filter of 450 nm. The concentration of DNA in the well
was plotted relative to the fluorescence measured. The panel on the left shows DNA concentrations In Vitro Cellular
Assays
up to 100 µg/ml; on the right, DNA concentrations of 0.1 to 10 µg/ml are shown.
12.2.11
Current Protocols in Pharmacology Supplement 12
 4. Plot the standard curve of micrograms reference DNA versus fluorescence and
perform linear regression analysis. Quantitate DNA content in experimental samples
using this standard curve (see Fig. 12.2.2).
This assay remains linear over a wide range of DNA concentrations. If the background is
set to zero and the maximum is set, for example, to 100 for 100 ìg/ml, the concentration
can be directly extrapolated from the fluorescence reading.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Alginate dissolving buffer

55 mM sodium citrate
150 mM NaCl
30 mM EDTA
Store up to 6 months at room temperature
Bovine (calf) thymus DNA or other reference DNA sample, 100 ìg/ml
Prepare solution by dissolving 100 µg/ml of DNA in water for a full day with rocking
at room temperature, then store at −20°C in aliquots for use as needed.
1,9-Dimethylmethylene blue (DMB) solutions
Stock solution (10×): Add 80 mg of 1,9-dimethylmethylene blue (DMB) powder
(Aldrich) and 12.5 ml ethanol to a 500-ml bottle. While stirring this solution, add
488 ml of formate buffer (see recipe). Store up to 6 months at room temperature in
a dark bottle.
DMB stains intensely, indiscriminately, and irreversibly. Wear gloves and lab coats whenever
handling DMB powder or solutions, and clean exposed bench surfaces immediately after-
wards.
When preparing the DMB stock solution, handle sodium formate and formic acid under a
fume hood.
Working solution (2×): Add 100 ml of 10× DMB stock solution to 400 ml of formate
buffer (see recipe). Stable for 2 months when stored at room temperature in the dark.
With prolonged storage, background increases and metachromasia declines.
DMEM/FBS/antibiotic-antifungal solution
To Dulbecco’s modified Eagle medium (DMEM; low glucose for bovine; high
glucose for other species including rabbit and human) containing antibiotic and
antifungal mixture for tissue culture (e.g., penicillin-streptomycin; Fungizone, Life
Technologies) at the appropriate dilution. Add fetal bovine serum (FBS) if needed
at 5% or 10% (v/v) as indicated. Store at 4°C until the expiration date for the given
batch of medium.
Formate buffer
4 g sodium formate
4 ml formic acid
Deionized water to 1 liter
Store up to 6 months at room temperature
NOTE: Handle sodium formate and formic acid under a fume hood.

Chondrocyte
Culture and Assay

12.2.12
Supplement 12 Current Protocols in Pharmacology
Hoechst dye solutions
Stock solution: Dissolve 10 mg Hoechst dye (bis-benzimide; Sigma) per milliliter
of water and pass through a 0.2-µm filter. Protect this 10 mg/ml stock solution from
light by wrapping in foil and store up to 1 year at 4°C.
Working solution:
10 µl 10 mg/ml Hoechst dye stock solution
5 ml 10× PBS (see recipe)
25 ml 3 M sodium chloride
5 ml TNE buffer (see recipe)
Bring to a final volume of 50 ml with deionized water. Prepare fresh on the day of the assay.
Discard working solution after use.
Phosphate-buffered saline (PBS), 10×
1 g/liter anhydrous CaCl2
2 g/liter KCl
2 g/liter KH2PO4
1 g/liter MgCl2 6H2O
80 g/liter NaCl
21.6 g/liter Na2HPO4⋅7H2O
Stable up to 6 months at room temperature.
TNE buffer
100 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)
1 M NaCl
10 mM EDTA
Store up to 3 to 4 months at room temperature.

COMMENTARY
Background Information
Chips in culture have long been used for
analysis of cartilage function, as for example
by Morales et al. (1984). The enzymic disso-
ciation of chondrocytes from cartilage for cell
culture manipulations was described in detail
by Kuettner et al. (1982), providing the basis
for experimental manipulations of isolated
chondrocytes. Thus, for example, foreign DNA
can be introduced into chondrocytes by trans-
fection or viral infection far more readily than
into intact cartilage, and RNA is easier to isolate
from cultured chondrocytes. Traditionally, dis-
sociated chondrocytes were cultured in mono-
layer, as is conventional for other cell culture
systems. It was recognized, however, that chon-
drocytes in this culture system lose their dis-
tinctive phenotype and adopt fibroblastic char-
acteristics such as the biosynthesis of collagen
I instead of cartilage-specific collagen II (Van
Osch et al., 1998).
Alginate culture was introduced to combine
favorable characteristics of these two culture
systems (Guo et al., 1989; Hauselmann et al.,
1992). Chondrocytes are dissociated and incor-
porated into an artificial, three-dimensional
matrix that maintains the chondrocyte pheno-
type for as long as 8 months (Hauselmann et
al., 1993). When needed, the matrix is readily
dissolved by addition of chelating agents, free-
ing chondrocytes for further experimental ma-
nipulations that are difficult when the cells are
embedded in a matrix. The chondrocyte pheno-
type is well maintained in this culture system
(Beekman et al., 1997; Liu et al., 1998). It
should be cautioned that the Ca2+ concentration
may not be at physiological levels and that the
alginate matrix does not perfectly simulate the
natural chondrocyte microenvironment. For
example, collagen ultrastructure and distribu-
tion may differ (Gregory et al., 1999).
Proteoglycans, especially large chondroitin
sulfate proteoglycan (aggregan), constitute an
essential component of cartilage, and the ability
of chondrocytes to synthesize proteoglycan
provides a measure of extracellular matrix
maintenance. Basic Protocol 2 derives from the
demonstration that chondrocytes add sulfate,
including 35S that may be present, to the gly-
cosaminoglycans that are attached to the pro- In Vitro Cellular
Assays

12.2.13
Current Protocols in Pharmacology Supplement 12
Table 12.2.2 Troubleshooting Guide for Chondrocyte or Cartilage Culture and Proteoglycan Assays

Observation Possible cause Recommendation

No proliferation in monolayer
No cell pellet after digestion of Nonviable cells
alginate beads
Slow monolayer proliferation
Peeling monolayers
Nonproliferating clumps of round Incomplete digestion by
chondrocytes interspersed with
monolayered chondrocytes
Microbial contamination
Alginate beads clump together,
leading to atypical and erratic
assay results
Low control 35S counts
Poor inhibition of incorporation
by protein synthesis inhibitors
Purple precipitate forms
prematurely during DMB assay
Erratic and/or excessively high
DMB assay readings
Chondroitin sulfate standard gives DMB sample may be faulty
low or negligible absorbance in
presence of DMB
Nonviable cells
Cells seeded too sparsely. Source If poor health, cells may recover in culture. If
cartilage in poor condition at time sparse seeding, use recommended seeding
of dissection.
Seeded too densely; excessive
matrix formation
collagenase during dissociation
Introduction of microbes during
tissue dissection.
Beads do not gel (polymerize)
separately when formed in
calcium chloride solution
Old 35S stock. Specific activity of Use fresh radioisotope. Use 35S with higher
35S may be too low.
Nonviable cells
Excessive 35S label
Too much free 35S remaining
Nonviable cells
Aggregation of substrate due to
excessive substrate or delay in
processing plates
No BSA or other blocking protein Make sure BSA is included in assay mixture
present
Bubbles may be present
(Some commercially supplied
samples of DMB do not work)
Use fresh tissue. Recheck medium
components. Verify absence of microbial
contamination. Include serum in medium.
Use fresh tissue. Recheck medium
components. Verify absence of microbial
contamination.
densities.
Use recommended seeding densities; reduce
densities further if needed.
Increase digestion time and/or reduce amount
of cartilage digested per unit of collagenase
Refer to UNIT 12.1. Observe indicated
precautions during dissection. Decontaminate
incubator if problem persists.
Make sure alginate droplets are well-dispersed
into medium. Allow them to gel thoroughly
before removing calcium chloride solution.
specific activity.
Use fresh tissue. Check medium components.
Use recommended seeding densities.
Reduce amount of label. If using cartilage
chips, allow longer label washout in saline.
Recheck column purification method.
If working with chips, make sure sufficient
washout time is allowed
Use fresh tissue. Check medium components.
Use recommended seeding densities.
Disregard affected wells. Read at lower
substrate concentrations. Read within ≤30 min
after adding DMB.
Pipet precisely without blowing out. If
problem occurs nonetheless, try centrifuging;
gently directing air or N2 air stream over
wells; touching bubbles gently with blotting
paper.
Mix chondroitin sulfate and DMB solution
1:1. If no pink color, obtain other samples of
DMB and retest.

teoglycan core protein. Virtually all free sulfate
is used in the sulfation of glycosaminoglycans,
so the amount of 35S incorporated by the bead
is proportional to the rate of proteoglycan syn-
thesis (Morales et al., 1984). Alternate Protocol
3 relies on the ability of dimethylmethylene
Chondrocyte
Culture and Assay blue to bind to sulfated glycosaminoglycans
and to change color from blue to purple upon
binding (Goldberg and Kolibas, 1990).
Either method of assay of proteoglycan syn-
thesis (Basic Protocol 2 or 3) is amenable to the
evaluation of drug or cytokine effects on pro-
teoglycan synthesis. For example, interleukin-
1, a chondrodestructive cytokine, prominently

12.2.14
Supplement 12 Current Protocols in Pharmacology
reduces proteoglycan synthesis within 24 to 48
hr (e.g., Taskiran et al., 1994). The ability of
drugs to ameliorate or block altogether the
inhibitory effect of IL-1 may indicate a mecha-
nistic basis for this action (e.g., Hauselmann et
al., 1994) and/or suggest a potential for the test
drug to exert chondroprotective activity (e.g.,
Yaron et al., 1999). Conversely, transforming
growth factor-β, which promotes extracellular
matrix formation, generally increases proteo-
glycan synthesis (Morales and Roberts, 1988),
although experimental conditions such as the
duration of culture may modify the observed
effects (van der Kraan et al., 1992). Four to
seven days of incubation are generally required
for this effect of transforming growth factor-β
to become detectable. Drugs or other sub-
stances that increase chondrocyte or cartilage
proteoglycan synthesis might be considered as
possible approaches to promote the repair of
cartilage.
The protocols in this unit for chondrocyte
culture are fundamental for the evaluation in
vitro of other chondrocyte characteristics and
their responses to physiological manipulations,
growth factors and experimental drugs. For
example, the release of proteoglycan fragments
into the culture medium is often an indication
of cartilage degradation and is readily detected
by subjecting culture medium to the DMB
staining protocol (e.g., Goldberg et al., 1993).
The inhibition of this release, which in many
cases is thought to be mediated by metallopro-
teinases, may indicate chondroprotective activ-
ity. Other assays of chondrocyte function may
include monitoring of collagen II synthesis,
detection of other extracellular matrix compo-
nents such as other known collagens and pro-
teoglycans, synthesis and release of chondrod-
estructive enzymes and analysis of message
levels for cartilage components.
Critical Parameters and
Troubleshooting
Articular cartilage from young bovines (2 to
6 months of age) yields significantly more
chondrocytes relative to the time and effort
expended than does human articular cartilage
or that from rabbits, rodents, or other small
laboratory animals. Cartilage is thicker in
younger than in mature individuals and respon-
siveness to experimental manipulations may
vary with both the species and the age of animal
or patient. Proteoglycan synthesis may also
vary according to the amount of extracellular
matrix already present in cultured chondro-
cytes, whether monolayered or in alginate.
Current Protocols in Pharmacology
Although Basic Protocol 1 (monolayer cul-
ture of chondrocytes) is compatible with con-
ventional cell culture procedures, the chondro-
cyte phenotype is best preserved in cartilage
chip culture (see Alternate Protocol 1) or algi-
nate culture (see Alternate Protocol 2). Any of
these in vitro culture protocols are amenable to
the evaluation of proteoglycan synthesis by the
35S incorporation method or to the sampling of
secreted products (such as nitric oxide or met-
alloproteinases) from culture medium. If gene
expression is to be evaluated, RNA extraction
is easier from monolayer or alginate cultures
than from cartilage, although methods have
recently been described that facilitate cartilage
RNA isolation (McKenna et al., 2000). The
DMB assay, and certain other cell stains, are
not compatible with alginate.
Monolayered chondrocytes need to be used
within several days of plating, lest the chondro-
cyte phenotype be lost. The use of cartilage
chips or alginate culture allows more time and
flexibility in the design and execution of ex-
periments.
Nonphysiological binding of 35S or nonspe-
cific staining of DMB constitutes assay “back-
ground” and should be determined, especially
when beginning a new series of experiments.
Because proteoglycan synthesis depends on de
novo protein synthesis, one approach to assess-
ing background levels is to treat samples with
a protein synthesis inhibitor such as cyclohexi-
mide. This treatment should reduce proteogly-
can synthesis by 3- to 10-fold or more (see Table
12.2.2). Another experimental issue is the pres-
ence or absence of serum. Conventional serum
supplementation (5% to 10%), in addition to
facilitating chondrocyte proliferation and
maintenance in culture, reliably increases pro-
teoglycan synthesis (Hascall et al., 1983). The
latter effect is favorable for studying inhibitors
of proteoglycan synthesis such as IL-1, but may
mask the ability of some treatments such as
TGF-β to increase proteoglycan synthesis and
may block the effects of experimental drugs or
other treatments that happen to be highly pro-
tein-bound. If the objective is to detect treat-
ment-induced increases in proteoglycan syn-
thesis, it is advisable to incubate the cultures
for several days to a week with the experimental
treatment in medium that is essentially serum-
free. Some treatments, such as cytokines, bind
nonspecifically to vessel walls in the absence
of soluble protein. If this might be a problem,
include a low concentration of BSA or FBS
(0.1% to 0.2%). By comparison with chondro- In Vitro Cellular
cyte cultures, cartilage chips are less dependent Assays
12.2.15
Supplement 12
 Table 12.2.3 Example of Data From 35S
Incorporation Assay of Proteoglycan Synthesis by
Monolayered Bovine Chondrocytesa

Treatment Conc. cpmb

None 0 78 ± 4
IL-1 10 ng/ml 24 ± 4
Cycloheximide 40 µM 19 ± 2
an = 6 per group. 10% FBS in medium.
bcpm values expressed as mean ± S.E.

Table 12.2.4 Example of Data Showing IL-1 and Cycloheximide Effects in the 35S
Incorporation Assay of Proteoglycan Synthesis by Alginate-Embedded Bovine
Chondrocytes

Treatmenta cpmb DNA (µg)b cpm/µg DNAb

Expt. 1c No IL–1 1161 ± 253 3.2 ± 0.8 371 ± 48
n=6 IL–1 281 ± 41 3.4 ± 0.7 85 ± 15
Expt. 2c No cycloheximide 2006 ± 209 2.9 ± 0.13 698 ± 91
n=2 Cycloheximide 411 ± 161 2.7 ± 0.35 181 ± 124
aMedium contained 10% FBS.
bValues expressed as mean ± S.E.
cSeparate experiments for IL–1 and cycloheximide.

Table 12.2.5 Example of Data Showing Effects of Growth Factors and Serum

Treatmenta Concentration cpmb DNA (µg)b cpm/µg DNAb

None — 172 ± 18 2.8 ± 0.2 80 ± 19

IGF-1 100 ng/ml 309 ± 29 2.6 ± 0.2 122 ± 14
TGF-β 50 ng/ml 603 ± 33 3.2 ± 0.2 195 ± 19
10% FBSc — 1112 ± 94 4.2 ± 0.2 253 ± 32
an = 6 per group.
bAll values expressed as mean ± S.E.
cFBS present in medium only as indicated.

Table 12.2.6 Example of Data Showing IL-1 and

Cycloheximide Effects in the 35S Incorporation Assay
of Proteoglycan Synthesis by Rabbit Cartilage Chips

Treatmenta Concentration cpmb

None — 8521 ± 2190

IL-1 5 ng/ml 3146 ± 768
Cycloheximide 10 µM 603 ± 33
an = 8
bAll values expressed as mean ± S.E.
Chondrocyte
Culture and Assay

12.2.16
Supplement 12 Current Protocols in Pharmacology
 Table 12.2.7 Example of Data From DMB Assay of
Proteoglycan Synthesis by Monolayered Bovine Chondrocytes
Treatmenta
Expt. 1 No IL-1
IL-1
Expt. 2 No cycloheximide
Cycloheximide
an = 4 per group. 10% FBS in medium.
bAbsorbance values expressed as mean ± S.E.
on serum supplementation for maintenance of
proteoglycan synthesis, probably because of
growth factors and accessory proteins bound to
or associated with cartilage.
Anticipated Results
From a well in a 6-well dish containing
bovine chondrocytes in monolayer with 10%
FBS medium, ∼100 to 200 cpm of 35S can be
expected; from a comparable well containing
20 to 25 alginate beads ∼1000 to 2500 cpm of
35S; from cartilage chips, several thousand cpm
of 35S. Quantitative values of DMB absorbance
vary with the apparatus used. Treatment of cells
with a protein synthesis inhibitor such as cy-
cloheximide should reduce 35S incorporation
or DMB staining to low or negligible values
(see Tables 12.2.3, 12.2.4, 12.2.6, and 12.2.7
for expected effects of cycloheximide and IL-1,
see Table 12.2.5 for the effects of TGF-β, IGF-
1, and FBS). In the absence of serum, incorpo-
ration of 35S by cultured chondrocytes should
be at least 2-fold lower, depending on experi-
mental conditions and the lot of serum. IL-1
should decrease both DMB-induced absor-
bance changes and 35S incorporation by ≥50%
when incubated in the presence of serum.
Time Considerations
Dissection of cartilage shavings from joints
takes ∼2 hr (see Support Protocol 1). Allow 5
to 6 hr (including ∼2 hr hands-on laboratory
work) to dissociate chondrocytes from carti-
lage, and an additional 1 hr of laboratory work
if they are to be embedded in alginate (see Basic
Protocol 1 and Alternate Protocol 1). Cartilage
chips require 1 hr for culture setup (see Alter-
nate Protocol 2). Assays of 35S from monolay-
ered or alginate-embedded chondrocytes entail
a 5-hr incubation followed by 2 hr hands-on
time in the early afternoon, an overnight incu-
bation and an additional ∼3 hr to separate free
from bound 35S (see Basic Protocol 2). Assays
of 35S incorporation by chips require approxi-
Current Protocols in Pharmacology
S-GAG (µg/ml)b
14.6 ± 0.6
5.9 ± 0.1
9.7 ± 0.5
0.02 ± 0.02
mately the same amount of time on the first day
but only ∼1 hr on the second day (see Basic
Protocol 2).
Assaying DNA content of protease-digested
samples (see Support Protocol 2) requires ∼1
hr; DMB assays (see Basic Protocol 3) take ∼1
hr in addition to buffer preparation and time
required to set up the spectrophotometer.
Literature Cited
Beekman, B., Verzijl, N., Bank, R.A., von der Mark,
K., and TeKoppele, J.M. 1997. Synthesis of col-
lagen by bovine chondrocytes cultured in algi-
nate: Posttranslational modifications and cell-
matrix interaction. Exp. Cell Res. 237:135-141.
Goldberg, R.L., Spirito, S., Doughty, J.R., and Di-
Pasquale, G. 1993. Release of cell surface pro-
teoglycan from chondrocytes by interleukin-1.
Agents Actions 39:C163-C165.
Gregory, K.E., Marsden, M.E., Anderson-MacKen-
zie, J., Bard, J.B., Bruckner, P., Farjanel, J., Rob-
ins, S.P., and Hulmes, D.J. 1999. Abnormal col-
lagen assembly, though normal phenotype, in
alginate bead cultures of chick embryo chondro-
cytes. Exp. Cell Res. 246:98-107.
Guo, J., Jourdian, G.W., and MacCallum, D.K.
1989. Culture and growth characteristics of
chondrocytes encapsulated in alginate beads.
Connect Tissue Res. 19:277-297.
Hascall, V.C., Handley, C.J., McQuillan, D.J., Has-
call, G.K., Robinson, H.C, and Lowther, D.A.
1983. The effect of serum on biosynthesis of
proteoglycans by bovine articular cartilage in
culture. Arch. Biochem. Biophys. 224:206-223.
Hauselmann, H.J., Fernandes, R.J., Block, J.A.,
Schmid, T.M., and Thonar, E.J.-M.A. 1993.
Adult articular chondrocytes retain their pheno-
type after 8 months of culture in alginate. Trans-
actions of the Orthopedic Research Society 39th
Annual Meeting, San Francisco, p. 624.
Hauselmann, H.J., Oppliger, L., Michel, B.A., Ste-
fanovic-Racic, M., and Evans, C. H. 1994. Nitric
oxide and proteoglycan biosynthesis by human
articular chondrocytes in alginate culture. FEBS
Lett. 352:361-364.
Liu, H., Lee, Y.-W., and Dean, M.F. 1998. Re-ex-
pression of differentiated proteoglycan pheno- In Vitro Cellular
type by dedifferentiated human chondrocytes Assays
12.2.17
Supplement 12
 during culture in alginate beads. Biochim. Bio-
phys. Acta. 1425:505-515.
McKenna, L.A., Gehrsitz, A., Soder, S., Eger, W.,
Kirchner, T. and Aigner, T. 2000. Effective iso-
lation of high-quality total RNA from human
adult articular cartilage. Anal. Biochem. 286:80-
85.
Morales, T.I. and Roberts, A. B. 1988. Transforming
growth factor beta regulates the metabolism of
proteoglycans in bovine cartilage organ cultures.
J. Biol. Chem. 263:12828-12831.
Taskiran, D., Stefanovic-Racic, M., Georgescu, H.,
and Evans, C. 1994. Nitric oxide mediates sup-
pression of cartilage proteoglycan synthesis by
interleukin-1. Biochem. Biophys. Res. Commun.
200:142-148.
van der Kraan, P., Vitters, E., and van den Berg, W.
1992. Differential effect of transforming growth
factor beta on freshly isolated and cultured chon-
drocytes. J. Rheumatol. 19:140-145.
von der Mark, K. 1986. Differentiation, modulation
and dedifferentiation of chondrocytes. Rheuma-
tology 10:272-315.
van Osch, G.J.V.M., Van der Veen, S.W., Buma, P.,
and Verwoerd-Verhoef, H.L. 1998. Effect of
transforming growth factor-beta on proteogly-
can synthesis by chondrocytes in relation to dif-
ferentiation stage and the presence of pericellular
matrix. Matrix Biol. 17:413-424.
Yaron, M., Shirazi, I., and Yaron, I. 1999. Anti-in-
terleukin-1 effects of diacerein and rhein in hu-
man osteoarthritic synovial tissue and cartilage
cultures. Osteoarthritis Cartilage 7:272-280.
Key References
Goldberg, R.L. and Kolibas, L.M. 1990. An im-
proved method for determining proteoglycans
synthesized by chondrocytes in culture. Connect
Tissue Res. 24:265-275.
This paper gives more details of the DMB assay
method that is presented.
Chondrocyte
Culture and Assay
12.2.18
Supplement 12
Hauselmann, H.J., Aydelotte, M.B., Schumacker,
B.L., Kuettner, K.E., Gitelis, S.H., and Thonar,
E.J.-M.A. 1992. Synthesis and turnover of pro-
teoglycans by human and bovine adult articular
chondrocytes cultured in alginate beads. Matrix
12:116-129.
Basic characteristics of the alginate culture system
are described in detail in this early paper.
Kuettner, K.E., Pauli, B.U., Gall, G., Memoli, V.A.,
and Shenk, R.K. 1982. Synthesis of cartilage
matrix by mammalian chondrocytes in vitro. Iso-
lation, culture characteristics and morphology. J.
Cell Biol. 93:743-750.
Chondrocyte cell dissociation from cartilage and
subsequent culturing is described in detail.
Morales, T.I., Wahl, L.M., and Hascall, V.C. 1984.
The effect of bacterial lipopolysaccharides on
the biosynthesis and release of proteoglycans
from calf articular cartilage cultures. J. Biol.
Chem. 259:6720-6729.
Cartilage chip cultures and the [35S] assay are
described.
Internet Resources
http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ah.htm
Office of Health and Safety, Center for Disease
Control, BMBL APPENDIX H. 1999. Working with
Human and Other Primate Cells.
Contributed by Jeffrey Liebman and
Ronald L. Goldberg
Novartis Institute of Biomedical Research
Summit, New Jersey
Current Protocols in Pharmacology