chondrocyte culture and assay-manual

UNIT 12.2Chondrocyte Culture and Assay

Chondrocytes are the cells solely responsible for the synthesis and maintenance ofcartilage. On the basis of comparisons of morphology and content of the matrix, threetypes of cartilage have been identified: elastic cartilage, fibrocartilage and hyalinecartilage (von der Mark, 1986). Elastic cartilage contains elastin and is found in the earand epiglottis. Fibrocartilage consists predominantly of type I collagen; it is found, forexample, in the vertebral nucleus pulposus and joint meniscus and is a transitional formbetween hyaline cartilage and dense connective tissue. Hyaline cartilage in mammals isuniquely characterized by a high content of collagen II and of aggrecan, a large,cartilage-specific proteoglycan species. It serves as a developmental precursor to boneand is present in adult mammals as nasal cartilage and intercostal cartilage. As skeletaldevelopment progresses and cartilagineous precursors are calcified to form bone, theendings of bones that are within articulated (moving) joints remain covered with hyalinecartilage. 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 ofarticular cartilage is to ensure the almost-frictionless movement of bone surfaces withinvertebrate joints. Osteoarthritis, a disease which is especially prevalent in the elderly, ischaracterized by defects in or deterioration of articular cartilage. Thus, articular cartilageis the focus of many pharmacological studies. Although the following protocols areapplicable to all types of hyaline cartilage, they are most commonly used for work witharticular cartilage, which in this unit is referred to as simply “cartilage”.

The structural integrity of cartilage derives from the fibrous network formed by thecartilage-specific protein, collagen II, which is immersed in a gel formed by the very largeproteoglycan 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 solelyby a single cell type, chondrocytes, which make up <10% of the total volume of humanarticular cartilage. Chondrocytes synthesize and secrete the extracellular matrix thatconstitutes cartilage, which contains a cartilage-specific collagen (collagen II; 70% ofcartilage dry weight), a very large proteoglycan (aggregan; 20% of dry weight), and, inlesser amounts, several smaller proteoglycan and collagen species that modulate thearchitecture of cartilage. This cell type also secretes numerous additional factors crucialto the maintenance and remodeling of cartilage, notably metalloproteinases, cytokines,and growth factors. A fundamental consideration for tissue culture is that the fullrepertoire of chondrocyte actions is maintained only when they are in 3-dimensionalmatrices that allow them to remain spherical (von der Mark, 1986).

This unit presents a method of isolating and culturing chondrocytes in monolayer fromarticular cartilage (see Basic Protocol 1) and two alternative culture systems: chondrocyteculture in alginate (see Alternate Protocol 1) and cartilage chip culture, also termed“explant culture” (see Alternate Protocol 2). Chondrocyte monolayer culture conforms toconventional cell culture procedures and facilitates the experimental manipulation ofchondrocytes. Alternate Protocol 1 and Alternate Protocol 2 each provide for 3-dimen-sional culture of chondrocytes, allowing them to retain their characteristic phenotype andrange of actions. Dissection of cartilage from joints is described (see Support Protocol1). Incorporation of 35S into chondrocytes or cartilage (see Basic Protocol 2) is a widelyused assay of proteoglycan synthesis, a basic measure of cartilage function. An alternative,staining-based assay, amenable to higher-throughput pharmacological studies, is alsopresented (see Alternate Protocol 3). Either of these assays is suitable for the evaluationof drug or cytokine effects on proteoglycan synthesis, which provides a measure of the

Supplement 12

Contributed by Jeffrey Liebman and Ronald L. GoldbergCurrent Protocols in Pharmacology (2001) 12.2.1-12.2.18Copyright © 2001 by John Wiley & Sons, Inc.

12.2.1

In Vitro CellularAssays

extent to which chondrocytes are actively synthesizing components of cartilage. Finally,an assay of DNA content is provided to allow normalization of proteoglycan synthesisrelative 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 cellculture conditions (UNIT 12.1). Medium should always contain both antibiotic and antifun-gal chemicals.

Unless otherwise indicated, perform all procedures including centrifugation and assaysat room temperature. Follow Universal Precautions whenever human tissue material isused (Office of Health and Safety, Center for Disease Control web page, 1999).

BASICPROTOCOL 1

CHONDROCYTE MONOLAYER CULTUREShavings 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 monolayersaccording 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 units150-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 stirrer20-µm nylon filter membrane (Spectra-Mesh, Spectrum Laboratories)Autoclaved glass funnel50-ml centrifuge tubes, sterileClinical centrifugeCulture vessels (see Table 12.2.1)

Dissociate chondrocytes from cartilage1. 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 todigest proteins that would otherwise impede digestion cartilage by bacterial collagenase.

Supplement 12 Current Protocols in Pharmacology

12.2.2

ChondrocyteCulture and Assay

2. Transfer the cartilage shavings (see Support Protocol 1) to a 150-mm plastic petridish containing 30 ml DMEM/antibiotic-antifungal solution.

3. Using two sterile stainless steel disposable scalpels (no. 21 or equivalent), mince thecartilage shavings into small chips (i.e., 0.5- to 1-mm across) and transfer to a 100-mlspinner flask equipped with side arms.

4. Add the filtered pronase E in DMEM solution from step 1 to the spinner flask, andplace the impeller assembly in the flask. Put the flask with the attached impeller ona magnetic stirrer in a 37°C, 5% CO2 humidified cell culture incubator and digestwith stirring for 1 hr.

Do not use a magnetic stir bar in place of the impeller assembly. The cells will be damagedas they pass between the stir bar and the bottom of the flask.

5. During the pronase E digestion, dissolve 350 mg of high clostripain bacterialcollagenase in 100 ml DMEM/5% FBS/antibiotic-antifungal solution and passsuccessively through 0.8-, 0.45-, and 0.22-µm filter units.

Do not substitute mammalian collagenase for bacterial collagenase. Bacterial collagenasedigests 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 andresume 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 duringdigestion and culture steps.

The chips should remain largely intact during pronase E digestion, but should shrink andbecome almost invisible during collagenase digestion. The collagenase digestion time mayneed 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 glassfunnel. Decant the collagenase solution from step 6 through this filter into 50-mlsterile centrifuge tubes.

The dissociated chondrocytes will pass through the filter, leaving undigested matrix behind.

Place chondrocytes in culture8. 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-dium).

Table 12.2.1 Recommended Chondrocyte MonolayerSeeding Densities

Vessel Vol. medium (ml) Cells (× 106)

96-well plate 0.1 0.0524-well plate 0.5 0.26-well plate 2.4 1 25-cm2 flask 6 2.5100-mm petri dish 14 5.775-cm2 flask 19 7.5

Current Protocols in Pharmacology Supplement 12

12.2.3


9. Wash the cells two times in DMEM (serum is not necessary), centrifuging after eachwash as in step 8.

10. Add ∼2 ml medium per hoof (the precise volume is not critical). Resuspend cells bypipetting up and down, then count cells.

A single bovine calf metacarpophalangeal joint should yield ∼3 × 106 cells, depending onhow 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 ofmedium. Change medium 1 day later.

Freshly isolated primary chondrocytes require up to 24 hr to attach completely. The mediumbecomes 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 daysof culture.

Unless collagen synthesis is of interest, monolayer chondrocyte cultures do not requireascorbic 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 notsubculture.

Primary monolayered bovine chondrocytes begin to assume a fibroblast phenotype afterseveral days in culture, and subculturing accelerates this undesirable dedifferentiationprocess. Therefore, if the chondrocyte phenotype is desirable or required, subculturingshould be avoided.

ALTERNATEPROTOCOL 1

CHONDROCYTE CULTURE IN ALGINATE

This protocol differs from Basic Protocol 1 in that chondrocytes are embedded in a3-dimensional matrix composed of alginate. The precautions indicated in Basic Protocol1 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% sodiumchloride solution (sterilize by autoclaving)

102 mM calcium chloride0.9% 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 syringe6-well culture dishes

1. Perform Basic Protocol 1, steps 1 to 10. Pellet cells by centrifugation for 10 min at250 × 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-wellculture dish (one dish per ∼4 × 106 cells).

3. Draw the cell suspension through a 20-G hypodermic needle into a sterile disposablesyringe large enough to accommodate the volume of suspension and dispensedropwise 25 drops into each well. Allow the beads to polymerize in this solution for≥10 min.


12.2.4


The droplets coalesce individually as they enter the solution and polymerize into discretebeads, 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, thusminimizing contact between the drops. Avoid contacting the calcium chloride solution withthe 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 andaspirate the calcium chloride solution from the other side of each well while retainingthe beads in the well.

5. Wash the beads by adding 5 ml of sterile saline to each well and rocking the platesgently to rinse the beads. Aspirate the saline and repeat the wash once. Gentlyresuspend the washed beads in DMEM/10% FBS/antibiotic-antifungal mix contain-ing 10 to 50 µg ascorbic acid/ml medium. Incubate under standard cell cultureconditions (see UNIT 12.1). Allow ≥7 to 14 days for the matrix to form before beginningexperiments 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 culturesare free of microbial contamination. At any time during culture, alginate can be dissolvedaccording to Basic Protocol 2 steps 2b to 4b, followed by step 6a.

ALTERNATEPROTOCOL 2

CARTILAGE CHIP CULTURE

This is a simple procedure for culturing intact cartilage. The precautions indicated in BasicProtocol 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 dishcontaining 1 ml of DMEM/10% FBS/antibiotic-antifungal solution and 10 to 50 µgascorbic 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 incubatorbefore beginning experiments that include, for example, addition of drugs or cytok-ines.

SUPPORTPROTOCOL 1

DISSECTION OF BOVINE ARTICULAR CARTILAGE

The following describes a widely used method for excising articular cartilage from freshlyobtained bovine metacarpophalangeal joints. Use analogous dissection procedures forfreshly 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 kneespecimens, which should be dissected within 2 days after donation. Frozen cartilage doesnot yield viable cells and cannot be used.


12.2.5


Materials

Calf hooves (from local slaughterhouse)70% ethanol1× 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. 21150-mm petri culture dishes

1. Obtain several calf hooves that also include the first joint above the hoof of ∼2- to6-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. Wipewith ethanol swabs or paper towels moistened with 70% ethanol.

3. Using a no. 21 disposable scalpel, make a lengthwise incision and dissect away theskin, taking care not to pierce the joint capsule. Wash the skinned limb with water,then 70% ethanol.

DC

BA

Figure 12.2.1 Dissection of cartilage from the bovine metacarpophalangeal joint. (A) The skin isremoved, exposing the outer joint capsule. (B) An incision through the anterior surface of the jointcapsule is made which spares the cartilage within. (C) After the joint has been opened and exposed,a scalpel is used to excise articular cartilage by shaving along the curved surface of the exposedjoint. (D) The same joint with some of the cartilage removed, revealing the underlying bone.


12.2.6


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 limbback at the joint.

The biological safety cabinet used should preferably be one that is not also used for routinecell 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 carenot to cut into cartilage (see Figure 12.2.1B), and continue the incision around thesides until the joint is exposed on three sides. Carefully sever the internal ligamentand open the joint fully (see Figure 12.2.1C), washing away the clear, viscoussynovial fluid with sterile 1× PBS.

If blood is visible in the metacarpophalangeal joint capsule when it is opened, the jointshould 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 closelyas possible so as to obtain long, uniformly shaped shavings, ideally in one or twolong pieces per joint surface.

Long pieces are especially desirable when cartilage is to be sectioned into chips (e.g., seeAlternate Protocol 2).

If uniformity of cartilage shavings is not critical (i.e., if chips are to be enzymaticallydigested to release chondrocytes for culture), material can also be excised from othercartilage surfaces within the joint to increase the overall yield of chondrocytes.

Excessive oozing blood from the shavings indicates that the incision was deep and includedbone, 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 cellculture hood.

Shavings from each hoof should be placed in a separate dish to minimize the possibility ofcross-contamination.

8. Wash the shavings two times with DMEM/antibiotic-antifungal mixture and immersein 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 for48 hr before chondrocyte dissociation is initiated (see Basic Protocol 1). Microbialcontamination typically presents as cloudy medium (bacteria) or visible mold.

BASICPROTOCOL 2

INCORPORATION OF 35S INTO CHONDROCYTES OR CARTILAGE

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 thesulfation of GAGs, so the amount of 35S incorporated is proportional to the rate ofproteoglycan (largely aggregan) synthesis, an indirect index of cartilage formation.


12.2.7


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 chipsPBS, calcium-free and magnesium-free0.25% trypsin, without calcium or magnesiumDMEM/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 chondrocytesonly)

HPLC-grade or molecular biology-grade waterScintillation cocktail0.9% salineAlginate dissolving buffer (see recipe)DMEM/antibiotic-antifungal solution (see recipe)100% ethanol

15-ml polypropylene centrifuge tubes56°C incubator (nonhumidified)Sephadex G-25 molecular sieve columns (PD-10, Pharmacia; for monolayered and

alginate-embedded chondrocytes)96-well scintillation counting plateScintillation counter with plate readerRocking platform at 4°C1.5-ml microcentrifuge tubes

CAUTION: Radioactive materials require special handling; radioactive waste must bedisposed of appropriately.

Assay monolayered chondrocytes1a. 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 cultureconditions (see UNIT 12.1).

If conditioned medium samples are needed, for example, to analyze the release of sulfatedglycosaminoglycans or other released components of cartilage, pipet the required volumesof 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. Subsequentsteps should be performed at the bench. More prolonged incubation of samples after benchwork may increase the risk of microbial contamination.

Adjust the amount of radionuclide correspondingly if vessels containing different volumesare used.

2a. After incubation, save the medium from one sample in a scintillation vial for laterquantification of radioactivity. Discard all other overlaying medium according toestablished radiation safety procedures.

Approximately 50,000 cpm per 10 �l should be detectable in the culture medium when fresh35S 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 letstand for 5 min at room temperature to release cells from dish surfaces.

Trypsin does not interfere with the recovery of extracellular matrix components.


12.2.8


5a. Add ∼2 ml of DMEM/10% FBS/antibiotic-antifungal solution to each well, transferto 15-ml polypropylene centrifuge tubes, and centrifuge for 10 min at 250 × g (1000rpm).

Pooling of samples from wells may be needed at this point to optimize assay sensitivitybecause monolayer culture yields relatively low counts as compared with alginate orcartilage 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 thefollowing 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 sulfatedglycosaminoglycans (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-wellscintillation counting plate, and perform scintillation counting. Normalize the countsto the relative DNA content of the samples (see Support Protocol 2).

Assay alginate-embedded chondrocytes1b. Follow steps 1a to 2a above.

2b. Wash the beads two times with 5 ml of 0.9% saline per well, aspirate saline, and add1 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 minat 250 × g (1000 rpm in a low-speed centrifuge).

5b. Follow steps 6a to 8a above.

Assay cartilage chips1c. 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 ina 37°C, 5% CO2 humidified incubator.

3c. Aspirate the radioactive medium and wash two times with 1× PBS, incubating ≥15min (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% ethanoleach 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.


12.2.9


7c. Add 1 ml of 2 mg/ml of 10× pronase E solution, vortex and incubate overnight at56°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 scintillationcocktail, and 100 µl protease-digested samples, mix well and quantify radioactivityusing a scintillation counter. Normalize 35S incorporation relative to DNA content(see Support Protocol 2).

BASICPROTOCOL 3

DIMETHYLMETHYLENE BLUE STAINING TO DETERMINEPROTEOGLYCAN CONTENT1,9-Dimethylmethylene blue (DMB) binds to sulfated glycosaminoglycans, causing achange from a blue to a purple color (i.e., metachromasia). Based on this principle, anassay is described that is more rapid and economical than the 35S incorporation assay (seeBasic Protocol 2), although potentially susceptible to greater variability between wells.This assay is suitable for monolayer chondrocyte cultures, but more difficult to apply toalginate cultures because of alginate interference. The assay is unsuitable for chip culturesbecause excessive background radioactivity is obtained in the digests.

Materials

Medium and protease digests from cartilage or chondrocyte culturesPBS-BSA: 1× phosphate-buffered saline (see recipe) containing 1% (w/v) purified

bovine serum albumin1 mg/ml chondroitin sulfate (CS) from shark cartilage (Sigma) with 0.005%

sodium azide as a preservative2× 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 6aor 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 tothe corresponding well.

3. Construct a standard absorbance curve by adding CS (as a standard) successively atconcentrations from 4 µg/well to 0.125 µg/well in successive 1:1 dilutions, in a finalvolume 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 between520 and 530 nm.

If this bandwidth is not accessible, a 580 to 600 nm filter may be used, but results tend tobe 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 theirabsorbance values are outside the linear assay range.


12.2.10


7. Normalize calculated proteoglycan absorbance values to DNA content values, ifavailable (see Support Protocol 2).

SUPPORTPROTOCOL 2

MEASUREMENT OF DNA CONTENT

Values for 35S incorporation (see Basic Protocol 2) or 1,9-dimethylmethylene blue (DMB)staining (see Basic Protocol 3) in monolayered or alginate-embedded chondrocytesshould be normalized for cell number. It is virtually impossible to count cells within intactcartilage or alginate matrix, but content of DNA is generally considered to correlate withcell number and can be used as a surrogate measure.

Materials

Hoechst dye working solution (see recipe), prepare fresh dailyLysed 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 bottomFluorescence plate reader

1. To duplicate wells of the cluster plate, add 200 µl of Hoechst dye working solutionand 50 µl of the lysed or protease-digested chondrocyte samples.

The Hoechst dye intercalates with DNA and emits detectable fluorescence, allowingdetection and quantitation of tissue DNA.

2. Prepare a standard curve using six serial dilutions (0.156 to 10 µg) of 100 µg/mlbovine (calf) thymus DNA or other reference DNA in TNE buffer. Add eachconcentration 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 diluentfor the reference DNA samples.

3. Measure fluorescence using a fluorescence plate reader set at 350 to 360 nm forexcitation and 450 to 460 nm for emission.

The minimum detectable level should be ∼100 ng/ml DNA. The assay is linear up to 100�g/ml (see Fig. 12.2.2).

1400012000100008000

Fl 3

60/4

50

600040002000

00 25 50

[DNA] (µg/ml)75 100

y = 151.25x + 12.258R2 = 0.9977

y = 127.37x − 101.86R2 = 0.9996

14001600

12001000

Fl 3

60/4

50

800600

200400

00 2 4 6 8 101 3 5 7 9

[DNA] (µg/ml)

Figure 12.2.2 Representative standard curves for assay of DNA content. Known concentrationsof DNA were serially diluted in a 96-well plate in buffer (indicated on the horizontal axis) and HoechstDye was added as described. The vertical axis of the figure indicates fluorescence as measuredwith an excitation filter of 360 and emission filter of 450 nm. The concentration of DNA in the wellwas plotted relative to the fluorescence measured. The panel on the left shows DNA concentrationsup to 100 µg/ml; on the right, DNA concentrations of 0.1 to 10 µg/ml are shown.


12.2.11


4. Plot the standard curve of micrograms reference DNA versus fluorescence andperform linear regression analysis. Quantitate DNA content in experimental samplesusing this standard curve (see Fig. 12.2.2).

This assay remains linear over a wide range of DNA concentrations. If the background isset to zero and the maximum is set, for example, to 100 for 100 �g/ml, the concentrationcan be directly extrapolated from the fluorescence reading.

REAGENTS AND SOLUTIONS

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

Alginate dissolving buffer55 mM sodium citrate150 mM NaCl30 mM EDTAStore up to 6 months at room temperature

Bovine (calf) thymus DNA or other reference DNA sample, 100 �g/mlPrepare solution by dissolving 100 µg/ml of DNA in water for a full day with rockingat room temperature, then store at −20°C in aliquots for use as needed.

1,9-Dimethylmethylene blue (DMB) solutionsStock 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, add488 ml of formate buffer (see recipe). Store up to 6 months at room temperature ina dark bottle.

DMB stains intensely, indiscriminately, and irreversibly. Wear gloves and lab coats wheneverhandling 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 afume hood.

Working solution (2×): Add 100 ml of 10× DMB stock solution to 400 ml of formatebuffer (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 solutionTo Dulbecco’s modified Eagle medium (DMEM; low glucose for bovine; highglucose for other species including rabbit and human) containing antibiotic andantifungal mixture for tissue culture (e.g., penicillin-streptomycin; Fungizone, LifeTechnologies) at the appropriate dilution. Add fetal bovine serum (FBS) if neededat 5% or 10% (v/v) as indicated. Store at 4°C until the expiration date for the givenbatch of medium.

Formate buffer4 g sodium formate4 ml formic acidDeionized water to 1 literStore up to 6 months at room temperature

NOTE: Handle sodium formate and formic acid under a fume hood.


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Hoechst dye solutionsStock solution: Dissolve 10 mg Hoechst dye (bis-benzimide; Sigma) per milliliterof water and pass through a 0.2-µm filter. Protect this 10 mg/ml stock solution fromlight by wrapping in foil and store up to 1 year at 4°C.

Working solution:10 µl 10 mg/ml Hoechst dye stock solution5 ml 10× PBS (see recipe)25 ml 3 M sodium chloride5 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 KCl2 g/liter KH2PO4

1 g/liter MgCl2 6H2O80 g/liter NaCl21.6 g/liter Na2HPO4⋅7H2OStable up to 6 months at room temperature.

TNE buffer100 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)1 M NaCl10 mM EDTAStore up to 3 to 4 months at room temperature.

COMMENTARY

Background InformationChips in culture have long been used for

analysis of cartilage function, as for exampleby Morales et al. (1984). The enzymic disso-ciation of chondrocytes from cartilage for cellculture manipulations was described in detailby Kuettner et al. (1982), providing the basisfor experimental manipulations of isolatedchondrocytes. Thus, for example, foreign DNAcan be introduced into chondrocytes by trans-fection or viral infection far more readily thaninto intact cartilage, and RNA is easier to isolatefrom cultured chondrocytes. Traditionally, dis-sociated chondrocytes were cultured in mono-layer, as is conventional for other cell culturesystems. 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 collagenI instead of cartilage-specific collagen II (VanOsch et al., 1998).

Alginate culture was introduced to combinefavorable characteristics of these two culturesystems (Guo et al., 1989; Hauselmann et al.,1992). Chondrocytes are dissociated and incor-

porated into an artificial, three-dimensionalmatrix that maintains the chondrocyte pheno-type for as long as 8 months (Hauselmann etal., 1993). When needed, the matrix is readilydissolved by addition of chelating agents, free-ing chondrocytes for further experimental ma-nipulations that are difficult when the cells areembedded in a matrix. The chondrocyte pheno-type is well maintained in this culture system(Beekman et al., 1997; Liu et al., 1998). Itshould be cautioned that the Ca2+ concentrationmay not be at physiological levels and that thealginate matrix does not perfectly simulate thenatural chondrocyte microenvironment. Forexample, collagen ultrastructure and distribu-tion may differ (Gregory et al., 1999).

Proteoglycans, especially large chondroitinsulfate proteoglycan (aggregan), constitute anessential component of cartilage, and the abilityof chondrocytes to synthesize proteoglycanprovides a measure of extracellular matrixmaintenance. Basic Protocol 2 derives from thedemonstration that chondrocytes add sulfate,including 35S that may be present, to the gly-cosaminoglycans that are attached to the pro-


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teoglycan core protein. Virtually all free sulfateis used in the sulfation of glycosaminoglycans,so the amount of 35S incorporated by the beadis proportional to the rate of proteoglycan syn-thesis (Morales et al., 1984). Alternate Protocol3 relies on the ability of dimethylmethyleneblue to bind to sulfated glycosaminoglycans

and to change color from blue to purple uponbinding (Goldberg and Kolibas, 1990).

Either method of assay of proteoglycan syn-thesis (Basic Protocol 2 or 3) is amenable to theevaluation of drug or cytokine effects on pro-teoglycan synthesis. For example, interleukin-1, a chondrodestructive cytokine, prominently

Table 12.2.2 Troubleshooting Guide for Chondrocyte or Cartilage Culture and Proteoglycan Assays

Observation Possible cause Recommendation

No proliferation in monolayer Nonviable cells Use fresh tissue. Recheck mediumcomponents. Verify absence of microbialcontamination. Include serum in medium.

No cell pellet after digestion ofalginate beads

Nonviable cells Use fresh tissue. Recheck mediumcomponents. Verify absence of microbialcontamination.

Slow monolayer proliferation Cells seeded too sparsely. Sourcecartilage in poor condition at timeof dissection.

If poor health, cells may recover in culture. Ifsparse seeding, use recommended seedingdensities.

Peeling monolayers Seeded too densely; excessivematrix formation

Use recommended seeding densities; reducedensities further if needed.

Nonproliferating clumps of roundchondrocytes interspersed withmonolayered chondrocytes

Incomplete digestion bycollagenase during dissociation

Increase digestion time and/or reduce amountof cartilage digested per unit of collagenase

Microbial contamination Introduction of microbes duringtissue dissection.

Refer to UNIT 12.1. Observe indicatedprecautions during dissection. Decontaminateincubator if problem persists.

Alginate beads clump together,leading to atypical and erraticassay results

Beads do not gel (polymerize)separately when formed incalcium chloride solution

Make sure alginate droplets are well-dispersedinto medium. Allow them to gel thoroughlybefore removing calcium chloride solution.

Low control 35S counts Old 35S stock. Specific activity of 35S may be too low.

Use fresh radioisotope. Use 35S with higherspecific activity.

Nonviable cells Use fresh tissue. Check medium components.Use recommended seeding densities.

Poor inhibition of incorporationby protein synthesis inhibitors

Excessive 35S label Reduce amount of label. If using cartilagechips, allow longer label washout in saline.Recheck column purification method.

Too much free 35S remaining If working with chips, make sure sufficientwashout time is allowed

Nonviable cells Use fresh tissue. Check medium components.Use recommended seeding densities.

Purple precipitate formsprematurely during DMB assay

Aggregation of substrate due toexcessive substrate or delay inprocessing plates

Disregard affected wells. Read at lowersubstrate concentrations. Read within ≤30 minafter adding DMB.

No BSA or other blocking proteinpresent

Make sure BSA is included in assay mixture

Erratic and/or excessively highDMB assay readings

Bubbles may be present Pipet precisely without blowing out. Ifproblem occurs nonetheless, try centrifuging;gently directing air or N2 air stream overwells; touching bubbles gently with blottingpaper.

Chondroitin sulfate standard giveslow or negligible absorbance inpresence of DMB

DMB sample may be faulty(Some commercially suppliedsamples of DMB do not work)

Mix chondroitin sulfate and DMB solution1:1. If no pink color, obtain other samples ofDMB and retest.


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reduces proteoglycan synthesis within 24 to 48hr (e.g., Taskiran et al., 1994). The ability ofdrugs to ameliorate or block altogether theinhibitory effect of IL-1 may indicate a mecha-nistic basis for this action (e.g., Hauselmann etal., 1994) and/or suggest a potential for the testdrug to exert chondroprotective activity (e.g.,Yaron et al., 1999). Conversely, transforminggrowth factor-β, which promotes extracellularmatrix formation, generally increases proteo-glycan synthesis (Morales and Roberts, 1988),although experimental conditions such as theduration of culture may modify the observedeffects (van der Kraan et al., 1992). Four toseven days of incubation are generally requiredfor this effect of transforming growth factor-βto become detectable. Drugs or other sub-stances that increase chondrocyte or cartilageproteoglycan synthesis might be considered aspossible approaches to promote the repair ofcartilage.

The protocols in this unit for chondrocyteculture are fundamental for the evaluation invitro of other chondrocyte characteristics andtheir responses to physiological manipulations,growth factors and experimental drugs. Forexample, the release of proteoglycan fragmentsinto the culture medium is often an indicationof cartilage degradation and is readily detectedby subjecting culture medium to the DMBstaining protocol (e.g., Goldberg et al., 1993).The inhibition of this release, which in manycases is thought to be mediated by metallopro-teinases, may indicate chondroprotective activ-ity. Other assays of chondrocyte function mayinclude 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 messagelevels for cartilage components.

Critical Parameters andTroubleshooting

Articular cartilage from young bovines (2 to6 months of age) yields significantly morechondrocytes relative to the time and effortexpended than does human articular cartilageor that from rabbits, rodents, or other smalllaboratory animals. Cartilage is thicker inyounger than in mature individuals and respon-siveness to experimental manipulations mayvary with both the species and the age of animalor patient. Proteoglycan synthesis may alsovary according to the amount of extracellularmatrix already present in cultured chondro-cytes, whether monolayered or in alginate.

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 cartilagechip culture (see Alternate Protocol 1) or algi-nate culture (see Alternate Protocol 2). Any ofthese in vitro culture protocols are amenable tothe evaluation of proteoglycan synthesis by the35S incorporation method or to the sampling ofsecreted products (such as nitric oxide or met-alloproteinases) from culture medium. If geneexpression is to be evaluated, RNA extractionis easier from monolayer or alginate culturesthan from cartilage, although methods haverecently been described that facilitate cartilageRNA isolation (McKenna et al., 2000). TheDMB assay, and certain other cell stains, arenot compatible with alginate.

Monolayered chondrocytes need to be usedwithin several days of plating, lest the chondro-cyte phenotype be lost. The use of cartilagechips or alginate culture allows more time andflexibility 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, especiallywhen beginning a new series of experiments.Because proteoglycan synthesis depends on denovo protein synthesis, one approach to assess-ing background levels is to treat samples witha protein synthesis inhibitor such as cyclohexi-mide. This treatment should reduce proteogly-can synthesis by 3- to 10-fold or more (see Table12.2.2). Another experimental issue is the pres-ence or absence of serum. Conventional serumsupplementation (5% to 10%), in addition tofacilitating chondrocyte proliferation andmaintenance in culture, reliably increases pro-teoglycan synthesis (Hascall et al., 1983). Thelatter effect is favorable for studying inhibitorsof proteoglycan synthesis such as IL-1, but maymask the ability of some treatments such asTGF-β to increase proteoglycan synthesis andmay block the effects of experimental drugs orother 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 culturesfor several days to a week with the experimentaltreatment in medium that is essentially serum-free. Some treatments, such as cytokines, bindnonspecifically to vessel walls in the absenceof 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-cyte cultures, cartilage chips are less dependent


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Table 12.2.3 Example of Data From 35SIncorporation Assay of Proteoglycan Synthesis byMonolayered Bovine Chondrocytesa

Treatment Conc. cpmb

None 0 78 ± 4

IL-1 10 ng/ml 24 ± 4

Cycloheximide 40 µM 19 ± 2an = 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 35SIncorporation Assay of Proteoglycan Synthesis by Alginate-Embedded BovineChondrocytes

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 ± 124aMedium 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 andCycloheximide Effects in the 35S Incorporation Assayof 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 = 8bAll values expressed as mean ± S.E.


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on serum supplementation for maintenance ofproteoglycan synthesis, probably because ofgrowth factors and accessory proteins bound toor associated with cartilage.

Anticipated ResultsFrom a well in a 6-well dish containing

bovine chondrocytes in monolayer with 10%FBS medium, ∼100 to 200 cpm of 35S can beexpected; from a comparable well containing20 to 25 alginate beads ∼1000 to 2500 cpm of35S; from cartilage chips, several thousand cpmof 35S. Quantitative values of DMB absorbancevary with the apparatus used. Treatment of cellswith a protein synthesis inhibitor such as cy-cloheximide should reduce 35S incorporationor DMB staining to low or negligible values(see Tables 12.2.3, 12.2.4, 12.2.6, and 12.2.7for 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 shouldbe at least 2-fold lower, depending on experi-mental conditions and the lot of serum. IL-1should decrease both DMB-induced absor-bance changes and 35S incorporation by ≥50%when incubated in the presence of serum.

Time ConsiderationsDissection of cartilage shavings from joints

takes ∼2 hr (see Support Protocol 1). Allow 5to 6 hr (including ∼2 hr hands-on laboratorywork) to dissociate chondrocytes from carti-lage, and an additional 1 hr of laboratory workif they are to be embedded in alginate (see BasicProtocol 1 and Alternate Protocol 1). Cartilagechips require 1 hr for culture setup (see Alter-nate Protocol 2). Assays of 35S from monolay-ered or alginate-embedded chondrocytes entaila 5-hr incubation followed by 2 hr hands-ontime in the early afternoon, an overnight incu-bation and an additional ∼3 hr to separate freefrom bound 35S (see Basic Protocol 2). Assaysof 35S incorporation by chips require approxi-

mately the same amount of time on the first daybut only ∼1 hr on the second day (see BasicProtocol 2).

Assaying DNA content of protease-digestedsamples (see Support Protocol 2) requires ∼1hr; DMB assays (see Basic Protocol 3) take ∼1hr in addition to buffer preparation and timerequired to set up the spectrophotometer.

Literature CitedBeekman, 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, inalginate 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 ofchondrocytes 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 ofproteoglycans by bovine articular cartilage inculture. 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 39thAnnual Meeting, San Francisco, p. 624.

Hauselmann, H.J., Oppliger, L., Michel, B.A., Ste-fanovic-Racic, M., and Evans, C. H. 1994. Nitricoxide and proteoglycan biosynthesis by humanarticular chondrocytes in alginate culture. FEBSLett. 352:361-364.

Liu, H., Lee, Y.-W., and Dean, M.F. 1998. Re-ex-pression of differentiated proteoglycan pheno-type by dedifferentiated human chondrocytes

Table 12.2.7 Example of Data From DMB Assay ofProteoglycan Synthesis by Monolayered Bovine Chondrocytes

Treatmenta S-GAG (µg/ml)b

Expt. 1 No IL-1 14.6 ± 0.6

IL-1 5.9 ± 0.1

Expt. 2 No cycloheximide 9.7 ± 0.5

Cycloheximide 0.02 ± 0.02an = 4 per group. 10% FBS in medium. bAbsorbance values expressed as mean ± S.E.


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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 humanadult articular cartilage. Anal. Biochem. 286:80-85.

Morales, T.I. and Roberts, A. B. 1988. Transforminggrowth factor beta regulates the metabolism ofproteoglycans 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 byinterleukin-1. Biochem. Biophys. Res. Commun.200:142-148.

van der Kraan, P., Vitters, E., and van den Berg, W.1992. Differential effect of transforming growthfactor beta on freshly isolated and cultured chon-drocytes. J. Rheumatol. 19:140-145.

von der Mark, K. 1986. Differentiation, modulationand 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 oftransforming growth factor-beta on proteogly-can synthesis by chondrocytes in relation to dif-ferentiation stage and the presence of pericellularmatrix. 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 cartilagecultures. Osteoarthritis Cartilage 7:272-280.

Key ReferencesGoldberg, R.L. and Kolibas, L.M. 1990. An im-

proved method for determining proteoglycanssynthesized by chondrocytes in culture. ConnectTissue Res. 24:265-275.

This paper gives more details of the DMB assaymethod that is presented.

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 articularchondrocytes cultured in alginate beads. Matrix12:116-129.

Basic characteristics of the alginate culture systemare 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 cartilagematrix by mammalian chondrocytes in vitro. Iso-lation, culture characteristics and morphology. J.Cell Biol. 93:743-750.

Chondrocyte cell dissociation from cartilage andsubsequent culturing is described in detail.

Morales, T.I., Wahl, L.M., and Hascall, V.C. 1984.The effect of bacterial lipopolysaccharides onthe biosynthesis and release of proteoglycansfrom calf articular cartilage cultures. J. Biol.Chem. 259:6720-6729.

Cartilage chip cultures and the [35S] assay aredescribed.

Internet Resourceshttp://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ah.htm

Office of Health and Safety, Center for DiseaseControl, BMBL APPENDIX H. 1999. Working withHuman and Other Primate Cells.

Contributed by Jeffrey Liebman and Ronald L. GoldbergNovartis Institute of Biomedical ResearchSummit, New Jersey


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chondrocyte culture and assay-manual

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