culture of chondrocytes in alginate beads

Culture of Chondrocytes in Alginate 15

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From: Methods in Molecular Medicine, Vol. 100: Cartilage and Osteoarthritis, Vol. 1: Cellular and Molecular ToolsEdited by: M. Sabatini, P. Pastoureau, and F. De Ceuninck © Humana Press Inc., Totowa, NJ

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Culture of Chondrocytes in Alginate Beads

Frédéric De Ceuninck, Christophe Lesur,Philippe Pastoureau, Audrey Caliez, and Massimo Sabatini

SummaryA classic method for the encapsulation and culture of chondrocytes in alginate beads is

described. Chondrocytes are released from cartilage matrix by collagenase/dispase digestionand mixed with a solution of 1.25% alginic acid until a homogenous suspension is obtained.The suspension is drawn into a syringe and pushed gently through a needle, so that drops fallinto a solution of calcium chloride. Beads form instantaneously and further polymerize after5 min in the calcium chloride solution. Chondrocytes from any species, including humanosteoarthritic chondrocytes, can be cultured with this technique. Under these conditions,chondrocytes maintain a high degree of differentiation. Beads can be dissolved by chelationof calcium with EDTA. In this way, chondrocytes can be recovered and further separated fromthe matrix by centrifugation. Almost all molecular and biochemical techniques, as well as anumber of biological assays, are compatible with the culture of chondrocytes in alginate.

Key Words: Chondrocyte; cartilage; alginate; differentiation; cell culture.

1. IntroductionAfter being released from their cartilaginous matrix by enzyme digestion,

chondrocytes have a tendency to dedifferentiate, especially if they are cul-tured at a low density in monolayer culture. The round cells rapidly lose theircartilage phenotype and transform into flattened fibroblast-like cells. Underthese conditions, certain specific markers of the chondrocytic phenotype aredownregulated, and some nonchondrocytic proteins such as type I collagenare synthesized (1). To prevent dedifferentation, culture at a high density(more than 5 × 104 cells/cm2) is recommended and passage culture must beavoided. These are important constraints, especially when working on humanosteoarthritic chondrocytes, which are often obtained in low amounts from asingle cartilage specimen.

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To maintain chondrocytes in their original phenotype, various methods havebeen developed, including culture in scaffolding materials, such as collagensponges, agarose, or alginate. Among these techniques, culture in alginate isoften used, as it offers many advantages over the others. Chondrocytes can behomogeneously dispersed and encapsulated in alginate beads and cultured formore than 8 mo without any apparent phenotype loss (2). Alginate beads allowthe reconstitution of a tridimensional scaffold closely resembling that of the car-tilage matrix (3). Chondrocytes express proteoglycans, especially aggrecan, andalso type II, but not type I collagen (2–8). Encapsulated chondrocytes are stillable to respond to growth factors and cytokines usually known to affect chon-drocyte metabolism (9–11). Beads are easy to handle, and many molecular, bio-chemical, and biological applications are compatible with this mode of culture(2–18). Cells can be recovered from beads by simple chelation of divalent ionswith ethylenediamine tetraacetic acid (EDTA) followed by centrifugation. Cul-ture in alginate beads is also useful to redifferentiate chondrocytes that havededifferentiated because of expansion in bidimensional culture (16–18). Theoriginal method of culture of chondrocytes in alginate beads was developed inthe late eighties by Guo et al. (19) and was then employed and improved bymany researchers. We describe here the method routinely used in our laboratory.

2. Materials

1. Articular cartilage explants from guinea pigs, rabbits, or rats or from human nor-mal or osteoarthritic cartilage.

2. Hanks’ balanced salt solution (HBSS; Gibco-BRL)3. Ham’s F-12 culture medium with Glutamax (Ham F-12; Gibco-BRL).4. Fetal calf serum (FCS).5. 10,000 U/mL Penicillin /10,000 µg/mL streptomycin stock solution (PS;

Gibco-BRL).6. Dispase/collagenase solution for guinea pig, rabbit, or rat cartilage: 2 mg/mL

dispase (from Bacillus polymixa; Gibco-BRL) and 3 mg/mL collagenase type I(Worthington) in HBSS. Sterilize through a 0.22 µm filter.

7. Dispase/collagenase solution for human cartilage: 2 mg/mL dispase (from B. poly-mixa; Gibco-BRL) and 3 mg/mL collagenase type I (Worthington) in Ham F-12supplemented with 10% FCS. Sterilize through a 0.22 µm filter.

8. Cell culture equipment, including 10-mm-diameter Petri dishes, pipets,multipipets, and appropriate tips or combitips.

9. 50-mL Capacity beaker sterilized by autoclaving.10. Cell strainer, 40 µm nylon (Falcon, cat. no. 2340).11. Blue max 50-mL sterile tubes (Falcon, cat. no. 2070).12. 21-Gage needle (Terumo, cat. no. NN-2125R) with syringe of 2, 5, or 10 mL

(Terumo).13. Sterile 0.9% NaCl solution.


14. Alginate solution: 1.25% alginic acid (Fluka, cat. no. 71238), 20 mM HEPES,150 mM NaCl, pH 7.4. First dissolve HEPES and NaCl powders in deionizedwater. Warm up the mixture at 60°C and add the alginate powder with constantstirring until the solution is homogeneous. It may take more than 1 h to achievecomplete dissolution. Let the solution cool down to room temperature and adjustto pH 7.4 (see Note 1) . Adjust the final volume of the solution with deionizedwater. Autoclave.

15. Polymerization solution: 102 mM CaCl2, 10 mM HEPES, pH 7.4. Pass through a0.22 µm-filter.

16. Dissolution solution: 55 mM EDTA, 10 mM HEPES, pH 7.4. Pass through a0.22 µm-filter.

17. Tissue culture incubator, set at 37°C, with water-saturated, 5% CO2 air.

3. Methods

3.1. Chondrocyte Isolation

Chondrocytes from any species may be used with this technique.

1. Guinea pig, rat, or rabbit cartilage explants: finely mince articular cartilage downto fragments of around 1 mm3. Transfer the fragments (0.25–0.5 g) into a Petridish containing 20 mL of dispase/collagenase solution in HBSS. Incubate at 37°Cfor 5 h, or until the explants are digested.

2. Human cartilage: finely mince articular cartilage down to fragments of around 1mm3 . Transfer the fragments (0.25–0.5 g) into a Petri dish containing 20 mL ofdispase/collagenase solution in Ham F-12 with 10% FCS. Incubate at 37°C for16 h or until the explants are digested (see Notes 2 and 3).

3. Pellet cells by 3-min centrifugation at 900g.4. Resuspend cell pellet in Ham F-12 medium supplemented with 10% FCS and 1%

PS. Count the cells on a hemacytometer, and centrifuge the suspension for 3 minat 900g. Discard the supernatant and keep the cell pellet.

3.2. Encapsulation of Chondrocytes in Alginate Beads

The overall method is depicted in Fig. 1 and described here in detail. Allsteps should be performed under a sterile hood.

1. Resuspend the cell pellet obtained in Subheading 3.1., step 4 by adding a vol-ume of alginate solution (Fig. 1A), so as to have 2 million cells/mL of alginatesolution (see Note 4).

2. Aspirate the suspension into a syringe and cap with a 21-gage needle.3. Gently push the piston of the syringe so that the solution is released dropwise

into 30 mL of polymerization solution, maintained under gentle stirring by amagnet, in a 50 mL sterile beaker (Fig. 1B).

4. Beads instantly polymerize when falling into the solution, entrapping thechondrocytes. Let beads polymerize completely under gentle agitation for an ad-ditional 10 min.


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5. Pour the solution containing the beads on the cell strainer laid on top of a 50-mLFalcon tube. Discard the filtered polymerization solution and carefully pick upthe beads with a spatula.

6. Transfer the beads into 30 mL of a sterile 0.9% NaCl solution (see Note 5) in a50-mL capacity beaker and wash beads by gentle stirring for 1–2 min.

7. Repeat step 6 three times (Fig. 1C) and finally rinse beads with complete culturemedium.

8. The gross appearance of alginate beads with entrapped rabbit chondrocytes cul-tured in FCS-containing medium is shown in Fig. 2. The granular aspect andincrease of density of beads at d 26 postencapsulation accounts for the prolifera-tion of chondrocytes and the synthesis of matrix components. By contrast, humanosteoarthritic chondrocytes do not proliferate in alginate beads (Fig. 3). However,they are still capable of synthetic activities.

Fig 1. (opposite page) Schematic representation of the encapsulation of chondrocytesin alginate beads. (A) Isolated cells are suspended at 2 million/mL of 1.25% alginicacid solution. (B) The mixture is aspirated in a syringe, and then released dropwise intoa calcium chloride solution by passage through a 21-gage needle. Beads form instantlyand completely polymerize by 10 min in this solution. (C) Beads undergo three washesin a 0.9% sterile NaCl solution and are then ready for culture.

Fig 2. (A) Morphological appearance of alginate beads with encapsulated rabbitchondrocytes. Beads were cultured in medium containing 10% FCS. The increase ofcell density was evident at d 12. Cell density was further increased at d 26. (B) Macro-scopic view of beads as a function of time. The volume of beads increased by about40% at d 26.


Fig. 3. (A) Morphological appearance of an alginate bead with encapsulated humanosteoarthritic chondrocytes 12 wk after encapsulation. Unlike the rabbit chondrocytesin Fig. 2, human osteoarthritic chondrocytes do not proliferate in alginate beads cul-tured in serum-containing medium. (B) Magnification (×5) of the bead shown in A.

9. Culture beads as long as required in Petri dishes in Ham F-12 medium containingGlutamax plus 10% FCS and 1% PS. Replace medium every 2 d. In our hands,chondrocytes need about 3 wk to reestablish a consistent pericellular and territo-rial extracellular matrix.

3.3. Experimental Procedures: Recommendations

1. Perform experiments in serum-free medium if required, after a prior 24-h wash-ing-out period to ensure that all interfering serum constituents have been removedfrom the beads.

2. For certain experimental procedures, it may be necessary to separate cells fromthe alginate matrix: for this, incubate beads in dissolution solution (at a ratio ofabout 200 µL/bead) for 5 min. Centrifuge. Recover cells in the pellet and thematrix in the supernatant.

3. Depending on the experiments, the results can be normalized relative to the weightof beads, the number of chondrocytes, or the DNA content within each bead.

4. Notes1. Care must be taken not to exceed the pH 7.4 limit since any attempt to bring back

pH to 7.4 with HCl may lead to precipitation.2. Digestion of human cartilage is slower than that of cartilage from rat, rabbit, or

guinea pig, even when the same digestion volume/cartilage weight is used. Toensure chondrocyte viability, the digestion is performed in culture mediumsupplemented with FCS, even if the presence of endogenous collagenase inhibi-tors in FCS may somewhat slow down the digestion. For reproducible digestionconditions, it is recommended to maintain the same enzyme/tissue ratio and todistribute the explants, if necessary, across several Petri dishes.


3. For a similar weight of explants before digestion, the number of cells obtainedfrom human cartilage will not be more than half that obtained from cartilage ofrat, rabbit, or guinea pig and will be highly dependent on the age of the donor andstage of osteoarthritis.

4. Expect about 200–300 drops, i.e., 200–300 beads starting from a 5-mL homog-enous suspension. This means that each bead will contain about 33–50 × 103

cells.5. Using other solutions (such as phosphate-buffered saline) to wash the beads may

lead to precipitation.

References

1. Benya, P. D. and Shaffer, J. D. (1982) Dedifferentiated chondrocytes reexpress thedifferentiated collagen phenotype when cultured in agarose gels. Cell 30, 215–224.

2. Hauselmann, H. J., Fernandes, R. J., Mok, S. S., et al. (1994) Phenotypic stabilityof bovine articular chondrocytes after long-term culture in alginate beads. J. CellSci. 107, 17–27.

3. Hauselmann, H. J., Masuda, K., Hunziker, E. B., et al. (1996) Adult humanchondrocytes cultured in alginate form a matrix similar to native human articularcartilage. Am. J. Physiol. 271, C742–C752.

4. Tamponnet, C., Ramdi, H., Guyot, J. B., and Lievremont, M. (1992) Rabbitarticular chondrocytes in alginate gel: characterization of immobilized prepara-tions and potential applications. Appl. Microbiol. Biotechnol. 37, 311–315.

5. Hauselmann, H. J., Aydelotte, M. B., Schumacher, B. L., Kuettner, K. E.,Gitelis, S. H., and Thonar, E., J. (1992) Synthesis and turnover of proteoglycansby human and bovine adult articular chondrocytes cultured in alginate beads.Matrix 12, 116–129.

6. Mok, S. S., Masuda, K., Hauselmann, H. J., Aydelotte, M. B., and Thonar, E. J.(1994) Aggrecan synthesized by mature bovine chondrocytes suspended in algi-nate; identification of two distinct metabolic matrix pools. J. Biol. Chem. 269,33021–33027.

7. Petit, B., Masuda, K., D’Souza, A. L., et al. (1996) Characterization of crosslinkedcollagens synthesized by mature articular chondrocytes cultured in alginate beads:comparison of two distinct matrix compartments. Exp. Cell Res. 225, 151–161.

8. Beekman, B., Verzijl, N., Bank, R. A., von der Mark, K., and TeKoppele, J. M.(1997) Synthesis of collagen by bovine chondrocytes cultured in alginate; posttrans-lational modifications and cell-matrix interaction. Exp. Cell Res. 237, 135–141.

9. Redini, F., Min, W., Demoor-Fossard, M., Boittin, M., and Pujol, J. P. (1997)Differential expression of membrane-anchored proteoglycans in rabbit articularchondrocytes cultured in monolayers and in alginate beads. Effect of transform-ing growth factor-beta 1. Biochim. Biophys. Acta 1355, 20–32.

10. Beekman, B., Verzijl, N., de Roos, J. A., and TeKoppele, J. M. (1998) Matrixdegradation by chondrocytes cultured in alginate: IL-1β induces proteoglycandegradation and proMMP synthesis but does not result in collagen degradation.Osteoarthritis Cartilage 6, 330–340.


11. Loeser, R. F., Todd, M. D., and Seely, B. L. (2003) Prolonged treatment of humanosteoarthritic chondrocytes with insulin-like growth factor-I stimulatesproteoglycan synthesis but not proteoglycan matrix accumulation in alginate cul-tures. J. Rheumatol. 30, 1565–1570.

12. Chubinskaya, S., Huch, K., Schulze, M., Otten, L., Aydelotte, M. B., and Cole, A.A. (2001) Gene expression by human articular chondrocytes cultured in alginatebeads. J. Histochem. Cytochem. 49, 1211–1219.

13. Stove, J., Fiedler, J., Huch, K., Gunther, K. P., Puhl, W., and Brenner, R. (2002)Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reportersystem in alginate beads. Osteoarthritis Cartilage 10, 212–217.

14. Knight, M. M., van de Breevaart Bravenboer, J., Lee, D. A., van Osch, G. J.,Weinans, H., and Bader, D. L. (2002) Cell and nucleus deformation in compressedchondrocyte-alginate constructs: temporal changes and calculation of cell modu-lus. Biochim. Biophys. Acta 1570, 1–8.

15. Enobakhare, B. O., Bader, D. L., and Lee, D. A. (1996) Quantification of sulfatedglycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethyl-methylene blue. Anal. Biochem. 243, 189–191.

16. Bonaventure, J., Kadhom, N., Cohen-Solal, L., et al. (1994) Reexpression of car-tilage-specific genes by dedifferentiated human chondrocytes cultured in alginatebeads. Exp. Cell Res. 212, 97–104.

17. Lemare, F., Steimberg, N., Le Griel, C., Demignot, S., and Adolphe, M. (1998)Dedifferentiated chondrocytes cultured in alginate beads: restoration of the dif-ferentiated phenotype and of the metabolic responses to interleukin-1β. J. Cell.Physiol. 176, 303–313.

18. Liu, H., Lee, Y. W., and Dean, M. F. (1998) Re-expression of differentiatedproteoglycan phenotype by dedifferentiated human chondrocytes during culturein alginate beads. Biochim. Biophys. Acta 1425, 505–515.

19. Guo, J. F., Jourdian, G. W., and MacCallum, D. K. (1989) Culture and growthcharacteristics of chondrocytes encapsulated in alginate beads. Connect. TissueRes. 19, 277–297.

culture of chondrocytes in alginate beads

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