Isolation and Culture of Chondrocytes From Human Adult Articular Cartilage By WILLIAM K. MANNINGAND WALTER M, BONNER, JR. in the concentration of acid mucopolysaccharides in cartilage matrix is characteristic of early osteoarthritis, It has been suggested that this might be due to the degradation of acid mucopolysaccharides by hyaluronidase activity.l Hyaluronidase activity has been found in human serum, synovial membrane, and synovial fluid, but the enzyme has not been found in extracts of articular cartilage.2 Another possibility is that in osteoarthritis there is an impairment of matrix production by the chondrocytes. This is suggested by the fact that the disease shows a familial tendency in humans and that inbreeding experiments have resulted in strains of mice predisposed to osteoarthriti~.~ We have attempted to study matrix production by isolated, viable chondrocytes in tissue culture. Such an approach will permit the study of matrix production while the influence of enzymes or other factors from different tissues is excluded. This paper describes methods for isolation and culture of viable chondrocytes from adult human articular cartilage. A DIMINUTION MATERIALS AND METHODS Articular cartilage was obtained at autopsy of 31 individuals aged 26 to 84, the time between death and autopsy not exceeding 8 hours. The knee was cleansed with alcohoI and opened across the patellar tendon. Cartilage was taken from the femoral condyles and the posterior surface of the patella by excision with a scalpel. The cartilage was cut into 2 mm. cubes and 500 mg. aliquots were suspended in 5 ml. of Cey’s From the Department of Medicine, Medical College of South Carolina, Charleston, South Carolina. Supported by U . S. P . H . S. Grant AM 9899-01. WILLIAMK. MANNING, AB: Student Research Balanced Salt Solution ( Microbiological Associates) containing 60 C units of collagenase (Cal Bio Chem), 100 units of Penicillin and 100 micrograms of Streptomycin per milliliter. The mixture was incubated at 37C. with gentle shaking for 18 hours. At the end of the incubation period, strands and clumps of undigested cartilage remained. Chondrocytes were liberated from these clumps by flushing through the nozzle of a 10 ml. syringe. The cells were filtered through a double thickness of lens paper to remove any undigested material, washed 3 times with balanced salt solution, and resuspended in a growth medium consisting of Eagle’s Minimum Essential Medium containing 20 per cent calf serum, L-glutamine, penicillin, and streptomycin. Cell counts were made with a Neubauer hemacytometer. Viability was determined with the vital stains Janus green and neutral red. Freshly liberated chondrocytes were smeared on glass slides, fixed with alcohol, and stained with toluidine blue. Monolayer cultures were prepared by placing approximately 1.5 x 106 cells in screw cap culture tubes and diluting to 4 ml. with additional growth medium. The tubes were inclined at 5 degrees in culture racks. Growth medium was changed every 3 days. Colonies of chondrocytes were localized and observed at different stages of incubation. Fixed slides of monolayer cultures were prepared by fixing and removing cells on a collodion membrane by the method of Manos.4 Slides were stained in 0.1 per cent aqueous toluidine blue, 0.1 per cent toluidine blue in 0.15 M acetate buffer at pH 3.8, hematoxylin and eosin, and Alcian blue. Organ cultures were prepared by centrifuging chondrocytes into a tight pellet. The pellet was transferred with a spatula onto a stainless steel grid in a plastic organ culture dish (Falcon Plastics). The growth medium described above was also used for organ culture and was changed every 3 days. Pellets were also transferred onto nutrient agar composed of 1.5 per cent agar in growth medium. Assistant, Medical College of South Carolina. WALTERM. BONNER,JR., M.D.: Assistant Professor of Medicine, Medical College of South Carolina. 235 ARTHRITIS AND RHEUMATISM,VOL. 10, No. 3 (June 1967) 236 MANNING AND BONNER To serve as a control in each experiment, a pellet was fixed prior to incubation in the growth medium. Pellets were harvested after 7 and 14 day intervals, fixed in 10 per cent formalin containing 0.3 per cent cetylpyridinium chloride, sectioned, stained, and examined for evidence of matrix production. Autoradiographs were made by adding 5 microcuries of S35 sodium sulfate (Abbott) to each milliliter of culture medium. After incubation for 18 hours the pellet was removed, washed with balanced salt solution, fixed, sectioned, and exposed to autoradiographic emulsion (Kodak NTB-3) for one week. The autoradiographs were counterstained with toluidine blue. Pellets were frozen, but otherwise handled similarly, to serve as controls. RESULTS Treatment of articular cartilage with collagenase, followed by gentle mechanical disruption of the cartilage remnants, consistently yielded large numbers of chondrocytes. The yield varied between 100,000 and 500,000 cells from each gram of cartilage, with osteoarthritic specimens giving higher yields. Nearly all cells appeared to be intact and normal, although a small number of naked nuclei could be found along with cells whose nuclei appeared to be pyknotic. Vital staining with neutral red and Janus green dyes indicated that at least 95 per cent of the cells were viable. Toluidine blue stains of freshly liberated chondrocytes reveal that the cells vary somewhat in size, have rounded or slightly oval nuclei, and have scant cytoplasm containing abundant fine granules (Fig. 1). Some cells or clumps of cells are surrounded by a halo of weakly-metachromatic material, probably unremoved matrix. We have noted no difference in the morphology of chondrocytes isolated from normal and abnormal cartilage, or from individuals of different ages. Bacterial contamination was noted in 2, and fungal contamination in 2 other preparations of isolated cells. Chondrocytes grown by the monolayer cell culture technique showed rapid at- Fig. 1.-Newly isolated chondrocytes. Toluidine blue stain, pH 3.8. x 1000. tachment to glass. Cytoplasmic spreading was evident within 48 hours and by 7 days all cells had become stellate in appearance (Fig. 2). Rapid proliferation of the stellate forms occurred after 48 to 72 hours. Monolayer cultures have been maintained for as long as 90 days before contamination with bacterial or fungal growth. Staining with toluidine blue showed no metachromatic material adjacent to the cells. Pellets of chondrocytes grown by organ culture methods for 7 to 14 days showed evidence of early matrix formation (Figs. 3-5). Many cells appeared to have become situated within lacunae, as are seen in intact cartilage. Individual cells or clumps of cells have been found to be surrounded by abundant metachromatic material (Fig. 6 ) . No dedifferentiation into stellate cells was observed and there was no evidence of proliferation of the cells. The autoradiographic study revealed concentration of the SS5 in and around the chondrocytes (Fig. 7). Control (frozen) pellets showed no accumulation of the isotope. ISOLATION AND CULTURE OF CHONDROCYTES 237 Fig. 2.-Dedifferentiated chondrocytes after two months in monolayer culture. Toluidine blue stain. X 100. Fig. %-Pellet of newly isolated chondrocytes. Stained with Alcian Blue, pH 2.5. X 250. Fig. 4.--Pellet of chondrocytes grown on stainless grid for Seven days. Alcian blue stain. There is more abundant Alcian bluepositive extracellular material. X 250. Fig. 5-Pellet of chondrocytes grown on agar medium for fourteen days. Toluidine blue stain, pH. 3.8 x 250. Granular material beneath the pellet is the agar matrix. The dark areas around the cells are metachromatic when seen in color. 238 MANNING AND BONNER large numbers of viable chondrocytes from adult human articular cartilage obtained at autopsy. That the chondrocytes remained viable after somatic death is not surprising, in view of the demonstration by Hagerty et al.5 that at least 98 per cent of the cells of costal cartilage are viable 6 hours after death of the individual. In another study,6 10-95 per cent of cells were found viable after storage of the costal cartilage for up to 14 days after autopsy. Digestion with trypsin has been used to extract viable cells from embryonic cartil a g e ' ~but ~ yielded only a few cells in our preliminary studies on adult cartilage specimens. The methods described in this paper are based on the studies of Smith,lo who used successive digestion with papain, Fig. 6.-Cells from pellet grown on agar for pronase and collagenase in extraction of fourteen days. Tohidine blue, pH 3.8. X 400. cells from adult mammals. We have found The accumulated extracellular material is met- that the use of collagenase alone is sufachromatic. ficient to release cells from the matrix of human articular cartilage, when followed by mild mechanical disruption of the partially digested fragments. Kawiakll has described the use of collagenase in extraction of viable cells from calf cartilage. Moscona7 has shown that isolated embryonic cells grown in a dispersed monolayer culture lose their histotypical features and histoformative tendencies while reaggregated cells retain their features and ability to reconstruct tissues. Studies by Holtzers confirm that embryonic chondrocytes behave in this manner. Embryonic chondrocytes grown in pellets form metachromatic capsules and incorporate S35sulfate into chondroitin sulfate. However, the same cells grown in monolayers lose their ability to synthesize matrix. The progeny of such cells fail to differentiate into cartilage cells when reconstituted into Fig. 7.-Autoradiograph, counterstainedwith pellets and are said to have become dedifaqueous toluidine blue. X 1000. f erentiated. The results reported herein show that DISCUSSION chondrocytes from adult human cartilage The development of an enzymatic behave like embryonic chondrocytes in method has made possible the isolation of monolayer and organ cultures. Matrix pro- 239 ISOLATION AND CULTURE OF CHONDROCYTES duction has been detected i n pellets cultured for 14 days, a n d it is anticipated that this procedure wilI permit histochemical or chemical assay of the polysaccharides pro- duced. These methods of isolation and culture should prove helpful in studying the functions of chondrocytes from normal and abnormal tissues. SUMMARY Viable chondrocytes have been isolated from human adult cartilage by digestion with collagenase followed by mild mechanical disruption. Monolayer cultures of chondrocytes showed dediff erentiation of the cells with faiIure to synthesize matrix. Aggregations of chondrocytes in organ cultures retained their capacity to produce matrix. The methods described should prove helpful in studying functions of chondrocytes from normal and abnormal cartilage. SUMMARIO IN INTERLINGUA Viabile chondrocytos esseva isolate a b cartilagine de humanos adulte per digestion con collagenase sequite de leve disruption mechanic. Culturas monostratal de chondrocytos monstrava disdifferentiation cellular con absentia de synthese matrical. Aggragatos de chondrocytos in culturas de organos reteneva lor capacitate de producer matrice. Le methodos describite promitte esser utile in studiar le functiones d e chondrocytos a b cartilagine normal e anormal. REFERENCES 1. Bollet, A. J., Handy, J. R., and Sturgill, B. C.: Chondroitin sulfate concentration and protein-polysaccharide composition of articular cartilage in osteoarthritis. J. CIin. Invest. 42:853, 1963. 2. Bollet, A. J., Bonner, W. M., Jr., and Nance, J. L.: The presence of hyaluronidase in various mammalian tissues. J. Biol. Chem. 238:3522, 1963. 3. Sokoloff, L., Crittenden, L. R., Yamamoto, R. S., and Jay, G. E., Jr.: The genetics of degenerative joint disease in mice. Arthritis Rheum. 5:531, 1962. 4. Manos, J. P.: Rapid removal of tissue culture monolayers by collodion for subsequent staining. Stain Techn. 40:75, 1965. 5. Hagerty, R. F., Calhoon, T. B., Lee, W. H., Jr., and Cuttino, J. T.: Characteristics of fresh human cartilage. Surg. Gynec. Obstet. 110:3, 1960. 6. Lee, W. H., Jr., Hagerty, R. F., and Braid, H. L.: Measurements of cellular viability; 7. 8. 9. 10. 11. A comparative study of neutral red and radioactive sulfate in the examination of the viability of cartilage. Plast. and Reconstr. Surg. 26:280, 1960. Moscona, A. : Rotation-method histogenetic aggregation of dissociated cells. Exp. Cell Res. 22:455, 1961. Holtzer, H., Abbott, J., Lash, J., and Holtzer, S.: The loss of phenotypic traits by differentiated cells in vitro, I. DedifFerentiation of cartilage cells. Proc. Nat. Acad. Sci. U.S.A. 46:1533, 1960. Thorp, F. K., and Dorfman, A,: The occurence of intracellular chondroitin sulfate. J. Cell Biol. 18:13, 1963. Smith, A. U.: Survival of frozen chondrocytes isolated from cartilage of adult mammals. Nature 205:782, 1965. Kawiak, J., Moskalewski, S., and Darzynkiewicz, Z.: Isolation of chondrocytes from calf cartilage. Exp. Cell Res., 39:59, 1965.