Calcified Tissue Research

Calc. Tiss. Res. 23, 61-66 (1977)

9 by Springer-Verlag 1977

Species Differences in Cell Culture of Mammalian Articular Chondrocytes Richard J. Webber, Charles J. Malemud, and Leon Sokoloff Department of Pathology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York, USA

Summary. Articular chondrocytes from eight mammalian species (rabbit, opossum, w o o d c h u c k , cat, dog, sheep, rhesus and cebus monkeys) were grown in monolayer culture using a single regimen. The animals were immature or young adult. H a m ' s F 1 2 medium supplemented with 10% fetal bovine serum was employed for the p r i m a r y cultures and Dulbecco-Vogt medium, for the secondary. M a r k e d species differences were found with respect to cell morphology, growth in p r i m a r y and secondary cultures, incorporation o f radiosulfate into macromolecules, adhesion to the flask surface, response to vitamin C, and ch,ondroid expression in spinner bottles. Under these particular conditio9s, r a b b i t chondrocytes grew most rapidly and incorporated several times m o r e sulfate than did the others. A d d i t i o n a l experiments carried out with other media on four of the species indicate that optimal conditions for culturing m a m m a l i a n chondrocytes must be determined for each species individually.

Key words: Cartilage, articular - - Tissue culture - Species specificity.

Introduction The bulk o f the literature concerning in vitro culture o f chondrocytes has been based on embryonic chick cells. A recent review concluded that " o n l y limited success has been achieved in the culture o f m a m m a l i a n c h o n d r o c y t e s " (Levitt and Dorfman, 1974). Evidence has been presented that articular chondrocytes grown from rabbits proliferate readily under m o n o l a y e r conditions (Sokoloff et al., 1970; Green, 1971; Corvol et Send offprint requests to Richard J. Webber, Department of Pathology, Health Sciences Center, State University of New York, Stony Brook, NY 11794, USA

al., 1972) and when they are then transferred to spinner bottles, synthesize large amounts of proteoglycan and collagen that faithfully recapitulate cartilage matrix (Srivastava et al., 1974b; N o r b y et al., 1977). The same culture methods fail when applied to h u m a n cartilage (Srivastava et al., 1974a). The following experiments were carried out to determine whether the latter phenomenon is peculiar to human cells or occurs more widely a m o n g m a m m a l i a n species. Since suitable human cartilage is not readily available, a similar problem in a kindred species might allow special cultural requirements for human cells to be identified.

Materials and Methods Chondrocytes were grown from the shoulder, hip, and knee joint cartilage of 8 mammalian species comprising examples of marsupial, lagomorph, rodent, carnivore, artiodactyl, and primate (Table 1). The species were selected to be large enough to minimize contamination of hYaline cartilage with flbrocartilaginous or other extraneous joint tissues (Hough and Sokoloff, 1975). The animals were immature or young adult and were killed variously by sedation with CO 2 and exsanguination or air embolism. The cartilages were grossly normal. Because the cellularity of articular cartilage is sparse, the samples from various joints were pooled to make up the requisite inoculum. Monolayer culture was carried out according to procedures described in detail elsewhere (Sokoloff et al., 1970; Green, 1971; Corvol et al., 1972; Srivastava et al., 1974a). The primary cells were grown at 37 ~ in Ham's F12 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (10/tg and 10 units respectively per ml). All media were purchased from Grand Island Biological Co., Grand Island, N.Y., and the serum, except in one experiment, from Associated Biomedic Systems, Buffalo, N.Y. Five to ten 75 cmz flasks (Falcon Plastics, Los Angeles, CA.) were each inoculated with 1.4 to 2.8 x 105 ceils. When the cells became confluent, they were trypsinized and approximately 104 cells per ml were inoculated into secondary culture flasks containing DulbeccoVogt medium (DMEM) supplemented as above. In addition to the standard procedure of using DMEM, two other nutrient regimens were employed in secondary cultures of 4 species (Table 1). These included substitution of F 12 for DMEM and supplementation with

62

Richard J. Webber et al.: Species Differences in Chondrocytes

Table 1. Monolayer culture findings in articular chondrocytes of eight mammalian species Species

Rabbit

(Oryctotagus cunieulus)

Animal no.

1 2

3 3

3 3

4 4

8 7

3

3

3

4

7

3 6-9 6-9

3 5 2

4 6 3

7 > 13 > 14

4-19 1 (Fells domesticus) 2

Cat

Opossum

3 1

6-9 3 Young 14 > 16

2

Young 15

3 5

> 14 >20

1

5

3

4

1

9

3

1

Several

1

Several

(Didelphis virginianus) Woodchuck

1

(Marmota monax) 2 Dog

Age 1~ Culture (days) a 2 ~ Culture (months) Attachment Spreading Confluent Medium out

Doubling 3sSO4 incorporation (dpm x 103/#g DI time (h) Medium Trypsin wash

14.6 DMEM 16.0 DMEM DMEM + ASC - 17.2 DMEM DMEM + ASC - -DMEM 14.2 c DMEM 14.2 c DMEM DMEM + ASC - -

2.6 + 0.47 (5) d 13.8 _+ 0.86 (6) 9.1 +0.87(6) 9.4 + 0.42 (6) 7.1+0.18(6) 11.2 _+ 0.90 (16) 0.6 + 0.02 (4) 1.1 _+0.10(5) 0.9 _+ 0.06 (5)

DMEM DMEM + ASC

> 108.0 ~ 3.7 i 0.89 (4) > 108.0 e - -

> 15

F 12 F12 + ASC DMEM DMEM F12 F I 2 + ASC DMEM

29.2 e 30.4 c 44.6 >72.0 c > 72.0 e 50.0 r 17.8

4

15

DMEM

22.6

1.2_+0.11(4)

3

3

13

DMEM

39.6

2.7 _+ 0.07 (4)

3

4

13

DMEM

33.4

3.2 + 0.03 (4)

--1.0 _+ 0.10 (4) 2.2 + 0.31 (4) --2.3 (2)

1.7 +_ 0.09 (6) 7.8_+0.80(6) 0.9 _+ 0.05 (6) 1.7_+0.08(6)

0.3+0.02(5) 0.3 _+ 0.02 (5)

i

( Canis familiaris) Sheep

(Ovis dalli) Rhesus monkey

(Macaea mulatta) Cebus monkey

( Cebus apella) All primary cultures were fed Ham's F 12 medium b Mean + SE (N) c Based on stained cell counts rather than D N A Different serum employed (see text): ASC, Na ascorbate Na ascorbate (40 #g per ml). The ascorbate was added to the medium immediately prior to each feeding of the cells. The cells were refed after 48 h and 2 days later were labeled for 20 h with 1.4 /.tCi Na235SOJml (600-800 mCi/mMole). The cells were harvested by trypsinization and centrifuged at 2400 rpm for 3 min. The population doubling time was measured in most experiments from the accumulation of DNA in other secondary culture flasks at intervals of 8 to 24 h beginning 1 day after inoculation. For this purpose, a total of 24 flasks was set up. At each of the stated intervals 6 flasks were harvested. These were divided into two pools of 3 flasks each. Thus each point on the growth curve from which the doubling time was computed represented the mean of the two groups of 3 flasks. In several cultures indicated in Table 1, insufficient material was available for measuring DNA chemically. The doubling time here was determined by staining and counting the cells in the flasks with an ocular reticule (Sokoloff et al., 1970). The cell mass in each flask was determined as the DNA content of the trypsinized pellet by a slight modification of Burton's method (Corvol et al., 1972). The medium was dialyzed against 0.1 M (NH~)zSO 4 for 4 h and then against running tap water overnight. An aliquot of the non-dialyzable portion was assayed for radioactivity. The behavior of cultured cells following transfer to suspension conditions (Srivastava et al., 1974a) was studied in 2 species. Twenty-five ml of medium containing approximately 7 x 104 trypsinized sheep chondrocytes per mI and 2 • 104 cat chondrocytes

per ml respectively were inoculated into 50 ml spinner bottles (Bellco, Vineland, N.J.). DMEM, modified by removing CaCI 2 and adding 0.1% Pluronic F68, was employed. The cells were refed at 48 h. The cells were centrifuged 20 h later and studied histologically for matrix production (fixed with 10% formalin, embedded in paraffin, sectioned and stained with safranin O-fast green and with aqueous toluidine blue at pH 4.2).

Results Primary each

and secondary

species.

cultures were obtained from

Attachment

of

an

unmeasured

but

relatively small proportion of freshly dissociated p r i m a r y cells o c c u r r e d in e a c h i n s t a n c e o v e r t h e c o u r s e o f 2 t o 5 d a y s ( T a b l e 1). T h e a t t a c h m e n t t o o k p l a c e m o s t r a p i d l y in t h e c a s e o f t h e o p o s s u m cells; w i t h i n 6 0 rain o f i n o c u l a t i o n , m o r e t h a n 9 0 % o f t h e cells w e r e e s t i m a t e d to a d h e r e t o t h e f l a s k s ( F i g . i). T h e r e w e r e s p e c i e s d i f f e r e n c e s in t h e r a t e a t w h i c h p r i m a r y cells spread out and became confluent. In most species, the chondrocytes were ameboid and migrated moderate d i s t a n c e s f r o m t h e cell c l u s t e r d u r i n g t h e p e r i o d o f

Richard J. Webber et al.: SpeciesDifferencesin Chondrocytes

63

Fig. 1. Attachment of freshly dissociated opossum chondrocytesto flask 60 rain after primary inoculation.(Phase contrast, 175x) growth (Fig. 2A); when confluent, these cells assumed a more polygonal appearance. The majority of the woodchuck cells remained more closely approximated and epithelioid at all times in primary culture (Fig. 2B). However, woodchuck cells did not thrive in secondary culture and became pleomorphic. A small but variable proportion of the cultured chondrocytes in all species was binucleate. Woodchuck cells in primary and monkey cells in secondary culture required two to three times longer for detachment from the flask by trypsin than did the other species. Incorporation of radiosulfate into macromolecules varied widely. This parameter of chondroid expression (Srivastava et al., 1974a) was best displayed by the rabbit cells and much less by the others (Table 1). It bore no relation to the population doubling time. In only one of 19 tests (Rabbit 1; Table 1) of radiosulfate incorporation by rabbit chondrocytes did the value fall within the range obtained by other species. The only explanation for this apparent anomaly was that this was the only experiment in which a different brand of serum was employed. Species variations were found with respect to the culture media used. Cat chondrocytes grew rapidly in DMEM but not in F12, the opposite occurred in the case of the opossum cells. Addition of Na ascorbate increased growth of lapine chondrocytes in two experiments (A = 62, 32%) and cat (A = 25%) as measured from the D N A content of the flasks of the final culture period. The differences between the means exceeded the standard error of the Controls four to nine-fold, but the small number of flasks in each instance (3) pre-

cluded more rigorous statistical evaluation. Ascorbate had no effect on total radiosulfate incorporation, but a greater proportion of the radioactivity was shifted from the culture medium to the cell-associated trypsin wash (Table I). Opossum chondrocytes did not respond to ascorbate. Histochemical examination of sheep chondrocytes in spinner culture revealed little extracellular deposit unlike the findings in rabbit cells (Srivastava et al., 1974a). The feline cells did not survive the procedure.

Discussion Under the basic culture regimen employed here and previously found to be successful in rabbits, marked species differences in the in vitro behavior of mammalian chondrocytes were found. There was no correlation between the rate of growth in primary or secondary culture, adhesion to the flask surface, cell morphology, incorporation of radiosulfate into macromolecules, chondroid expression in spinner bottles, or position in the taxonomic scale. The primate cells (Old and New-World monkeys) grew readily unlike the human chondrocytes reported previously (Srivastava et al., 1974b). Previously it was shown that DMEM results in greater sulfate incorporation by rabbit cells than does F 12 (Sokoloff et al., 1970). In the present experiments, the response of chondrocytes to different media (DMEM, F12, and supplementation with Na ascorbate) varied widely among the species studied. In rabbits, results have been quite reproducible and the

64

Richard J. Webberet al.: SpeciesDifferencesin Chondrocytes

Fig. 2. Speciesdifferencesin appearance of monolayercultured chondrocytes.(A) Rabbit cells, 5 days in primary culture, have an ameboid appearance. (B) Woodchuck cells, 7 days in primary culture, are epithelioid (Phase contrast, 175x) previously reported population doubling time (Green, 1971; Malemud and Sokoloff, 1974) was comparable to that reported in the present study. In addition to these and the previously mentioned chick embryo cells, chondrocytes have also been grown from bovine joints (Sokoloff et al., 1970), urodele (Chiakulas et al., 1966), embryonic mouse (Callerio-Babudieri and Callerio, 1973), fetal guinea pig (Lohmander et al., 1976), and neonatal rat cartilage (unpublished data). Postnatal pig chondrocytes have been maintained in primary suspension culture (Wiebkin and Muir, 1973). Rabbit

chondrocytes express their phenotype under suspension conditions (Srivastava et at., 1974a; Norby et al., 1977) but this was not true of the sheep and cat cells reported here. Although much is known about requirements for growth and phenotypic expression of individual cell types, there is little systematic information on the sources of species differences in cell culture. Aside from direct genetic specificities, these sources must include extrinsic factors such as latent infections. Several phylogenetic requirements are readily understood, e.g.,

Richard J. Webber et al.: Species Differences in Chondrocytes osmotic and temperature optima in poikilothermic animals (Clark, 1972). Genetically-governed differences between cells cultured from various species are well documented in host-range specificities for viruses (Purchase et al., 1971). Even within one species, genetic differences are commonly found with respect to viral infection (Hartley et al., 1970) and specific enzyme activities. Certain rodent cells remain diploid for m a n y more passages than do human cells; whether these species differences reflect inherent patterns of senescence or some subtle transformation of the cells has not been resolved (Hay, 1970). In the present study, the rodent (woodchuck) chondrocytes displayed no special vigor and thus were quite different from the rat chondrocytes we have grown previously. In trying to explain the failure of human cells to thrive as the other primate cells did, it is unlikely that the surgical procedures employed were at fault. Simmons and Lesker (1975) have reported that anesthesia of rats with ether for only 4 to 5 rain greatly reduces several enzymatic activities of articular cartilage. Volatile as well as non-volatile anesthetics have deleterious effects on cell growth in vitro (Ishii and Corbascio, 1971; Jackson and Epstein, 1971); these compounds presumably were eliminated during dissociation of the cells. Tourniquets applied to the limbs are known to alter the pH of joint fluid. This also was probably of no consequence because articular cartilage, being avascular, has a relatively low pH and predominantly glycolytic metabolism. There are many biological similarities among various mammalian hyaline joint cartilages and it is well known that this tissue is uniquely resistant to allograft rejection. All the cells are chondrocytes, albeit probably heterogeneous. The bulk of the matrix consists o f type II collagen and proteoglycans. The molecular constitution of both these components is similar if not identical among mammalian and often other vertebrate and even sometimes invertebrate species (Miller and Matukas, 1974; Sandson et al., 1970; Stanescu et al., 1973). Recent observations have extended immunologic cross-reactivity not only to the core but the glycoprotein link of the proteoglycan (Keiser and Sandson, 1974). There are, however, established genetic differences among chondrocytes. A species-specific d e t e r m i n a n t - "new component" - - is present in the proteoglycan of human articular cartilage (Keiser and Sandson, 1974). The failure of cartilage allografts to be rejected is related to the shielding of donor cells from the recipient tissues by the intercellular matrix. When, however, articular chondrocytes are dissociated from their matrix, transplantation antigens are unmasked (Elves, 1974). Isolated syngeneic chondrocytes from rat embryos are accepted and allogeneic chondrocytes rejected by different strains of rats (Heyner, 1969). It would be unwarranted

65 to speculate whether any of these antigenic differences are related biologically to the difficulty experienced in culturing human chondrocytes. The likelihood is that some special growth requirement will have to be arrived at by another route. Recently several other laboratories have reported greater success in growing human articular chondrocytes by employing variously inactivated human sera (rather than fetal calf serum), a supplement of a-ketoglutarate, high environmental p H (8.0) and F12 media (Schwartz et al., 1974; 1976; Lust et al., 1976; Schindler et al., 1976).

Acknowledgements. We thank Sheldon Scher, Assistant Director of the Division of Laboratory Animal Resources, for the animal specimens; and Dr. Clyde J. Dawe for critical review of the manuscript. This study was supported by Grant AM 17258-01 from the National Institutes of Health and the New York State Chapter of the Arthritis Foundation.

References Callerio-Babudieri, D., Callerio, C.: Comportamento del fisozima su cellule cartilagine di embione di topo coltivate in vitro. Boll. Ist Sieroter. Milan 52, 468-474 (1973) Chiakulas, J.J., Scheving, L.E., Tsai, T.H.: The long-term in vitro cultivation of urodele cartilage cells on a glass substratum. Anat. Rec. 154, 455 (1966) Clark, H.F.: Cultivation of cells from poikilothermic vertebrates. In: Growth, nutrition and metabolism of cells in culture (G.H. Rothblat and V.J. Cristofalo, eds.), Vol. 2, pp. 287-325. New York and London: Academic Press 1972 Corvol, M.-T., Malemud, C.J., Sokoloff, L.: A pituitary growthpromoting factor for articular chondrocytes in monolayer culture. Endocrinology 90, 262-271 (1972) Elves, M.W.: A study of the transplantation antigens on chondrocytes from articular cartilage. J. Bone Joint Surg. 56B, 178185 (1974) Green, W.T., Jr.: Behavior of articular chondrocytes in cell culture. Clin. Orthop. 75, 248-260 (1971) Hartley, J.W., Rowe, W.P., Huebner, R.J.: Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Viro[. 5, 221-225 (1970) Hay, R.J.: Cell strain senescence in vitro: Cell culture anomaly or an expression of a fundamental inability of normal cells to survive and proliferate. In: Aging in cell and tissue culture (E. Holeckov/t and V.J. Cristofalo, eds.), pp. 7-24. New York and London: Plenum Press 1970 Heyner, S.: The significance of the intercellular matrix in the survival of cartilage allografts. Transplantation 8, 666-677 (1969) Hough, A.J., Sokoloff, L.: Tissue sampling as a potential source of error in experimental studies of cartilage. Conn. Tiss. Res. 3, 27-31 (1975) Ishii, D., Corbascio, A.N.: Some metabolic effects of halothane on mammalian tissue culture cells in vitro. Anesthesiology 34, 427-438 (1971) Jackson, S.H., Epstein, R.A.: The metabolic effects of nonvolatile anesthetics on mammalian hepatoma cells in vitro. I. Inhibition of cell replication. Anesthesiology 34, 409-414 (1971) Keiser, H., Sandson, J.I.: Immunodiffusion and gel-electrophoretic studies of human articular cartilage proteoglycan. Arthr. Rheum. 17, 219-228 (1974)

66 Levitt, D., Dorfman, A.: Concepts and mechanisms of cartilage differentiation. Curr. Topics Devel. Biol. 8, 103-149 (1974) Lohmander, S., Moskalewski, S., Madsden, K., Thyberg, J., Friberg, U.: Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chow drocytes. Exp. Cell Res. 99, 333-345 (1976) Lust, G., Nuki, G., Seegmiller, J.E.: Inorganic pyrophosphate and proteoglycan metabolism in cultured human articular chondrocytes and fibroblasts. Arthr. Rheum. 19, 479-487 (1976) Malemud, Cal., Sokoloff, L.: Some biological characteristics of a pituitary growth factor (CGF) for cultured lapine articular chondrocytes. J. Cell. Physiol. 84, 171-179 (1974) Miller, E.J., Matukas, V.J.: Biosynthesis of collagen: The biochemist's view. Fed. Proc. 33, 1197-1204 (1974) Norby, D.P., Malemud, C.J., Sokoloff, L.: Differences in the collagen types synthesized by lapine articular chondrocytes in spinner and monolayer culture. Arthr. Rheum. 20, 709 (1977) Purchase, H.G., Burmester, B.R., C unningham, C.H.: Responses of cell cultures from various avian species to Marek's disease virus and herpesvirus of turkeys. Amer. J. Vet. Res. 32, 1811-1823 (1971) Sandson, J., Damon, H., Mathews, M.B.: Molecular localization of a cross-reactive antigenic determinant of cartilage proteoglycan. In: Chemistry and molecular biology of the intercellular matrix (E.A. Balazs, ed.), Vol. 3, pp. 1563-1567. New York and London: Academic Press 1970 Schindler, F.H., Ose, M.A., Solursh, M.: The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell cultare. In Vitro 12, 44-47 (1976)

Richard J. Webber et al.: Species Differences in Chondrocytes Schwartz, E.R., Kirkpatrick, P.R., Thompson, R.C~: Sulfate metabolism in human chondrocyte cultures. J. Clin. Invest. 54, 1056-1063 (1974) Schwartz, E.R., Kirkpatrick, P.R., Thompson, R.C.: The effect of environmental pH on glycosaminoglycan metabolism by normal human chondrocytes. J. Lab. Clin. Med. 87, 198-205 (1976) Simmons, D.J., Lesker, P.A.: Effects of short term ether and pentothal anesthesia on bone and cartilage metabolism. Cand. J. Physiol. Pharm. 53, 33-37 (1975) Sokoloff, L., Malemud, C.J., Green, W.T., Jr.: Sulfate incorporation by articular chondrocytes in monolayer culture. Arthr. Rheum. 13, 118-124 (1970) Srivastava, V.M.L., Malemud, C.J., Hough, A.J., Bland, J.H., Sokoloff, L.: Preliminary experience with cell culture of human articular chondrocytes. Arthr. Rheum. 17, 165-169 (1974a) Srivastava, V.M.L., Malemud, C.J., Sokoloff, L.: Chondroid expression by lapine articular chondrocytes in spinner culture following monolayer growth. Conn. Tiss. Res. 2, 127-136 (1974b) Stanescu, V., Maroteaux, P., Sobczak, E.: Gel electrophoresis of the proteoglycans of the growth and of the articular cartilage from various species. Biomedicine 19, 460-463 (1973) Wiebkin, O.E., Muir, H.: The inhibition of sulphate incorporation in isolated adult chondrocytes by hyaluronic acid. FEBS Lett. 37, 42-46 (1973)

Received July 30 / Accepted November 9, 1976