Clones of embryonic chick chondrocytes have been isolated and collagen biosynthesis has been followed as the clones grow and eventually lose division capacity. Analysis of collagen type at each successive subculture until the time of cellular senescence has shown that a change in synthesis occurs from the cartilage-specific Type II collagen (chain composition Ial(II)H) to a mixture of Type I collagen (chain composition [al(I) MATERIALS AND METHODS Materials. F-10 medium containing twice the usual concentrations of amino acids and pyruvate (F-10 2 X), trypsin (2.5%), bovine-serum albumin (Fraction V), fetal calf serum, Ca++_, and Mg++-free saline, and glutamine were obtained from the Grand Island Biological Co. The radioactive precursor [2-3H]glycine (6.9 Ci/mmol) was obtained from the New England Nuclear Corp. Carrier Type I and Type II chick collagens were prepared as described previously (6). Ascorbic acid, f3-aminopropionitrile fumarate, and BrdUrd were purchased from the Sigma Chemical Co.Cell Culture and Cloning Procedures. Chondrocytes isolated from the sterna of 13-day chick embryos were grown for 4-5 days without feeding in medium F-10 2 X plus 10% fetal calf serum (vol/vol) and 1% bovine-serum albumin (wt/vol) as described previously (7). To isolate clones, the cells selected as "floaters" (4) were first centrifuged from the medium and incubated for 10 min at 370 in Ca++-and Mg++-free saline containing 0.06% trypsin. After centrifugation, cells were resuspended in F-10 2 X and a finely-drawn glass micropipette was used to withdraw a single cell, which was placed within a drop of medium located at the center of a 60 mm tissue culture dish (Falcon Plastics) (4, 8). Several dishes were prepared and each dish was incubated at 370 for 4-6 hr until all cells attached. Medium (3 ml) was added to dishes and the morphology and growth characteristics of each clone were observed daily. Only those clones that grew rapidly and initially possessed the distinct polygonal morphology of chondrocytes were retained. By these criteria, successful clones were obtained from 20-30% of all single cells. Occasionally clones of fibroblasts were observed (5/208 chondrocyte clones) and these were also retained. After about 3 weeks, cells in the chondrocyte clones were enveloped with matrix and some of the cells began to float away from the central mass of chondrocytes and to form secondary colonies. At this time the cells were dissociated with 0.1% trypsin in Ca++-and Mg++-free saline for 45 min. At the first subculture cells were replated into two or more 60 mm tissue culture dishes at 50,000/ml, and at subsequent subcultures were replated into 100 mm dishes at 100,000/ml. When a high cell density was achieved after each subculture, the cells in one or more of the dishes were incubated for 24 hr with [2-3H]glycine (50-100 ,uCi/ml) in the presence of (3-aminopropionitrile (100 ug/ml) and ascorbic acid (50 ,g/ml) in order to label newly-synthesized collagen.Isolation of Collagen from Cell Cultures. The procedures used to i...

Five different collagen chains and one smaller collagenous fragment have been isolated from the collagens found in the combined cell layer and medium of rhesus monkey aortic smooth muscle cell cultures. The collagen chains which can be identified are alpha1 (III), alpha1(I), alpha2, A and B. The smaller collagenous peptide exhibits an apparent molecular weight of 45 000 and has been designated CP45 (Mayne, R., et al. (1977), Arch. Biochem. Biophys. 181, 462). Smooth muscle cells continue to synthesize the collagens from which these components are derived for at least eight passages in culture. At each passage the alpha1 (III) chain consistently represents about one-half of the total collagen which is recovered after initial fractionation by agarose gel chromatography. The results show that smooth muscle cells derived from rhesus monkey thoracic aorta are phenotypically stable for many generations in vitro.

Chick embryo chondrocytes, in primary culture, initially synthesize only Type II collagen (chain composition, [al(II)J3), as judged by two criteria: (i) carboxymethylcellulose chromatography of the denatured collagen, and (ii) carboxymethyl-cellulose chromatography of the cyanogen bromide peptides derived from the isolated chains. After a period of growth in 5-bromo-2'-deoxyuridine, however, synthesis of two different types of collagen could be detected after differential salt precipitation of the newly synthesized native collagens from neutral salt solutions at 2.2 M NaCl and subsequently at 0.01 M Na2HPO4. By the criteria indicated above, the collagen precipitating at 2.2 M NaCI was identified as Type I collagen (chain composition, [al(I)Jsa2), whereas the collagen subsequently precipitated at 0.01 M Na2HPO4 was found to be comprised entirely of al(I) chains, indicating a chain composition, [al(I)13. We propose to designate the latter type of molecule as the Type I trimer.By careful selection of a culture medium, embryonic chick chondrocytes will maintain the cartilage phenotype in vitro for as many as 35 doublings (1). If, however, the cells are grown in media containing embryo extract (2, 3) or 5-bromo-2-deoxyuridine (BrdUrd) (4-7), or if a clone of chondrocytes is allowed to grow until there is loss of division capacity (8), the cells will flatten and lose their ability to accumulate metachromatic matrix. Such cells have been variously described as dedifferentiated, "altered," or fibroblast-like (8-10). When it was realized that the large amount of unsulfated glycosaminoglycan synthesized by chondrocyte cultures grown in embryo extract could be identified as hyaluronic acid (11, 12), it was proposed that any instability on the part of the chondrocyte phenotype might also be evident in the synthesis of collagen chains other than the al(II). chain. This collagen chain is a normal and specific gene product synthesized exclusively by cartilaginous structures (13,14). Previous chromatographic analyses of the collagen chains synthesized by control chondrocyte cultures and cultures grown in the presence of either embryo extract or BrdUrd strongly suggested that the cells normally elaborate al(II) chains (Type II collagen), but that in the presence of embryo extract or BrdUrd al(I) and a2 chains (Type I collagen) were synthesized as well (3,9).The present communication examines in greater detail both the collagen chains and the native collagens synthesized by chondrocytes grown in the presence of BrdUrd. The data indicate that there is a definite switch in collagen biosynthesis involving the cessation of Type II collagen synthesis, the initiation of Type I collagen synthesis, and the assembly of molecules comprised of only a1(I) chains.MATERIALS AND METHODS Materials. F-10 medium containing 2X amino acids and 2X pyruvate (F-10 2X), trypsin (2.5%), bovine serum albuAbbreviations: CM-cellulose, carboxymethyl-cellulose; CNBr, cyanogen bromide; BrdUrd, 5-bromo-2'-deoxyuridine. All cultures were fed daily for a...

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