Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro

The effect of glucocorticoids on sulfated proteoglycan synthesis by rabbit costal chondrocyte cultures exposed to serum-free conditions has been examined. Low density cultures of rabbit costal chondrocytes were maintained on dishes coated with extracellular matrix produced by bovine corneal endothelial cells and exposed to a 9:1 mixture (v/v) of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with transferrin, high density lipoproteins, fibroblast growth factor, and insulin (Medium A). Chondrocytes maintained in the presence of Medium A supplemented with 10(-7) M hydrocortisone reorganized, at confluence, into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix in which abundant thin collagen fibrils characteristic of type II collagen were observed. The cell ultrastructure and fibrils of the pericellular matrix were similar to those seen in vivo. In contrast, cells maintained in the presence of Medium A alone, once they reached confluence, formed a fibroblastic multilayer and produced thick collagen bundles. The level of 35SO4(2-) incorporated into large cartilage-specific proteoglycans in glucocorticoid-supplemented cultures was 33-fold higher than that of glucocorticoid-free cultures. The level of 35SO4(2-) incorporated into small ubiquitous proteoglycans was only 4-fold higher than that of glucocorticoid-free cultures. On the other hand, the level of [3H]glucosamine incorporated into hyaluronate in glucocorticoid-supplemented cultures was 4.5-fold lower than that of glucocorticoid-free cultures. Within 24 h of their addition to confluent cultures, hydrocortisone or dexamethasone markedly stimulated proteoglycan synthesis. This effect was not mimicked by androgens, estrogens, progesterone, or an inactive form of glucocorticoids such as deoxycorticosterone. This suggests that glucocorticoids have a direct and specific stimulatory effect on cartilage-specific proteoglycan synthesis and are essential for the maintenance of this synthesis in low density chondrocyte cultures.     

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

Growth of kidney epithelial cells in hormone-supplemented,serum-free medium

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors – insulin, transferrin, prostaglandin E₁, hydrocortisone and triiodothyronine – as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E₁ in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over-growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.     

Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco’s modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium

The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.     

The Persistence in the Synthesis of Type H Proteoglycan and Type II Collagen by Chondrocytes Cultured in the Presence of Chick Embryo Extract1

The effect of chick embryo extract on the phenotypic expression of differentiated chondrocytes has been studied in consideration of the fact that these cells are well characterized by certain specific cell products, such as type H proteochondroitin sulfate and type II collagen. In this study, we utilized floating chondrocytes derived from chick embryonic sterna, which can be cultured in suspension with no apparent change in the type of cell products for at least a period of eight weeks, as described in a previous paper (1). In the presence of chick embryo extract in the medium, the floating chondrocytes became attached to the bottom of the culture dish, and the attached cells took on a fibroblast-like appearance. Biochemical analyses of the proteochondroitin sulfate and collagen synthesized by the attached cells revealed that if the culture medium was renewed everyday, the cells having a fibroblast-like appearance continued to synthesize type H proteochondroitin sulfate and type II collagen. When however, the medium was replaced every other day, the synthesis of both proteochondroitin sulfate and collagen by the attached cells switched from the chondrocyte type to the fibroblast type, i.e. the synthesis of type M proteochondroitin sulfate and type I collagen, with little change in the fibroblast-like appearance. The results show that the morphological features of chondrocytes are not necessarily associated with the biochemical properties of these cells, and further suggest that, in chick embryo extract, there is no modulator capable of acting directly on the chondrocytes to bring about phenotypic changes with respect to the synthesis of collagen and proteoglycans.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Replacement of serum by insulin, transferrin, albumin, phosphatidyl choline, cholesterol, and some trace elements in cultures of mouse myeloid leukemia cells sensitive to inducers of differentiation

In serum-supplemented culture conditions, mouse myeloid leukemia cells (Ml) could be induced by various inducers including dexamethasone to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzymes, and change into forms that were morphologically similar to macrophages and granulocytes. Ml cells grew in the absence of serum in synthetic medium supplemented with insulin, transferrin, and several trace elements. The cells were maintained for more than 6 months in this medium. Induction of differentiation by dexamethasone was obtained with the cells that had been grown in serum-free medium for several days. However, the ability to be induced to differentiate in this medium was then lost with further growth of the cells in this medium. These cells could be induced to differentiate by dexamethasone with the aid of albumin, phosphatidyl choline, and cholesterol, but not by the inducer alone. The cells were leukemogenic in syngeneic mice. Macrophage-like cells that developed spontaneously from Ml cells were continuously maintained in this environment without morphological alteration or loss of their differentiated properties such as formation of Fc receptors, phagocytosis, and lysozyme synthesis.     

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.     

Synergistic Action of Transforming Growth Factor-β and Insulin-like Growth Factor-I Induces Expression of Type II Collagen and Aggrecan Genes in Adult Human Articular Chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor β (TGF-β) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-β1 or TGF-β2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-β and IGF-I.     

Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60

The human promyelocytic leukemia cell line HL-60 can be grown in serum-free synthetic medium supplemented with insulin and transferrin alone. Growth of HL-60 in this defined medium is at a rate approx. 80% of that in medium containing serum. Moreover, the distinct morphological and histochemical myeloid characteristics of HL-60 are maintained in such serum-free medium. The HL-60 promyelocytes are induced by DMSO to differentiate to mature granulocytes equally well in both serum-supplemented and serum-free medium. However, this defined medium does not support colony growth of HL-60 in semi-solid medium such as methylcellulose.     

Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Summary Chronic treatment (more than 3 d) of GH₃ cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH₃ cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH₃ cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH₃ in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.     

In vitro insulin-like growth factor I interaction with cartilage cells derived from postnatal animals

This paper reports data on the in vitro effects of insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) on the phenotypic expression of epiphyseal chondrocytes grown in serum-free (SF) culture medium. bFGF mostly stimulates chondrocyte DNA and inhibits sulfated proteoglycan synthesis and type II collagen mRNA. On the contrary, IGF-I is poorly mitogenic but strongly stimulates protein synthesis and type II collagen mRNA. In addition, IGF-I prevents the expression of type I collagen gene. Lastly, chondrocytes cultured in SF medium are able to locally produce IGF-I peptides. In conclusion, IGF-I and bFGF have opposite effects on the phenotypic expression of chondrocytes in vitro: bFGF is mostly mitogenic and IGF-I appears to be a differentiating factor.