Summary
A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (μTμM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This μTμM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d μTμM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the μTμM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation.
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Ahrens, P. B.; Solursh, M.; Reiter, R. S. Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60:69–82; 1977.
Bader, D.; Masaki, T.; Fischman, D. A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J. Cell Biol. 95:763–770; 1982.
Barnes, D.; Sato, G. Methods for the growth of cultured cells in serum-free medium. Anal. Biochem. 102:255–270; 1980.
Barnes, D.; Sato, G. Serum-free cell culture: A unifying approach. Cell 22:649–655; 1980.
Brunk, C. F.; Jones, K. C.; James, T. W. Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem. 92:497–500; 1979.
Caplan, A. I. Effects of the nicotinamide-sensitive teratogen 3-acetylpyridine on chick limb cells in culture. Exp. Cell Res. 62:341–355; 1970.
Chacko, S.; Holtzer, S.; Holtzer, H. Suppression of chondrogeneic expression in mixtures of normal chondrocytes and BUDR-altered chondrocytes grown in vitro. Biochem. Biophys. Res. Commun. 34:183–189; 1969.
Goetinck, P. F.; Pennypacker, J. P.; Royal, P. P. Proteochondroitin sulfate synthesis and chondrogenic expression. Exp. Cell Res. 87:241–248; 1974.
Guntakatta, M.; Matthews, E. J.; Rundell, J. O. Development of a mouse embryo limb bud cell culture system for the estimation of chemical teratogenic potential. Teratog. Carcinog. Mutagen. 4:349–364; 1984.
Hagiwara, Y.; Saito, K.; Atsumi, S., et al. Iron supports myogenic differentiation to the same degree as does iron-bound transferrin. Dev. Biol. 120:236–244; 1987.
Hajek, A. S.; Solursh, M. The effect of ascorbic acid on growth and synthesis of matrix components by cultured chick embryo chondrocytes. J. Exp. Zool. 200:377–388; 1977.
Hamburger, V.; Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49–92; 1951.
Hassell, J. R.; Horigan, E. A. Chondrogenesis: A model developmental system for measuring the teratogenic potential of compounds. Teratog. Carcinog. Mutagen. 2:325–331; 1982.
Ii, I.; Kimura, I.; Ozawa, E. A myotrophic protein from chick embryo extract: Its purification, identity to transferrin, and indispensability for avian myogenesis. Dev. Biol. 94:366–377; 1982.
Karasawa, K.; Kimata, K.; Ito, K., et al. Morphological and biochemical differentiation of limb bud cells cultured in chemically defined medium. Dev. Biol. 70:284–305; 1979.
Kujawa, M. J.; Caplan, A. I. Hyaluronic acid bonded to cell-culture surfaces stimulates chondrogenesis in stage 24 limb mesenchyme cell cultures. Dev. Biol. 114:504–518; 1986.
Lev, R.; Spicer, S. S. Specific staining of sulfate groups with Alcian blue at low pH. J. Histochem. Cytochem. 12:309; 1964.
Lowry, O. H.; Rosebrough, N. J.; Farr, A., et al. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.
McQuillan, D. J.; Handley, C. J.; Campbell, M. A., et al. Stimulation of proteoglycan biosynthesis by serum and insulinlike growth factor-I in cultured bovine articular cartilage. Biochem. J. 240:423–430; 1986.
Nameroff, M.; Holtzer, H. Contact-mediated reversible suppression of myogenesis. Dev. Biol. 19:380–396; 1969.
Owens, E. M.; Solursh, M. In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos. Dev. Biol. 88:297–311; 1981.
Parker, C. L.; Paulsen, D. F.; Rosebrock, J. A., et al. Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells. Exp. Cell Res. 130:21–30; 1980.
Paulsen, D. F.; Parker, C. L.; Finch, R. A. Region-dependent capacity for limb chondrogenesis: Patterns of chondrogenesis in cultures from different regions of the developing chick wing. Differentiation 14:159–165; 1979.
Pennypacker, J. P. The growth of embryonic chick limb mesenchyme cells in serum-free medium. In: Kelley, R. O.; Goetinck, P. F.; MacCabe, J. A., eds. Limb development and regeneration, part B. New York: Alan R. Liss; 1983:167–174.
Sasse, J.; Horowitz, A.; Pacifici, M., et al. Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody. J. Cell Biol. 99:1856–1866; 1984.
Solursh, M. Use of tissue culture in the analysis of limb chondrogenesis. Mechanistic approaches to developmental toxicology. In: McLachlin, J. M.; Pratt, R. M.; Markert, C. L., eds. Developmental toxicology: mechanisms and risk, Banbury Report no. 26. New York: Cold Spring Harbor Laboratory; 1987.
Solursh, M.; Ahrens, P. B.; Reiter, R. S. A tissue culture analysis of the steps in limb chondrogenesis. In Vitro 14:51–61; 1978.
Swalla, B. J.; Owens, E. M.; Linsenmayer, T. F., et al. Two distinct classes of prechondrogenic cell types in the embryonic limb bud. Dev. Biol. 97:59–69; 1983.
Swalla, B. J.; Solursh, M. Independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds. Dev. Biol. 116:31–38; 1986.
Syftestad, G. T.; Triffitt, J. T.; Urist, M. R., et al. An osteoinductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes. Calcif. Tissue Int. 36:625–627; 1984.
Thal, G.; Sasse, J.; Holtzer, H., et al. Differential survival of cartilage and muscle cells in chick limb-bud cell cultures maintained in chemically defined and serum-containing media. Differentiation 31:20–28; 1986.
Umansky, R. The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev. Biol. 13:31–56; 1966.
Wedden, S. E.; Lewin-Smith, M. R.; Tickle, C. The patterns of chondrogenesis of cells from facial primordia of chick embryos in mocromass culture. Dev. Biol. 117:71–82; 1986.
Wilk, A. L.; Greenburg, J. H.; Horigan, E. A., et al. Detection of teratogenic compounds using differentiating embryonic cells in culture. In Vitro 16:269–276; 1980.
Paulsen, D. F.; Solursh, M. Retinoic acid enhances limb chondrogenesis in high-density microtiter cultures grown in defined medium. J. Cell Biol. 105:293A; 1987.
Swalla, B. J.; Solursh, M. Inhibition of limb chondrogenesis by fibronectin. Differentiation 26:42–48; 1984.
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This work was supported by grants RR08006-13 (DFP) and HD05505 and HD18577 (MS) from the National Institutes of Health, Bethesda, MD. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa 52242 (maintained by NIH grant NO1-HD62915).
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Paulsen, D.F., Solursh, M. Microtiter micromass cultures of limb-bud mesenchymal cells. In Vitro Cell Dev Biol 24, 138–147 (1988). https://doi.org/10.1007/BF02623891
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DOI: https://doi.org/10.1007/BF02623891