Microtiter micromass cultures of limb-bud mesenchymal cells |
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Authors: | Douglas F Paulsen Michael Solursh |
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Institution: | (1) Department of Biology, University of Iowa, 52242 Iowa City, Iowa;(2) Department of Anatomy, Morehouse School of Medicine, 30310 Atlanta, Georgia |
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Abstract: | 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.
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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Keywords: | limb-bud mesenchyme microtiter plates chondrogenesis myogenesis serum-free medium ELISA |
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