Micromass Cultures in Teratology

Rafael A. Ponce1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 13.3
DOI:  10.1002/0471140856.tx1303s07
Online Posting Date:  May, 2001
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This unit describes methods for culture of undifferentiated midbrain (mesencephalon) and limb bud cells from gestation day 12 rat embryos. When grown over 5 days in vitro, these mixed cell populations express many morphological, biochemical, molecular, and immunophenotypic characteristics observed during in vivo differentiation. These cultures can be used in a wide variety of studies designed to investigate normal cellular ontogeny, the teratogenic potential of test agents, or the mechanisms underlying the cellular response to environmental stress.

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Table of Contents

  • Basic Protocol 1: Preparing Neuroepithelial and Limb Bud Cell Micromass Culture
  • Alternate Protocol 1: Preparing Cells from Embryos of Pretreated Dams
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparing Neuroepithelial and Limb Bud Cell Micromass Culture

  • 70% ethanol
  • Earle's balanced salt solution (EBSS; Life Technologies) with and without calcium and magnesium (CMF‐EBSS), sterile, 37°C
  • 4 to 8 time‐mated, gestation day 12 to 12.5 rats (where gestation day 12 represents the 12th day after confirming a positive pregnancy)
  • 50% (v/v) heat‐inactivated horse serum (see recipe), 37°C
  • Trypsin solution (see recipe), 37°C
  • Sterile culture medium (see recipe), 37°C
  • Collagen solution (see recipe, optional)
  • Sterile dissecting instruments: 2 dissecting forceps and 1 pair tissue dissecting scissors (straight operating scissors)
  • Sterile 100‐mm petri dishes
  • Plastic waste bags
  • Disposable absorbent pads
  • Laminar flow hood
  • Dissecting microscope(s) with light source
  • Sterile microdissecting instruments (1 to 2 sets):
  •  2 Dumont (watchmaker's) forceps, pattern no. 5 (Fine Science Tools)
  •  Pair microdissecting Wecker scissors (Fine Science Tools)
  •  Slotted Moria spoon (Fine Science Tools)
  • Data recording sheet
  • 15‐ml sterile tubes, labeled (labeled as follows: CNS1, CNS2, CNS3, LB1, LB2, LB3, CM, trypsin)
  • Sterile incubator trays
  • Pulled‐glass pipets (i.d. ∼0.7 mm), sterile
  • 10‐ml disposable syringes, sterile
  • 13‐mm Swinney filters (Millipore) with a 10‐µm nylon mesh membrane filter (Spectrum), sterile
  • Hemacytometer and hand‐held counter
  • Repeater pipettor (Eppendorf)
  • Sterile 0.5‐ml tips for repeater pipettor
  • 35 × 10–mm sterile primaria‐coated tissue culture dishes (Falcon)
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Literature Cited

Literature Cited
   Brown, L.P., Flint, O.P., Orton, T.C., and Gibson, G.G. 1986a. Chemical teratogenesis: Testing methods and the role of metabolism. Drug Metab. Rev. 17:221‐260.
   Brown, L.P., Flint, O.P., Orton, T.C., and Gibson, G.G. 1986b. In vitro metabolism of teratogens by differentiating rat embryo cells. Food Chem. Toxicol. 24:737‐742.
   Faustman, E.M. 1988. Short‐term tests for teratogens. Mutat. Res. 205:355‐384.
   Faustman, E.M. and Sweeney, C. 1994. Effects of ethylnitrosourea on expression of proto‐oncogene pp60c‐src and high‐molecular‐weight neurofilament protein in rodent embryo central nervous system cells in vitro. Toxicol. Appl. Pharmacol. 128:182‐188.
   Flint, O.P. 1983. A micromass culture method for embryonic neural cells. J. Cell. Sci. 61:247‐262.
   Flint, O.P. and Ede, D.A. 1982. Cell interactions in the developing somite: In vitro comparisons between amputated (am/am) and normal mouse embryos. J. Embryol. Exp. Morph. 67:113‐125
   Flint, O.P. and Orton, T.C. 1984. An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol. 76:383‐395.
   Flint, O.P., Orton, T.C., Ferguson, R.A. 1984. Differentiation of rat embryo cells in culture: Response following acute maternal exposure to teratogens and non‐teratogens. J. Appl. Toxicol. 4:109‐116.
   Kidney, J.K. and Faustman, E.M. 1995. Modulation of nitrosourea toxicity in rodent embryonic cells by O6‐benzylguanine, a depletor of O6‐methylguanine‐DNA methyltransferase. Toxicol. Appl. Pharmacol. 133:1‐11.
   Ou, Y.C., Thompson, S.A., Kirchner, S.C., Kavanagh, T.J., and Faustman, E.M. 1997. Induction of growth arrest and DNA damage‐inducible genes Gadd45 and Gadd153 in primary rodent embryonic cells following exposure to methylmercury. Toxicol. Appl. Pharmacol. 147:31‐38.
   Ou, T.C., White, C.C., Kresja, C.M., Ponce, R.A., Kavanagh, T.J., and Faustman, E.M. 1999a. The role of intracellular glutathione in methylmercury‐induced toxicity in embryonic neural cells. Neurotoxicology 20:793‐804.
   Ou, Y.C., Thompson, S.A., Ponce, R.A., Schroeder, J., Kavanagh, T.J., and Faustman, E.M. 1999b. Induction of the cell cycle regulatory gene p21 (Waf1, Cip1) following methylmercury exposure in vitro and in vivo. Toxicol. Appl. Pharmacol. 157:203‐212.
   Ponce, R.A., Kavanagh, T.J., Mottet, N.K., Whittaker, S.G., and Faustman, E.M. 1994. Effects of methyl mercury on the cell cycle of primary rat CNS cells in vitro. Toxicol. Appl. Pharmacol. 127:83‐90.
   Ribeiro, P.L. and Faustman, E.M. 1990. Chemically induced growth inhibition and cell cycle perturbations in cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 104:200‐211.
   Sawyer, L.M. and Goetinck, J.P. 1981. Chondrogenesis in the mutant nanomelia. Changes in the fine structure and proteoglycan synthesis in high density limb bud cell cultures. J. Exp. Zool. 216:121‐131.
   Seeley, M.R. and Faustman, E.M. 1995. Toxicity of four alkylating agents on in vitro rat embryo differentiation and development. Fundam. Appl. Toxicol. 26:136‐42.
   Solursh, M., Jansen, K.L., Singly, C.T., Linsenmayer, T.F., and Reiter, R.S. 1982. Two distinct regulatory steps in cartilage differentiation. Dev. Biol. 94:311‐325.
   Strum, K. and Tam, P.P.L. 1993. Isolation and culture of whole postimplantation embryos and germ layer derivatives. Meth. Enzymol. 225:164‐190.
   Sweeney, C., Kirby, Z., and Faustman, E.M. 1992. Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto‐oncogene pp60c‐src and high‐molecular‐weight neurofilament protein. Cell Biol. Toxicol. 8:113‐128.
   Walum, E. and Flint, O.P. 1990. Midbrain micromass cultures: a model for studies of teratogenic and sub‐teratogenic effects on CNS development. Acta Physiol. Scand. Suppl. 592:61‐72.
   Whittaker, S.G. and Faustman, E.M. 1991. Effects of albendazole and albendazole sulfoxide on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 109:73‐84.
   Whittaker, S.G. and Faustman, E.M. 1992. Effects of benzimidazole analogs on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 113:144‐151.
   Whittaker, S.G., Wroble, J.T., Silbernagel, S.M., and Faustman, E.M. 1993. Characterization of cytoskeletal and neuronal markers in micromass cultures of rat embryonic midbrain cells. Cell Biol. Toxicol. 9:359‐375.
   Wilk, A.L., Greenburg, J.H., Horigan, E.A., Pratt, R.M., and Martin, G.R. 1980. Detection of teratogenic compounds using differentiating embryonic cells in culture. In Vitro 16:269‐276.
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