Rat Embryo Cultures for In Vitro Teratology

Alan G. Fantel1, Rudolf Bechter2, David Beckman3

1 University of Washington, Seattle, Washington, 2 Novartis Pharma Ltd., Basel, Switzerland, 3 Novartis Pharmaceuticals Corporation, East Hanover, New Jersey
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 13.2
DOI:  10.1002/0471140856.tx1302s06
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Mammalian embryos provide a particular challenge to those who wish to study developmental processes because development takes place inside the mother's body, thus limiting the investigator's ability to directly affect and observe the embryonic stages of development. Fortunately, as this unit illustrates, methods have been developed for in vitro culture of rodent embryos during early postimplantation (gestation days 9 to 11) and early fetal (gestation days 12 to 14) stages, which are the periods during which the major systems of the embryo are established. Embryos at these stages are particularly suitable for screening studies to determine the effects of teratological agents.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Culture of Early Postimplantation Embryos (gd 9 through gd 11)
  • Basic Protocol 2: Culture of Early Fetal Stages (gd 12 to 14)
  • Support Protocol 1: Collecting Serum for Embryo Culture
  • Support Protocol 2: Preparation of Liver Homogenates for Studies of Biotransformation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Culture of Early Postimplantation Embryos (gd 9 through gd 11)

  Materials
  • Time‐mated gravidas (≤250‐g rats, gestational day 8 to 10)
  • Anesthetic: ether/O 2, halothane/N 2O/O 2 or isoflurane/O 2
  • Normal saline or Hanks' balanced salt solution (HBSS), ice‐cold (see appendix 2A for both recipes)
  • Embryo culture medium (see recipe), prewarmed to 37°C
  • 20 mM potassium phosphate buffer, pH 7.4 (optional; appendix 2A)
  • Compounds to be tested for effects on embryos, dissolved in DMSO if necessary
  • S‐9 supernatant from rat liver (optional; see protocol 4)
  • NADPH (for use with S‐9 supernatant; optional)
  • Glucose 6‐phosphate (G6P; for use with S‐9 supernatant; optional)
  • Gas mixtures (depending on stage and length of culture):
  •  5% O 2/5% CO 2/90% N 2 (gd 9)
  •  20% O 2/5% CO 2/75% N 2 (gd 10)
  •  40% O 2/5% CO 2/55% N 2 (optional late gd 10)
  •  95% O 2/5% CO 2 (gd 11 and later)
  • Petri dishes, sterile
  • Dissecting microscope
  • Dissecting equipment, sterile, including:
  •  Forceps
  •  Watchmakers forceps
  •  Fine iris scissors
  • Tissue culture vessels (e.g., 125‐ml medium bottles, 10‐ml test tubes) with gas‐tight seals, sterile
  • Rotator apparatus for use within incubator:
  •  Powered bars for round bottles
  •  Motorized plate for test tubes
NOTE: All solutions and equipment coming into contact with embryos must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Culture of Early Fetal Stages (gd 12 to 14)

  Materials
  • Time‐mated gravidas (≤250‐g rats, gestational day 12 to 14)
  • Fetal culture medium (see recipe), prewarmed to 37°C and fully flushed with 95% O 2/CO 2
  • Gas mixture: 95% O 2/5% CO 2
  • Compounds to be tested for effects on embryos, dissolved in DMSO if necessary
  • S‐9 supernatant from rat liver (optional; see protocol 4)
  • NADPH (for use with S‐9 supernatant; optional)
  • Glucose 6‐phosphate (G6P; for use with S‐9 supernatant; optional)
  • Flat‐sided 625‐ml tissue culture flasks
  • Rubber stoppers to fit tissue culture flasks
  • 18‐G hypodermic needles
  • Gas regulators and tubing
  • Side‐arm Ehrlenmeyer flask
  • 1‐ml syringes
  • Sintered‐glass diffuser
  • Dissecting microscope
  • Dubnoff metabolic incubator (Precision Scientific)
NOTE: All solutions and equipment coming into contact with fetuses must be sterile, and aseptic technique should be used accordingly.

Support Protocol 1: Collecting Serum for Embryo Culture

  Materials
  • Nonpregnant rats (often retired breeders or older males)
  • Anesthetic: ether/O 2, halothane/N 2O/O 2 or isoflurane/O 2
  • 18‐G needle
  • Sterile, nonheparinized syringe
  • Refrigerated centrifuge
  • 50‐ml conical centrifuge tubes
  • 56°C water bath

Support Protocol 2: Preparation of Liver Homogenates for Studies of Biotransformation

  Materials
  • Adult rats
  • Polychlorinated biphenyls (PCB, Araclor 1254)
  • Corn oil
  • 0.05 mM potassium phosphate buffer, pH 7.4
  • 3‐ml syringe and 18‐G needle
  • Dounce homogenizer with Teflon pestle
  • Refrigerated centrifuge
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abir, R., Ornoy, A., Hur, H.B., Jaffe, P., and Pinus, H. 1993. IgG exchange as a means of partial correction of anomalies in rat embryos in vitro, induced by sera from women with recurrent abortion. Toxicol. In Vitro 7:817‐826.
   Allen, R.G. and Venkatraj, V.S. 1992. Oxidants and antioxidants in development and differentiation. J. Nutr. 122:631‐635.
   Barber, C.V., Carda, M.B., and Fantel, A.G. 1993. A new technique for culturing rat embryos in vitro between gestation day 14 and 15. Toxicol. In Vitro 7:695‐700.
   Beaudoin, A.R. and Fisher, D.L. 1981. An in vivo/in vitro evaluation of teratogenic action. Teratology 23:57‐61.
   Bechter, R. and Terlouw, G.D.C. 1990. Xenobiotic metabolism in the isolated conceptus. Toxicol. In Vitro 4:480‐492.
   Bechter, R., Bouis, P., and Fischer, V. 1989. Primary hepatocyte culture as an activating system for xenobiotica tested in the rat whole embryo in vitro. In Alternative Methods in Toxicology. Vol. 7 In Vitro Toxicology New Directions (A.M. Goldberg and M.L. Principe, eds.) pp. 313‐326. Mary Ann Liebert, New York.
   Bechter, R., Terlouw, G.D.C., Lee, Q.P., and Juchau, M.R. 1991. Effects of QA 208‐199 and its metabolite 209‐668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses. Teratog. Carcinog. Mutagen. 11:185‐194.
   Beckman, D.A., Pugarelli, J.E., Jensen, M., Koszalka, T.R., Brent, R.L., and Lloyd, J.B. 1990. Sources of amino acids for protein synthesis during early organogenesis in the rat. 1. Relative contributions of free amino acids and of proteins. Placenta 11:109‐121.
   Beckman, D.A., Pugarelli, J.E., Koszalka, T.R., Brent, R.L., and Lloyd, J.B. 1991. Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac. Placenta 12:37‐46.
   Boveris, A., Oshino, N., and Chance, B. 1972. The cellular production of hydrogen peroxide. Biochem. J. 128:617‐630.
   Brent, R.L., Beckman, D.A., Jensen, M., and Koszalka, T.R. 1990. Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis. Teratology 41:405‐413.
   Brown, N.A. and Fabro, S. 1981. Quantitation of rat embryonic development in vitro: A morphological scoring system. Teratology 24:65‐78.
   Brown, N.A., Clarke, D.O., and McCarthy, A. 1991. Adaptation of postimplantation embryos to culture: Membrane lipid synthesis and response to valproate. Reprod. Toxicol. 5:245‐53.
   Chatot, C.L., Klein, N.W., Piatek, J., and Pierro, L.J. 1980. Successful culture of rat embryos in human serum: Use in the detection of teratogens. Science 207:1471‐1473.
   Chen, E.Y., Fujinaga, M., and Giaccia, A.J. 1999. Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development. Teratology 60:215‐225.
   Fantel, A.G. 1996. Reactive oxygen species in developmental toxicity: Review and hypothesis. Teratology 53:196‐217.
   Fantel, A.G., Greenaway, J.C., Juchau, M.R., and Shepard, T.H. 1979. Teratogenic bioactivation of cyclophosphamide in vitro. Life Sci. 25:67‐72.
   Fantel, A.G., Greenaway, J.C., Shepard, T.H., Juchau, M.R., and Selleck, S.B. 1981. The teratogenicity of cytochalasin D and its inhibition by drug metabolism. Teratology 23:223‐231.
   Freeman, S.J. and Brown, N.A. 1994. Inhibition of yolk sac function in late gastrulation rat conceptuses as a cause of teratogenesis: An in vivo/in vitro study. Reprod. Toxicol. 8:137‐143.
   Freeman, S.J. and Lloyd, J.B. 1983. Inhibition of proteolysis in rat yolk sac as a cause of teratogenesis. Effects of leupeptin in vitro and in vivo. J. Embryol. Exp. Morphol. 78:183‐193.
   Freeman, S.J. and Lloyd, J.B. 1986. Evidence that suramin and aurothiomalate are teratogenic in rat by disturbing yolk sac–mediated embryonic protein nutrition. Chem. Biol. Interact. 58:149‐160.
   Freeman, S.J., Brent, R.L., and Lloyd, J.B. 1982. The effect of teratogenic antiserum on yolk‐sac function in rat embryos cultured in vitro. J. Embryol. Exp. Morphol. 71:63‐74.
   Jones, C., Greiner, B., Wächter, F., Terlouw, G.D.C., and Bechter, R. 1993. An immunohistochemical investigation into the presence and localization of cytochrome P‐450 isozymes in organogenesis‐stage rat conceptal tissue. Toxicol. In Vitro 7:685‐693.
   Kitchin, K.T., Schmid, B.P., and Sanyal, M.K. 1981. Teratogenicity of cyclophosphamide in a coupled microsomal activating/embryo culture system. Biochem. Pharmacol. 30:59‐64.
   Klein, N.W., Plenefisch, J.D., Carey, S.W., Fredrickson, W.T., Sackett, G.P., Burbacher, T.M., and Parker, R.M. 1982. Serum from monkeys with histories of fetal wastage causes abnormalities in cultured rat embryos. Science 215:66‐69.
   Klug, S., Lewandowski, C., and Neubert, D. 1985. Modification and standardization of the culture of early postimplantation embryos for toxicological studies. Arch. Toxicol. 58:84‐88.
   Kochar, D.M. 1975. The use of in vitro procedures in teratology. Teratology 11:273‐287.
   Miki, A., Fujimoto, E., Ohsaki, T., and Mizoguti, H. 1988. Effects of oxygen concentration on functions of the rat visceral yolk sac endoderm in vitro. A histochemical study using whole‐embryo culture technique. Kobe J. Med. Sci. 34:55‐70.
   Morriss, G.M. and New, D.A.T. 1979. Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos. J. Embryol. Exp. Morphol. 54:17‐35.
   New, D.A.T. 1966. Development of rat embryos cultured in blood sera. J. Reprod. Fertil. 12:509‐524.
   New, D.A.T. 1967. Development of explanted rat embryos in circulating medium. J. Embryol. Exp. Morphol. 17:513‐525.
   New, D.A.T. 1971. Methods for the culture of post‐implantation rodents. In Methods in Mammalian Embryology (J.C. Daniel, ed.) pp. 305‐319. W.H. Freeman, San Francisco.
   New, D.A.T. 1979. Whole‐embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53:81‐122.
   Oglesby, L.A., Ebron‐McCoy, M.T., Logsdon, T.R., Copeland, F., Beyer, P.E., and Kavlock, R.J. 1992. In vitro embryotoxicity of a series of para‐substituted phenols: Structure, activity, and correlation with in vivo data. Teratology 45:11‐33.
   Ozolins, T.R., Oglesby, L.A., Wiley, M.J., and Wells, P.G. 1995. In vitro murine embryotoxicity of cyclophosphamide in embryos co‐cultured with maternal hepatocytes: Development and application of a murine embryo‐hepatocyte co‐culture model. Toxicology 102:259‐274.
   Robkin, M.A. and Shepard, T.H. 1972. In vitro culture of somite stage rat embryos: A new technique for maintaining growth and continuously monitoring heart rate. Applications in metabolic and teratologic studies. In Vitro 8:151‐160.
   Schmid, B.P., Trippmacher, A., and Bianchi, A. 1982. Teratogenicity induced in cultured rat embryos by the serum of procarbazine treated rats. Toxicology 25:53‐60.
   Terlouw, G.D.C. and Bechter, R. 1992. Comparison of the metabolic activity of yolk sac tissue in the whole embryo and isolated yolk sac culture. Reprod. Toxicol. 6:85‐92.
   Terlouw, G.D.C. and Bechter, R. 1993. Cytochrome P‐450‐dependent biotransformation of QA 208‐199 in cultured rat conceptuses. Toxicol.In Vitro 7:247‐258.
   Terlouw, G.D.C., Namkung, M.J., Juchau, M.R., and Bechter, R. 1993. In vitro embryotoxicity of N‐methyl‐N‐7‐propoxynaphthalene‐2‐ethylhydroxylamine QAB: Evidence for N‐dehydroxylated metabolite as a proximate dysmorphogen. Teratology 48:431‐439.
   Zhao, J., Krafft, N., Terlouw, G.D.C., and Bechter, R. 1993. A model combining the whole embryo culture with human liver S‐9 fraction for human teratogenic prediction. Toxicol. In Vitro 7:827‐831.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library