Organ Culture of Midfacial Tissue and Secondary Palate

Barbara D. Abbott1

1 U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 13.6
DOI:  10.1002/0471140856.tx1306s16
Online Posting Date:  August, 2003
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Palatal organ culture provides an in vitro model for the study of the formation of the secondary palate, which forms the roof of the mouth in the developing fetus. The protocol describes the steps for culture of the mid‐facial region of the fetal mouse or rat. In culture the secondary palatal shelves proceed through stages of growth, elevation and fusion in a manner analogous to that occurring in utero. This model provides a tool for studies of mechanisms of normal and abnormal palatogenesis and has applications for developmental biology and toxicology.

Keywords: cleft palate; palatogenesis; palate organ culture

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Table of Contents

  • Basic Protocol 1: Submerged Palatal Organ Culture of Mouse Midfacial Tissues
  • Alternate Protocol 1: Submerged Palatal Organ Culture of Rat Midfacial Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Submerged Palatal Organ Culture of Mouse Midfacial Tissues

  • Supplemented custom‐modified Biggers BGJ medium (see recipe)
  • Dimethylsulfoxide (DMSO)
  • Chemical to be tested
  • Penicillin/streptomycin solution: 10,000 U penicillin G with 10 mg streptomycin/ml
  • Medical gas mixture tank of 50% O 2, 5% CO 2, 45% N 2
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; see recipe)
  • Time‐mated, gestation day (GD) 12 pregnant female mice (mated overnight, next morning plug‐positive, confirmed mating = GD 0)
  • Rocker platform with adjustable tilt rate (side‐to‐side motion) to fit in incubator
  • 37°C culture incubator
  • 25‐cm2 (75‐cm2 for rat) tissue‐culture flasks with tight‐fitting caps (e.g., 70‐ml capacity, Corning)
  • 0.2‐µm syringe filters
  • 1‐, 5‐, and 10‐ml syringes
  • Two‐stage gas regulator, suitable for use with pressurized oxygen
  • Laminar flow hood (must be operable with door open or have ports for microscopes)
  • Disposable 1‐ml plastic transfer pipets with bulbs, sterile
  • Dissecting stereo microscope(s)
  • Fiber‐optic illuminator(s)
  • Gauze pads
  • Dissection pad (cut a black rubber stopper, size no.12 or other suitable dark rubber pad, horizontally to provide ½‐in. height, sterilized by storing in a beaker of 70% ethanol)
  • 60‐mm petri dishes
  • Dumont forceps no. 5, stainless steel (at least 2 pairs, have extra on hand and be aware that these points are easily damaged even in routine use)
  • 3‐in. Vannas ultra‐microscissors straight (Roboz Surgical or equivalent spring‐loaded iridectomy scissors with very fine small blades)
  • Disposable no. 11 scalpels
  • 5‐in. micro‐dissecting spatula (Roboz or equivalent)
  • 4‐in. dissecting forceps serrated, curved
  • 4¼‐in. curved, blunt‐tip microdissecting scissors
  • 4‐in. curved, sharp‐tip microdissecting scissors
  • 5‐in. straight hemostatic forceps, delicate, flat mosquito jaw (Roboz or equivalent)
  • Vacuum flask and vacuum hose
  • 5‐, 10‐, and 25‐ml disposable pipets, sterile
  • Vacuum pipettor

Alternate Protocol 1: Submerged Palatal Organ Culture of Rat Midfacial Tissues

  • Time‐mated, gestation day (GD) 14 rats (mated overnight, next morning plug‐positive, confirmed mating = GD 0)
  • 75‐cm2 tissue‐culture flasks, canted‐neck plug seal (caps must form gas‐tight seal, no ventilated filter caps)
  • Vannas ultra‐microscissors (spring‐loaded iridectomy scissors), with slightly larger blades than used for mouse
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Literature Cited

Literature Cited
   Abbott, B.D. and Pratt, R.M. 1987a. Human embryonic palatal epithelial differentiation is altered by retinoic acid and epidermal growth factor in organ culture. J. Craniofac. Genet. Dev. Biol. 7:241‐265.
   Abbott, B.D. and Pratt, R.M. 1987b. Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture. J. Craniofac. Genet. Dev. Biol. 7:219‐240.
   Abbott, B.D. and Birnbaum, L.S. 1990. Rat embryonic palatal shelves respond to TCDD in organ culture. Toxicol. Appl. Pharmacol. 103:441‐451.
   Abbott, B.D. and Birnbaum, L.S. 1991. TCDD exposure of human embryonic palatal shelves in organ culture alters the differentiation of medial epithelial cells. Teratology 43:119‐132.
   Abbott, B.D. and Buckalew, A.R. 1992. Embryonic palatal responses to teratogens in serum‐free organ culture. Teratology 45:369‐382.
   Abbott, B.D., Lau, C., Buckalew, A.R., Logsdon, T.R., Setzer, W., Zucker, R.M., Elstein, K.H., and Kavlock, R.J. 1993. Effects of 5‐fluorouracil on embryonic rat palate in vitro: Fusion in the absence of proliferation. Teratology 47:541‐554.
   Abbott, B.D., Logsdon, T.R., and Wilke, T.S. 1994. Effects of methanol on embryonic mouse palate in serum‐free organ culture. Teratology 49:122‐134.
   Abbott, B.D., Birnbaum, L.S., and Diliberto, J.J. 1996. Rapid distribution of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro. Toxicol. Appl. Pharmacol. 141:256‐263.
   Abbott, B.D., Probst, M.R., Perdew, G.H., and Buckalew, A.R. 1998. AH receptor, ARNT, glucocorticoid receptor, EGF receptor, EGF, TGF alpha, TGF beta 1, TGF beta 2, and TGF beta 3 expression in human embryonic palate, and effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Teratology 58:30‐43.
   Abbott, B.D., Buckalew, A.R., Diliberto, J.J., Wood, C.R., Held, G., Pitt, J.A., and Schmid, J.E. 1999. AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture. Toxicol. Sci. 47:62‐75.
   Al‐Obaidi, N., Kastner, U., Merker, H.J., and Klug, S. 1995. Development of a suspension organ culture of the fetal rat palate. Arch. Toxicol. 69:472‐479.
   Donovan, J. and Brown, P. 1995. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, E.M. Shevach, and W. Strober, eds.), pp.1.8.1‐1.8.4. John Wiley & Sons, New York.
   Ferguson, M.W., Honig, L.S., and Slavkin, H.C. 1984. Differentiation of cultured palatal shelves from alligator, chick, and mouse embryos. Anat. Rec. 209:231‐249.
   Lewis, C.A., Thibault, L., Pratt, R.M., and Brinkley, L.L. 1980. An improved culture system for secondary palatal elevation. In Vitro 16:453‐460.
   National Birth Defects Prevention Network. 1997. Birth defects surveillance data from selected states. Teratology 56:115‐175.
   Pisano, M.M. and Greene, R.M. 2000. Palate development. In vitro procedures. Methods Mol. Biol. 137:267‐274.
   Shiota, K., Kosazuma, T., Klug, S., and Neubert, D. 1990. Development of the fetal mouse palate in suspension organ culture. Acta. Anat. 137:59‐64.
   Shuey, D.L., Buckalew, A.R., Wilke, T.S., Rogers, J.M., and Abbott, B.D. 1994. Early events following maternal exposure to 5‐fluorouracil lead to dysmorphology in cultured embryonic tissues. Teratology 50:379‐386.
   Thesleff, I. 1981. Use of organ culture techniques in craniofacial developmental biology. Proc. Finn. Dent. Soc. 77:159‐169.
   Tyler, M.S. and Koch, W.E. 1975. In vitro development of palatal tissues from embryonic mice. I. Differentiation of the secondary palate from 12‐day mouse embryos. Anat. Rec. 182:297‐301.
   Tyler, M.S. and Pratt, R.M. 1980. Effect of epidermal growth factor on secondary palatal epithelium in vitro: Tissue isolation and recombination studies. J. Embryol. Exp. Morphol. 58:93‐106.
   Whitby, K.E. 1987. Teratological research using in vitro systems. III. Embryonic organs in culture. Environ. Health Perspect. 72:221‐223.
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