The Syrian Hamster Embryo (SHE) Low pH Cell Transformation Assay

Kamala Pant1, Marilyn J. Aardema2

1 BioReliance, Rockville, Maryland, 2 The Procter and Gamble Company, Miami Valley Innovation Center, Cincinnati, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.3
DOI:  10.1002/0471140856.tx2003s35
Online Posting Date:  February, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The Syrian hamster embryonic (SHE) cell transformation assay (CTA) at pH 6.7 can determine the ability of a test article to induce morphological transformation (MT) in cultured SHE cells. The assay uses SHE cells prepared at gestation day ∼13 and frozen. Target cells are seeded onto a layer of feeder cells (X‐ray irradiated SHE cells) and treated with test article for 24 hr or 7 days. After a growth period of 7 days, the cells are fixed, stained, and evaluated for MT. Normal colonies typically contain a monolayer of cells with an organized, often flowing, pattern of growth and minimal cell criss‐crossing at a confluent density. Transformed colonies contain randomly oriented, stacked cells, with cell criss‐crossing throughout the colony. The transformed cells are frequently more basophilic than their normal counterparts, with increased nuclear/cytoplasmic ratios. This assay provides a valuable tool for evaluating the carcinogenic potential of a test article. Curr. Protoc. Toxicol. 35:20.3.1‐20.3.16. © 2008 by John Wiley & Sons, Inc.

Keywords: SHE; morphological transformation; clonal transformation; foci formation; colony transformation; in vitro carcinogenicity prediction

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

Table of Contents

  • Introduction
  • Basic Protocol 1: SHE Cell Transformation Assay (CTA)
  • Support Protocol 1: Preparation of Syrian Golden Hamster (SHE) Cell Isolates
  • Support Protocol 2: Testing of New Cell Isolates, Serum, and New Media Prior to Use in the Assay
  • Support Protocol 3: Preparation of Test Article Dosing Solution and Treatment
  • Support Protocol 4: Preliminary Cytotoxicity Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: SHE Cell Transformation Assay (CTA)

  Materials
  • 1 × 106cell/ml Syrian hamster embryo (SHE) cells, cryopreserved stock ( protocol 2)
  • Complete Dulbecco's modified Eagle's medium, LeBoeuf's modification (complete DMEM‐L; see recipe), pretested ( protocol 3)
  • Calcium‐ and magnesium‐free Hanks' balanced salt solution (CMF‐HBSS, GIBCO; or see appendix 2A)
  • Detachment solution (see recipe)
  • Fetal bovine serum (FBS)
  • 0.4% (w/v) trypan blue
  • Test article dosing solutions ( protocol 4)
  • Dulbecco's phosphate‐buffered saline (DPBS, GIBCO; or see appendix 2A)
  • Methanol
  • Giemsa stain
  • 25‐, 150‐, and/or 225‐cm2 flasks, sterile
  • 60‐mm tissue culture dishes, sterile
  • X‐ray machine, low energy (Faxitron or equivalent)
  • 15‐ and 50‐ml centrifuge tubes, sterile
  • Hemacytometer with glass coverslip
  • Stereomicroscope, 10× to 30× magnification (e.g., Stereomaster, Fisher)

Support Protocol 1: Preparation of Syrian Golden Hamster (SHE) Cell Isolates

  Materials
  • Three to four Syrian golden hamsters, females at ∼13 days gestation, from an approved source (e.g., Charles River Labs)
  • 70% (v/v) ethanol
  • Wash solution (see recipe)
  • Dissociation solution (see recipe)
  • Fetal bovine serum, grade‐characterized (FBS; Hyclone)
  • Penicillin/streptomycin solution (10,000 U/ml and 10,000 µg/ml, respectively; GIBCO)
  • Complete Dulbecco's modified Eagle's medium, LeBoeuf's modification (complete DMEM‐L, 625‐ml bottle; see recipe)
  • 0.4% (w/v) trypan blue
  • Calcium‐ and magnesium‐free Hanks’ balanced salt solution (CMF‐HBSS, GIBCO; or see appendix 2A)
  • Detachment solution (see recipe)
  • 2× cryoprotective medium (see recipe)
  • CO 2 tank
  • Absorbent pads
  • Surgical instruments (scissors, forceps, scalpels), sterilized
  • 100‐mm plastic petri dishes (not tissue culture treated), sterile
  • Class II biosafety cabinet
  • 250‐ml conical, glass, trypsinization flask, sterile
  • Magnetic stir bars (sterile) and stirrer
  • 50‐ml centrifuge tubes, sterile
  • 150‐, 162‐ or 225‐cm2 tissue culture flasks, sterile
  • Hemacytometer with glass coverslip
  • 1‐ml cyrovials, sterile
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Berwald, Y. and Sachs, L. 1963. In vitro transformation with chemical carcinogens. Nature 200:1182‐1184.
   Custer, L., Gibson, D.P., Aardema, M.J., and LeBoeuf, R.A. 2000. A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay. Mut. Res. 455:129‐139.
   Isfort, R.J., Cody, D.B., Doerson, C., Kerckaert, G.A., and LeBoeuf, R.A. 1994. Alterations in cellular differentiation, mitogenesis, cytoskeleton and growth characteristics during Syrian hamster embryo cell multistep in vitro transformation. Int. J. Cancer 59:114‐125.
   Kerckaert, G.A., Isfort, R.J., Carr, G.J., Aardema, M.J., and LeBoeuf, R.A. 1996. A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70. Mut. Res. 356:65‐84.
   LeBoeuf, R.A. and Kerckaert, G.A. 1986. The induction of transformed‐like morphology and enhanced growth in Syrian hamster embryo cells grown at acidic pH. Carcinogenesis 1986:1431‐1440.
   LeBoeuf, R.A. and Kerckaert, G. 1987. Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH. Carcinogenesis 1987:680‐697.
   LeBoeuf, R.A., Kerckaert, G.A., Poiley, J.A., and Raineri, R. 1989. An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH. Mutat. Res. 222:205‐218.
   LeBoeuf, R.A., Kerckaert, G.A., Aardema, M.J., and Gibson, D.P. 1990. Multistage neoplastic transformation of Syrian hamster embryo cells cultured at pH 6.70. Cancer Res. 50:3722‐3729.
   OECD. 2006. Detailed review paper on cell transformation assay for detection of chemical carcinogens. OECD Environment, Health, and Safety Publications Series on Testing and Assessment No. 31, Environment Directorate. OECD Publishing.
   Pienta, R.J., Poiley, J.A., and Lebherb, W.G. III. 1977. Morphological transformation of early passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens. Int. J. Cancer 19:642‐655.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library