Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis

Brynn Levy1, Vaidehi Jobanputra1, Dorothy Warburton1

1 College of Physicians and Surgeons of Columbia University, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.5
DOI:  10.1002/0471142905.hg0805s60
Online Posting Date:  January, 2009
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Abstract

In clinical settings, chromosome studies are usually performed on solid tissue other than solid tumors for one of two reasons: the tissue biopsy is the only tissue available from the patient, or tissues other than the standard peripheral blood lymphocytes must be examined because of suspected mosaicism. This unit describes methods for culturing tissue samples to be used for preparation of metaphase chromosomes or for biochemical or DNA analysis. Protocols include an efficient enzymatic cell disruption procedure and a mechanical disruption procedure. In addition, a procedure for processing uncultured cells for analysis by fluorescence in situ hybridization (FISH) is provided. Finally, a method is presented that can sometimes be applied to placental tissue involving the use of mitotically active chorionic villus tissue to prepare metaphase chromosome spreads directly, without cell culture. Two support protocols describe methods for collecting tissue samples. Curr. Protoc. Hum. Genet. 60:8.5.1‐8.5.11. © 2009 by John Wiley & Sons, Inc.

Keywords: products of conception; spontaneous abortions; chorionic villi

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enzymatic Disruption and Culture of Tissues for Metaphase Chromosome Analysis
  • Alternate Protocol 1: Mechanical Disruption and Culture of Tissues for Metaphase Chromosome Analysis
  • Alternate Protocol 2: Enzymatic Disruption and Slide Preparation for Fish Analysis
  • Support Protocol 1: Preparation of Human Products of Conception for Culture
  • Support Protocol 2: Preparation of Skin or Other Tissue Biopsies for Culture
  • Alternate Protocol 3: Direct Preparation of Chorionic Villi
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Enzymatic Disruption and Culture of Tissues for Metaphase Chromosome Analysis

  Materials
  • Tissue of interest
  • Trypsin/EDTA solution ( appendix 3G)
  • Collagenase solution (see recipe)
  • Complete culture medium: containing 15% to 20% (v/v) fetal bovine serum (FBS; see appendix 3G), preferably FBS‐supplemented Ham F‐10 or α‐MEM (GIBCO, Invitrogen)
  • 35‐mm petri plates
  • 15‐ml centrifuge tube
  • 20‐G needle and 1‐ml syringe or fine Pasteur pipet
  • Clinical centrifuge (e.g., IEC, Thermo Scientific)
  • 22‐mm square glass coverslip in 35‐mm petri dish, presterilized (e.g., Coverslip Kit, MatTek)
  • Inverted microscope
  • Additional reagents and equipment for preparing metaphase chromosome spreads by the in situ method (unit 8.4), analyzing chromosome banding (unit 4.2), and preparing karyotypes ( appendix 4A)

Alternate Protocol 1: Mechanical Disruption and Culture of Tissues for Metaphase Chromosome Analysis

  Materials
  • Tissue sample: 2‐ to 3‐mm3 fetal tissue, 2‐mm‐deep × 2‐mm‐diameter skin biopsy, or 3‐mm3 tissue biopsy, fresh or stored at 4°C (see Support Protocols protocol 41 and protocol 52)
  • HBSS ( appendix 2D)/5× antibiotics (e.g., GIBCO PSN antibiotic mixture, Invitrogen): add antibiotic solution at 5× the normal concentration of antibiotic and store up to 1 month at 4°C
  • Complete culture medium: containing 15% to 20% (v/v) fetal bovine serum (FBS; see appendix 3G), preferably FBS‐supplemented Ham F‐10 or α‐MEM (GIBCO, Invitrogen)
  • 35‐mm petri plates
  • Dissection instruments: needles, scissors, fine forceps, and scalpel, sterile
  • 25‐cm2 plastic tissue culture flasks with caps
  • Additional reagents and equipment for preparing metaphase chromosome spreads by the flask method (unit 8.4), analyzing chromosome banding (unit 4.2), and preparing karyotypes ( appendix 4A)

Alternate Protocol 2: Enzymatic Disruption and Slide Preparation for Fish Analysis

  Materials
  • Hypotonic solution: 0.54% (w/v) KCl
  • Enzymatically digested, uncultured chorionic villi ( protocol 6)
  • Fixative (3:1 methanol/acetic acid)
  • Fluorescence in situ hybridization (FISH) probes (e.g., see unit 4.3)
  • 15‐ml centrifuge tube
  • Clinical centrifuge (e.g., IEC, Thermo Scientific)
  • Glass microscope slides, precleaned
  • Slide warmer, 45°C

Support Protocol 1: Preparation of Human Products of Conception for Culture

  Materials
  • Products of conception
  • HBSS ( appendix 2D) or other balanced salt solution
  • Dissecting instruments: needles, scissors, fine forceps, and scalpel, sterile
  • Dissecting microscope (optional)
  • Plastic petri plate or stainless steel container
IMPORTANT NOTE: Although most samples will not be sterile, so that real sterile technique is impossible, all dishes and instruments should be clean and alcohol sterilized.

Support Protocol 2: Preparation of Skin or Other Tissue Biopsies for Culture

  Materials
  • HBSS ( appendix 2D) or other balanced salt solution or unsupplemented tissue culture medium
  • Dissecting instruments: sterile scissors and forceps, or scalpel, sterile
  • Centrifuge tubes of appropriate size with screw caps, sterile
  • Additional reagents and equipment for performing a biopsy
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Figures

Videos

Literature Cited

   Byrne, J., Warburton, D., Kline, J., Blanc, W., and Stein, Z. 1985. Morphology of early fetal deaths and their chromosomal characteristics. Teratology 32:297‐315.
   Eiben, B., Bartels, I., Barh‐Porsch, S., Borgmann, S., Gatz, G., Gellert, G., Goebel, R., Hamanns, W., Hentemann, M., Osmers, R., Rauskolb, R., and Hansmann, I. 1990. Cytogenetic analysis of 750 spontaneous abortions with the direct preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am. J. Hum. Genet. 47:656‐663.
   Jobanputra, V., Sobrino, A., Kinney, A., Kline, J., and Warburton, D. 2002. Multiplex interphase FISH as a screen for common aneuploidies in spontaneous abortions. Hum. Reprod. 17:1166‐1170.
   Kalousek, D.K. 1990. Confined placental mosaicism and intrauterine development. Pediatr. Path. 10:69‐77.
   Kalousek, D.K., Barrett, I.J., and Gartner, A.B. 1992. Spontaneous abortion and confined chromosomal mosaicism. Hum. Genet. 88:642‐646.
   Kalousek, D.K., Langlois, S., Barrett, I., Yam, I., Wilson, D.R., Howard‐Peebles, P.N., Johnson, M.P., and Giorgiutti, E. 1993. Uniparental disomy for chromosome 16 in humans. Am. J. Hum. Genet. 52:8‐16.
   Levy, B., Hirschhorn, K., and Kardon, N.B. 2009. Chromosome abnormalities in spontaneous abortions. In Cytogenetics in Reproductive Medicine (D. Wells, ed.) pp. 59‐66. Landes Bioscience, Austin, Tex.
   Menasha, J., Levy, B., Hirschhorn, K., and Kardon, N.B. 2005. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: New insights from a 12‐year study. Genet. Med. 7:251‐263.
   Pagon, R.A., Hall, J.G., Davenport, S.L., Aase, J., Norwood, T.H., and Hoehn, H.W. 1979. Abnormal skin fibroblast cytogenetics in four dysmorphic patients with normal lymphocyte chromosomes. Am. J. Hum. Genet. 31:54‐61.
   Tjio, J.H. and Levan, A. 1956. The chromosome number in man. Hereditas 42:1‐6.
   Warburton, D., Anyane‐Yeboa, K., and Francke, U. 1987. Mosaic tetrasomy 12p: Four new cases and confirmation of the chromosomal origin of the supernumerary chromosome in one of the original Pallister‐Mosaic syndrome cases. Am. J. Med. Genet. 27:275‐283.
Key References
   Jobanputra et al., 2002. See above.
  Describes the FISH strategy for detecting aneuploidies in spontaneous abortions.
   Levy et al., 2007. See above.
  Reviews the incidence and spectrum of the various chromosome abnormalities found in miscarriage specimens.
   Priest, J.H. 1997. General Cell Culture Principles and Fibroblast Culture. In The AGT Cytogenetics Laboratory Manual (M.J. Barch, T. Knutsen and J.L. Spurbeck, eds.) pp. 173‐197. Lippincott‐Raven, New York.
  Although tissue culture is more like cooking than science (researchers have personal idiosyncrasies in technique and preferences for media and other reagents), these three chapters effectively outline the underlying principles of the techniques, insofar as they are understood, and describe many variations.
   Priest, J.H. and Rao, K.W. 1997. Prenatal Chromosome Diagnosis. In The AGT Cytogenetics Laboratory Manual. (M.J. Barch, T. Knutsen and J.L. Spurbeck, eds.) pp. 199‐215. Lippincott‐Raven, New York.
  Contains pictures providing a useful guide to the many possible forms of specimens from spontaneous abortion and may aid in sorting out which tissues to culture.
   Rooney, D.E. and Czepalkowski, B.H. Prenatal diagnosis and tissue culture. 1992. In Human Cytogenetics: A Practical Approach, 2nd ed., Vol. 1 (D.E. Rooney and B.H. Czepalkowski, eds.) pp. 55‐89. IRL Press at Oxford University Press, Oxford.
   Warburton, D., Byrne, J., and Canki, N. 1991. Chromosomal Anomalies and Prenatal Development: An Atlas. Oxford University Press, New York.
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