Establishment and Culture of Human Skin Fibroblasts

Jacob Villegas1, Michael McPhaul1

1 University of Texas Southwestern Medical Center, Dallas, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.3
DOI:  10.1002/0471142727.mb2803s71
Online Posting Date:  August, 2005
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Abstract

The establishment of skin fibroblast strains provides a vehicle by which biochemical and genetic studies may be applied. In addition to providing genetic material to study the basis of disease, fibroblast cell lines established from skin biopsies may provide the investigator with a model in which to study specific disease states or normal physiology. This unit provides methods for the biopsy, establishment, and culture maintenance of skin fibroblasts to be used in further scientific study.

Keywords: Skin fibroblast; Patient-derived primary culture; Tissue culture

     
 
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Table of Contents

  • Basic Protocol 1: Genital Skin Biopsy for Generation of Fibroblast Cultures
  • Basic Protocol 2: Establishment of Primary Cultures of Skin Fibroblasts
  • Basic Protocol 3: Establishment and Maintenance of Skin Fibroblast Cell Lines
  • Basic Protocol 4: Cryogenic Preservation of Skin Fibroblast Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Genital Skin Biopsy for Generation of Fibroblast Cultures

  Materials
  • Betadine solution
  • Sterile saline solution: 0.9% (w/v) NaCl in deionized, distilled water
  • 1% sterile lidocaine solution
  • Biopsy culture medium (see recipe)
  • Scalpel and forceps, sterile (autoclaved)
  • 50‐ml conical tubes
  • Silver nitrate sticks (silver nitrate applicators, 75% w/w, Arzol Chemical)
  • 4‐0 Vicryl suture

Basic Protocol 2: Establishment of Primary Cultures of Skin Fibroblasts

  Materials
  • Biopsy (see protocol 1)
  • Dulbecco's modified phosphate‐buffered saline (DPBS) without calcium or magnesium (Cellgro or see recipe)
  • Primary culture medium (see recipe)
  • 25‐cm2 tissue culture–treated flasks with 0.22‐µm filter caps
  • Forceps, scissors, and scalpels, sterile (autoclaved)
  • 6‐cm tissue culture dish
  • 1‐ml tuberculin syringe (disposable) with 23‐G needle
  • 37°C, 5% CO 2 humidified circulating incubator
  • Inverted microscope

Basic Protocol 3: Establishment and Maintenance of Skin Fibroblast Cell Lines

  Materials
  • Explants grown in 25‐cm2 tissue culture–treated flasks (see protocol 2)
  • Dulbecco's modified phosphate‐buffered saline (DPBS) without calcium or magnesium (Cellgro or see recipe)
  • 0.25% trypsin/EDTA solution: 2.5 g/liter porcine trypsin and 0.38 g/liter EDTA⋅4Na+ in Hanks' balanced salt solution (HBSS; commercially available as a 1× solution)
  • Fibroblast culture medium (see recipe)
  • 75‐cm2 tissue culture–treated flasks with 0.22‐µm filter cap
  • 10‐ml pipets

Basic Protocol 4: Cryogenic Preservation of Skin Fibroblast Cell Lines

  Materials
  • Fibroblast culture medium (see recipe)
  • Dimethyl sulfoxide (DMSO), sterile
  • Established fibroblast cell lines in 75‐cm2 tissue culture flasks (see protocol 3)
  • Dulbecco's modified phosphate‐buffered saline (DPBS) without calcium or magnesium (Cellgro or see recipe)
  • 0.25% trypsin/EDTA solution: 2.5 g/liter porcine trypsin and 0.38 g/liter EDTA⋅4Na+ in Hanks' balanced salt solution (HBSS; commercially available as a 1× solution)
  • 15‐ml conical tube
  • Sterile freezing vials (1.5‐ to 2‐ml ampules)
  • Cryogenic freezing container (e.g., Cryo 1°C Freezing Containers, “Mr. Frosty,” Nalgene Labware) or liquid nitrogen freezer
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Figures

Videos

Literature Cited

Literature Cited
   Avila, D.M., Wilson, C.M., Nandi, N., Griffin, J.E., and McPhaul, M.J. 2002. Immunoreactive AR and genetic alterations in subjects with androgen resistance and undetectable AR levels in genital skin fibroblast ligand‐binding assays. J. Clin. Endocrinol. Metab. 87:182‐188.
   Basu, S.K., Goldstein, J.L., and Brown, M.S. 1978. Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts. J. Biol. Chem. 253:3852‐3856.
   Carrel, A. 1912. On the permanent life of tissues outside the organism. J. Exp. Med. 15:516‐528.
   Catalano, S., Avila, D.M., Marisco, S., Wilson, J.D., Glickman, S.E., and McPhaul, M.J. 2002. Virilization of the female spotted hyena cannot be explained by alterations in the amino acid sequence of the androgen receptor (AR). Mol. Cell Endocrinol. 194:85‐94.
   Freshney, R.I. 2000. The Culture of Animal Cells: A Manual of Basic Technique, 4th ed., pp. 158‐161. Wiley‐Liss, New York.
   Harrison, R.G. 1907. Observations on the living developing nerve fiber. Proc. Soc. Exp. Biol. Med. 4:140‐143.
   Migeon, B.R., Der Kaloustain, V.M., Nyhan, W.L., Young, W.J., and Childs, B. 1968. X‐linked hypoxanthine‐guanine phosphoribosyl transferase deficiency: Heterozygote has two clonal populations. Science 160:425‐427.
   Quigley, C.A., DeBellis, A., Marchke, K.B., El‐Awady, M.K., Wilson, E.M., and French, F.S. 1995. Androgen receptor defects: Historical, clinical, and molecular perspectives. Endocr. Rev. 16:298‐300.
   Sherman, J.K. 1965. Pretreatment with protective substances as a factor in freeze‐thaw survival. Cryobiology 1:249‐304.
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