Establishment of Fibroblast Cultures

Akira Takashima1

1 University of Texas Southwest Medical Center, Dallas, Texas
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.1
DOI:  10.1002/0471143030.cb0201s00
Online Posting Date:  May, 2001
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Abstract

Fibroblasts are fairly easily isolated from a variety of tissues, in this case skin. The cells are fast growing and can be rapidly expanded from small samples. They are suitable for behavioral, functional, biochemical, and genomic studies. Skin fibroblast cultures are started from explants in which the epidermis is enzymatically removed to prevent contamination of the fibroblasts with epidermal cells. Alternatively, the dermis can be enzymatically dissociated to provide a suspension of fibroblasts.

     
 
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Table of Contents

  • Basic Protocol 1: Skin Explant Culture
  • Alternate Protocol 1: Dissociated Fibroblast Culture
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Skin Explant Culture

  Materials
  • Skin specimen (see Commentary)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.5% (w/v) dispase II (Boehringer‐Mannheim) in PBS (store up to 3 months at −20°C; optional)
  • 0.3% (w/v) trypsin (from bovine pancreas; Sigma) in PBS (store up to 3 months at −20°C; optional)
  • Complete growth medium (DMEM or RPMI; see recipe)
  • Trypsin/EDTA solution (see recipe)
  • 0.4% (w/v) trypan blue in PBS (store up to 6 months at room temperature)
  • Freezing medium: 10% DMSO/90% FBS or 10% DMSO/90% complete DMEM
  • 15‐ml and 50‐ml polypropylene centrifuge tubes
  • Lids from 100‐mm tissue culture dishes
  • Eye forceps (2 pairs)
  • Surgical scalpel with disposable no. 22 blade
  • 35‐mm tissue culture dishes or 6‐well plates
  • 22‐mm glass coverslips (wrap several coverslips in aluminum foil and sterilize by autoclaving)
  • Hemacytometer
  • 25‐cm2 tissue culture flasks
  • 1.5‐ml cryotubes (e.g., Nunc)
  • Liquid nitrogen freezer
CAUTION: When working with human blood, cells, or infectious agents, appropriate biosafety practices must be followed.NOTE: Use Milli‐Q water or equivalent in all protocol steps and for preparing all solutions.

Alternate Protocol 1: Dissociated Fibroblast Culture

  • 1000 U/ml collagenase type IA in recipePBS (see recipe for PBS; store enzyme solution up to 3 months at −20°C)
  • Nylon mesh (85‐µm mesh; Tetko; cut into 5‐cm square, wrap in aluminum foil, and sterilize by autoclaving)
CAUTION: When working with human blood, cells, or infectious agents, appropriate biosafety practices must be followed.NOTE: Use Milli‐Q water or equivalent in all protocol steps and for preparing all solutions.
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Figures

Videos

Literature Cited

Literature Cited
   Nolta, J.A., Konn, J.B. 1997. Human hematopoietic cell culture, transduction, and analyses. In Current Protocols in Human Genetics (N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp13.7.1‐13.7.35. John Wiley & Sons, New York.
   Pandya, A.G., Sontheimer, R.D., Cockerell, C.J., Takashima, A., Piepkorn, M. 1995. Papulonodular mucinosis associated with systemic lupus erythematosis: Possible mechanisms of increased glycosaminoglycan accumulation. J. Am. Acad. Dermatol. 32:199‐205.
   Schuhmachers, G., Xu, S., Bergstresser, P.R., and Takashima, A. 1995. Identity and functional properties of novel skin‐derived fibroblast lines (NS series) that support the growth of epidermal‐derived dendritic cell lines. J. Invest. Dermatol. 105:225‐230.
   Suhonen, J., Ray, J., Blömer, U. and Gage, F.H. 1996. Ex vivo and in vivo gene delivery to the brain. In Current Protocols in Human Genetics (N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp. 13.3.1‐13.3.24. John Wiley & Sons, New York.
   Xu, S., Ariizumi, K., Caceres‐Dittmar, G., Edelbaum, D., Hashimoto, K., Bergstresser, P.R., and Takashima, A. 1995. Successive generation of antigen‐presenting, dendritic cell lines from murine epidermis. J. Immunol. 154:2697‐2705.
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