Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells

Christian Unger1, Ulrika Felldin1, Agneta Nordenskjöld1, M. Sirac Dilber1, Outi Hovatta1

1 Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.7
DOI:  10.1002/9780470151808.sc01c07s5
Online Posting Date:  June, 2008
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Abstract

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arouse. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures of research grade human skin fibroblast lines. We also describe how to make feeder layers for hESC using these fibroblasts. Curr. Protoc. Stem Cell Biol. 5:1C.7.1‐1C.7.10. © 2008 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells (hESCs); human foreskin fibroblasts; derivation and expansion; freezing and thawing; feeder cell layer

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells
  • Support Protocol 1: Vital Freezing, Thawing, and the Recovery of Feeder Cells
  • Support Protocol 2: Mitotic Inactivation by γ‐Irradiation to Prepare Human Fibroblast Feeder Cells for hESC Culture
  • Support Protocol 3: Mitotic Inactivation by Mitomycin C Treatment to Prepare Human Fibroblast Feeder Cells for hESC Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells

  Materials
  • Human foreskin tissue from young boys (not older than 2 years), a few mm3
  • Disinfectant soap
  • Phosphate‐buffered saline (D‐PBS) plus antibiotics (see recipe)
  • Blood sample from the donor for basic pathogen screening
  • Heat‐inactivated fetal bovine serum (FBS; Invitrogen, cat. no. 10500‐064 or Hyclone, cat. no. SH30070)
  • Derivation/culture medium (see recipe)
  • D‐PBS without calcium and magnesium (Invitrogen, cat. no. 14190)
  • TrypLE Express (Invitrogen)
  • Trypan blue (Invitrogen)
  • Sterile transportation tube
  • Laminar flow hood (Class II biosafety cabinet)
  • BD Primaria 6‐well‐plates (BD Biosciences) or easy grip cell culture dish with Primaria surface treatment (BD)
  • 10‐cm petri dish
  • Dissecting forceps and fine scissors, sterilized by autoclaving
  • 12‐ml tube (i.e., BD Falcon)
  • Inverted microscope
  • Hemacytometer (e.g., from Marienfeld GmbH, Germany)
  • Tissue culture–treated plasticware: 6‐well plates, 25‐cm2 and 75‐cm2 flasks
  • Additional reagents and equipment for counting viable cells using a hemacytometer and trypan blue staining (unit 1.3)
CAUTION: Take special care when working with human primary tissue!

Support Protocol 1: Vital Freezing, Thawing, and the Recovery of Feeder Cells

  Materials
  • Early passage (≤3) human foreskin fibroblasts ( protocol 1)
  • Heat‐inactivated fetal bovine serum, ice cold
  • Freezing medium (see recipe), ice cold
  • 70% ethanol
  • Derivation/culture medium (see recipe)
  • 12‐ml tubes
  • Centrifuge with swing‐out rotor, able to spin 12‐ml tubes
  • 2‐ml cryovials
  • Freezing container (Styrofoam box or Mr. Frosty; Nalgene)
  • −80°C freezer
  • −150°C freezer or nitrogen tank
  • 37°C water bath
  • 75‐cm2 culture flask
  • Additional reagents and equipment for washing and detaching the cells ( protocol 1)

Support Protocol 2: Mitotic Inactivation by γ‐Irradiation to Prepare Human Fibroblast Feeder Cells for hESC Culture

  Materials
  • Confluent cultures of human foreskin fibroblasts ( protocol 1)
  • Derivation/culture medium (see recipe)
  • γ source (if available; e.g. Cs‐137 irradiator, Gammacell 2000)
  • 6‐well plates
  • Additional reagents and equipment for washing and detaching the cells ( protocol 1)

Support Protocol 3: Mitotic Inactivation by Mitomycin C Treatment to Prepare Human Fibroblast Feeder Cells for hESC Culture

  Materials
  • Cultures of human skin fibroblasts (see protocol 1)
  • Derivation/culture medium (see recipe)
  • Mitomycin C solution (see recipe)
  • D‐PBS without calcium and magnesium (Invitrogen, cat. no.14190)
  • TrypLE Express (Invitrogen)
  • Trypan blue (Invitrogen)
  • 12‐ml tube
  • Hemacytometer
  • 6‐well plates
  • Additional reagents and equipment for viable cell counting using a hemacytometer and trypan blue (unit 1.3)
CAUTION: Mitomycin C is toxic.
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Figures

  •   FigureFigure 1.C0.1 Phase contrast picture of confluent primary human foreskin fibroblast cells (A) at low (4×) and (B) higher (10×) magnification.

Videos

Literature Cited

Literature Cited
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