Defined, Feeder‐Independent Medium for Human Embryonic Stem Cell Culture

Tenneille Ludwig1, James A. Thomson2

1 WiCell Research Institute, Madison, Wisconsin, 2 University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.2
DOI:  10.1002/9780470151808.sc01c02s2
Online Posting Date:  September, 2007
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Abstract

The developmental potential of human ES cells makes them an important tool in developmental, pharmacological, and clinical research. For human ES cell technology to be fully exploited, however, culture efficiency must be improved, large‐scale culture enabled, and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers, which limit the scale, present biological variability, and expose the cells to potential contaminants. Defined, feeder‐independent culture systems improve the safety and efficiency of ES cell technology, enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture, transfer, and passage of cells grown in these feeder‐independent conditions. They provide a basis for routine feeder‐independent culture, and a starting point for additional optimization of culture conditions. Curr. Protoc. Stem Cell Biol. 2:1C.2.1‐1C.2.16. © 2007 by John Wiley & Sons, Inc.

Keywords: feeder‐independent culture; human ES cells; defined medium; bFGF

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transferring Human Embryonic Stem Cells to and Culturing in Feeder‐Independent Conditions
  • Basic Protocol 2: Passaging Human Embryonic Stem Cells in Feeder‐Independent Conditions
  • Alternate Protocol 1: Passaging with Dispase
  • Alternate Protocol 2: Passaging with Collagenase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transferring Human Embryonic Stem Cells to and Culturing in Feeder‐Independent Conditions

  Materials
  • Human ES cells in standard (MEF or feeder‐free) culture, in 6‐well plates
  • mTeSR culture medium (see recipe)
  • Matrigel‐coated 6‐well plates (see recipe)

Basic Protocol 2: Passaging Human Embryonic Stem Cells in Feeder‐Independent Conditions

  Materials
  • Human ES cell culture in Matrigel coated 6‐well plates ( protocol 1)
  • mTeSR culture medium (see recipe)
  • Washing medium (see recipe)
  • EDTA splitting medium (see recipe)
  • Inverted microscope with marking objective (Nikon)
  • Pasteur pipets (Fisher Scientific)
  • 15‐ml conical tube (optional)
  • Glass serological pipets (Fisher Scientific)

Alternate Protocol 1: Passaging with Dispase

  Materials
  • Human ES cell culture in Matrigel‐coated 6‐well plates (for transfer or from protocol 1)
  • mTeSR culture medium (see recipe)
  • Dispase splitting medium (see recipe)
  • Warmed DMEM/F‐12 (Invitrogen)
  • Inverted microscope with marking objective (Nikon)
  • Pasteur pipets (Fisher Scientific)
  • 37°C incubator
  • 15‐ml conical tube (optional)
  • Glass serological pipets (Fisher Scientific)

Alternate Protocol 2: Passaging with Collagenase

  Materials
  • Human ES cell culture on MEFs (for transfer to feeder‐independent systems)
  • mTeSR culture medium (see recipe)
  • Collagenase splitting medium (see recipe)
  • Warmed DMEM/F‐12 (Invitrogen)
  • Inverted microscope with marking objective
  • Pasteur pipets (Fisher Scientific)
  • 15‐ml centrifuge tube (optional)
  • Glass serological pipets (Fisher Scientific)
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Figures

Videos

Literature Cited

Literature Cited
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   Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T., Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R., Piekarczyk, M.S., Llanas, R.A., and Thomson, J.A. 2006b. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24:185‐187.
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