Culture of Mouse Embryonic Stem Cells

Gabi Tremml1, Matthew Singer1, Richard Malavarca1

1 Millipore Corporation, Bioscience Division, Billerica, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.4
DOI:  10.1002/9780470151808.sc01c04s5
Online Posting Date:  April, 2008
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Abstract

In this unit standard culture conditions for mouse embryonic stem cells (mESCs) on primary murine embryonic fibroblast (PMEF or MEF) monolayers, culture conditions without MEF for feeder‐independent mESCs, and culture conditions in chemically defined media for both feeder‐independent mESCs and feeder‐dependent mESCs are described. For expansion of an mESC line, it is crucial that cells maintain their undifferentiated state and their self‐renewal capacity, and that they remain karyotypically normal, all of which are necessary for successful chimerization of the germ line upon blastocyst injection. Derivation and culture conditions for the original mESCs have been described (notably Robertson, 1987; Smith, 1991; Nagy et al., 2003), however, as there are more and more mESC lines available, it becomes evident that culture conditions are cell‐line specific to some extent, and there is a constant demand for culturing details for mESC lines derived from different mouse strains. Curr. Protoc. Stem Cell Biol. 5:1C.4.1‐1C.4.19. © 2008 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cell (mESC); murine embryonic fibroblasts (MEF); fetal bovine serum (FBS); leukemia inhibitory factor (LIF); trypsin/EDTA; chemically defined media; ESGRO complete; Accutase; enzyme‐free dissociation solution

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Standard Mouse Embryonic Stem Cell Culture on MEFs
  • Support Protocol 1: Serum Testing
  • Alternate Protocol 1: mESC Culture Without MEFs in Standard Medium
  • Alternate Protocol 2: mESC Culture Using Chemically Defined Media: Sequential Adaptation
  • Alternate Protocol 3: mESC Culture Using Chemically Defined Media: Direct Adaptation
  • Support Protocol 2: Cryopreservation of mESCs
  • Support Protocol 3: Counting mESCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Standard Mouse Embryonic Stem Cell Culture on MEFs

  Materials
  • 0.1% gelatin (Millipore cat. no. SF008)
  • Hygromycin‐resistant MEFs (Millipore cat. no. PMEF‐H)
  • Neomycin‐resistant MEFs (Millipore cat. no. PMEF‐N)
  • MEF medium (see recipe), prewarmed
  • mESC medium (see recipe), prewarmed
  • 129SvEv – ES cells (Millipore cat. no. CMTI‐1)
  • C57BL6/J – ES cells (Millipore cat. no. CMTI‐2)
  • D‐PBS without Ca2+ and Mg2+ (CMF‐DPBS; Millipore cat. no. BSS‐1006‐A)
  • D‐PBS (Millipore cat. no. SM‐2002‐C)
  • 0.05% trypsin/EDTA (Millipore cat. no. SM‐2002‐C)
  • 100‐mm tissue culture dishes (25‐cm2 or 75‐cm2, Falcon)
  • 37°C water bath
  • 15‐ and 50‐ml tubes (Falcon)
  • Swing‐out tissue culture centrifuge
  • High‐quality microscope with 10× and 20× oculars

Support Protocol 1: Serum Testing

  Materials
  • mESCs in culture (see protocol 1)
  • Samples of three to five FBS serum lots
  • Additional reagents and equipment for culturing mESCs (see protocol 1)

Alternate Protocol 1: mESC Culture Without MEFs in Standard Medium

  • Standard mESC medium (see recipe), prewarmed
  • mESC lines: E14Tg2, R1, D3

Alternate Protocol 2: mESC Culture Using Chemically Defined Media: Sequential Adaptation

  • mESC medium (see recipe)
  • ESGRO Complete clonal‐grade medium (Millipore cat. no. SF001)
  • Accutase (Millipore cat. no. SF006)
  • Enzyme‐free dissociation solution (Millipore cat. no. SF009)
  • ESGRO Complete basal medium (Millipore cat. no. SF002)

Alternate Protocol 3: mESC Culture Using Chemically Defined Media: Direct Adaptation

  • Feeder‐free mESC cultures (see protocol 4)

Support Protocol 2: Cryopreservation of mESCs

  Materials
  • mESCs
  • DPBS without Ca2+ and Mg2+ (CMF‐DPBS; Millipore cat. no. BSS‐1006‐A)
  • 0.05% trypsin/EDTA (Millipore cat. no. SM‐2002‐C), Accutase (Millipore cat. no. SF006), or enzyme‐free dissociation solution (Millipore cat. no. SF009)
  • mESC medium (see recipe) or ESGRO complete basal medium (see Alternate Protocols protocol 42 and protocol 53)
  • 2× ES‐qualified freezing medium (Millipore cat. no. ES002D)
  • Liquid nitrogen tank
  • ESGRO complete freezing medium (serum‐free; Millipore cat. no. SF005)
  • Cryovials (inside thread; Nunc)
  • 15‐ml tubes
  • Freezing containers (Nunc)
  • Styrofoam
  • 37°C water bath
CAUTION: Freezing solutions contain DMSO, see MSDS for handling, storage, and disposal.

Support Protocol 3: Counting mESCs

  Materials
  • mESCs to be counted
  • Appropriate mESC medium
  • 0.4% (w/v) Trypan blue (Invitrogen cat. no. 15250‐061)
  • Hemacytometer
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Figures

Videos

Literature Cited

   Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. 1984. Formation of germ‐line chimaeras from embryo‐derived teratocarcinoma cell lines. Nature 309: 255‐256.
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   Conner, D.A. 2000. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 51: 23.2.1‐23.2.7.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87: 27‐45.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154‐156.
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Internet Resources
   http://mailman.ic.ac.uk/mailman/listinfo/transgenic‐list
  This Web site hosts an electronic discussion forum. The list members include active researchers in transgenesis from novice to the experts in the field. Major keywords cover a wide spectrum of disciplines: homologous recombination, targeted mutagenesis, inducible expression, ES cells, microinjection, mouse genetics, and animal husbandry.
   http://www.informatics.jax.org/mgihome/lists/lists.shtml
  This Web site hosts an electronic discussion forum for topics in mouse genetics.
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