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Cytotechnology

, 63:227 | Cite as

Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells

  • Johan StenbergEmail author
  • Maria Elovsson
  • Raimund Strehl
  • Eva Kilmare
  • Johan Hyllner
  • Anders Lindahl
Method in Cell Science

Abstract

Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.

Keywords

Human embryonic stem cell In vitro differentiation Embryoid body Agarose In vitro culture 

Abbreviations

hESC

Human embryonic stem cell

EB

Embryoid body

EBs

Embryoid bodies

SCID

Severe combined immunodeficiency

3-D

Three dimensional

MEF

Mouse embryonic fibroblast

PBS

Phosphate buffered saline

AFP

Alpha fetoprotein

DAPI

4′,6-diamidino-2-phenylindole

Bfgf

Basic fibroblast growth factor

PPD11

p-Phenylenediamine anti-fade solution

IgG

Immuno globulin G

Notes

Acknowledgments

The study was funded by: Swedish Research Council grant no. 2005-7544, The ALF/LUA research grant from the Sahlgrenska University Hospital. The Inga Britt and Arne Lundberg Research Foundation.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Johan Stenberg
    • 1
    Email author
  • Maria Elovsson
    • 1
  • Raimund Strehl
    • 2
  • Eva Kilmare
    • 2
  • Johan Hyllner
    • 2
  • Anders Lindahl
    • 1
  1. 1.Department of Clinical Chemistry and Transfusion Medicine, Institute of BiomedicineThe Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University HospitalGothenburgSweden
  2. 2.Cellartis ABGothenburgSweden

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