Faculty Collaboration Database

Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts. Stem Cells Dev 2008 Apr;17(2):383-7

Date

05/02/2008

Pubmed ID

18447652

DOI

10.1089/scd.2007.0138

Scopus ID

2-s2.0-43049149421 (requires institutional sign-in at Scopus site)   8 Citations

Abstract

Highly enriched, pure populations of pluripotent mouse embryonic stem (mES) cells are a prerequisite to downstream experimental manipulations. However, the existing preplating method does not allow complete removal of co-cultured mouse embryonic fibroblast (MEF) feeder cells. The primary objective of the current investigation was to develop and validate a rapid, single-step separation technique for the complete removal of MEF feeder cells from mES cells. A discontinuous density gradient was prepared using Histopaque 1119 at incremental percentages from the top to bottom of a test tube (20, 40, 60, and 100% in culture medium). A suspension of mES cells and MEF feeder cells was layered on top of the gradient. After centrifugation at 400 x g, ES cells and MEF feeder cells were segregated discretely in separate layers at the 40/20% and 100/60% density interfaces, respectively. The mES cells were enriched to a purity of greater than 99% with a recovery rate of greater than 90%. The separation did not alter the viability or the differentiation potential of mES cells. This study validates a simple technique that enables the preparation of highly enriched mES cells that are essentially free of contaminating MEF feeder cells. The discontinuous density gradient separation method is inexpensive, efficient, rapid, and reproducible. The method can be readily scaled-up to accommodate large batch preparations, enabling a broad range of processing needs. Overall, this simple technique significantly expedites the recovery and enrichment of mES cells from MEFs.

Author List

Li Z, Barron MR, Lough J, Zhao M

Authors

Matthew R. Barron PhD Research Scientist I in the Surgery department at Medical College of Wisconsin
John W. Lough PhD Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Cell Count
Cell Differentiation
Cell Separation
Cell Survival
Cells, Cultured
Coculture Techniques
Embryonic Stem Cells
Fibroblasts
Mice
Pluripotent Stem Cells
Time Factors