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Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation

Ellerström, Catharina LU ; Strehl, Raimund; Noaksson, Karin; Hyllner, Johan and Semb, Henrik LU (2007) In Stem Cells 25(7). p.1690-1696
Abstract
Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up... (More)
Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
enzymatic, single cells, human embryonic stem cell, human feeders, dissociation, expansion
in
Stem Cells
volume
25
issue
7
pages
1690 - 1696
publisher
AlphaMed Press
external identifiers
  • wos:000247722100012
  • scopus:34547158898
ISSN
1549-4918
DOI
10.1634/stemcells.2006-0607
language
English
LU publication?
yes
id
392be802-2212-459b-ac41-9237c3edcde0 (old id 646084)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17379766&dopt=Abstract
date added to LUP
2007-12-11 20:08:38
date last changed
2017-10-22 04:45:09
@article{392be802-2212-459b-ac41-9237c3edcde0,
  abstract     = {Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations.},
  author       = {Ellerström, Catharina and Strehl, Raimund and Noaksson, Karin and Hyllner, Johan and Semb, Henrik},
  issn         = {1549-4918},
  keyword      = {enzymatic,single cells,human embryonic stem cell,human feeders,dissociation,expansion},
  language     = {eng},
  number       = {7},
  pages        = {1690--1696},
  publisher    = {AlphaMed Press},
  series       = {Stem Cells},
  title        = {Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation},
  url          = {http://dx.doi.org/10.1634/stemcells.2006-0607},
  volume       = {25},
  year         = {2007},
}