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TECHNOLOGY DEVELOPMENT

Facilitated Expansion of Human Embryonic Stem Cells by Single-Cell Enzymatic Dissociation

^aCellartis AB, Göteborg, Sweden;
^bStem Cell Center, Lund University, Lund, Sweden

Key Words. Human embryonic stem cell • Human feeders • Single cells • Enzymatic dissociation • Expansion

Correspondence: Henrik Semb, Ph.D., Stem Cell Center, Lund University, BMC, B10, SE-221 84 Lund, Sweden. Telephone: 46-46-222-31-59; Fax: 46-46-222-36-00; e-mail: henrik.semb{at}med.lu.se

Received on September 28, 2006; accepted for publication on March 13, 2007.

First published online in STEM CELLS EXPRESS  March 22, 2007.


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.

Disclosure of potential conflicts of interest is found at the end of this article.

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