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TISSUE-SPECIFIC STEM CELLS

Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays

^aLaboratory of Stem Cell Bioengineering, Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland;
^bMax Planck Institute of Immunobiology, Department of Molecular Embryology, Freiburg, Germany

Key Words. Neural stem cells • Neurospheres • Microwell arrays • Hydrogels • Cell culture • Clonal assays

Correspondence: Correspondence: Matthias P. Lutolf, Ph.D.,Institute of Bioengineering, Laboratory of Stem Cell Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Bldg. AI 3138, Station 15, CH-1015 Lausanne, Switzerland. Telephone: +41 21 693 18 76; Fax: +41 21 693 96 65; e-mail: matthias.lutolf{at}epfl.ch

Received on May 21, 2008; accepted for publication on July 13, 2008.

First published online in STEM CELLS EXPRESS  July 31, 2008.

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801–806; Stem Cells 2007;25:871–874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics.

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