title: Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.
abstract: 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.
authors.affiliation: Institute of Bioengineering, Laboratory of Stem Cell Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Myriam
authors.initials: M
authors.lastname: Cordey
authors.forename: Monika
authors.initials: M
authors.lastname: Limacher
authors.forename: Stefan
authors.initials: S
authors.lastname: Kobel
authors.forename: Verdon
authors.initials: V
authors.lastname: Taylor
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Lutolf
mesh_terms: D000818:Animals; D001681:Biological Assay; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards*; D019070:Cell Lineage / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010641:Phenotype; D011092:Polyethylene Glycols / Q000494:pharmacology; D015203:Reproducibility of Results; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects; D046888:Tissue Array Analysis / Q000295:instrumentation*
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols