ABSTRACT
Conventional in situ hydrogel micropatterning techniques work successfully for relatively stiff hydrogels, but they often result in locally damaged surfaces upon demolding in the case of soft and fragile polymer networks formed at low precursor concentration. To overcome this limitation, we have developed a versatile method, termed soft embossing, for the topographical micropatterning of fragile chemically cross-linked polymer hydrogels. Soft embossing is based on the imprinting of a microstructured template into a gel surface that is only partially cross-linked. Free functional groups continue to be consumed and upon complete cross-linking irreversibly confine the microstructure on the gel surface. Here we identify and optimize the parameters that control the soft embossing process and show that this method allows the fabrication of desired topographies with good fidelity. Finally, one of the produced gel micropatterns, an array of microwells, was successfully utilized forculturing and analyzing live single hematopoietic stem cells. Confining the stem cells to their microwells allowed for efficient quantification of their growth potential during in vitro culturing.
Subject(s)
Hydrogels/chemistry , Polymers/chemistry , Animals , Bone Marrow Cells/metabolism , Cell Culture Techniques , Cell Proliferation , Cross-Linking Reagents/chemistry , Dimethylpolysiloxanes/chemistry , Eyeglasses , Hematopoietic Stem Cells/cytology , Kinetics , Mice , Mice, Inbred C57BL , Microscopy, Confocal/methods , Polyethylene Glycols/chemistry , Rheology/methods , Sulfhydryl Compounds/chemistry , Surface Properties , Time FactorsABSTRACT
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.
