Clusters of neural stem/progenitor cells cultured on a soft poly(vinyl alcohol) hydrogel crosslinked by gamma irradiation

• We prepared the gamma-crosslinked poly(vinylalcohol) (PVA) hydrogels.
• Neural stem/progenitor cells (NSPCs) from the fetal mouse brain was cultured on the PVA hydrogels.
• Cells adhered to the surface and then proliferated forming a cluster of cells in the DMEM/F12 medium to keep the immature undifferentiated state.
• Gamma-crosslinked PVA hydrogel is a useful substrate to amplify the NSPCs.

Neural stem/progenitor cells (NSPCs) in the central nervous system (CNS) have the capacity to self-renew by proliferation and are multipotent, giving rise to neurons, astrocytes, and oligodendrocytes. NSPCs can be amplified in neurosphere suspension cultures for cell transplantation therapy to treat CNS diseases as well as for in vitro pharmacological/toxicological assays; however, these suspension cultures have certain limitations, including the inconvenience of changing the culture medium as well as difficulty of live imaging. In the present study, we prepared a gamma-crosslinked poly(vinyl alcohol) (PVA) hydrogel and assessed its suitability as a substrate for adherent NSPC cultures. Differentiation was determined by evaluating the expression of the markers nestin (progenitors), βIII tubulin (neurons), and glial fibrillary acidic protein and S100β (glia) by immunocytochemistry and quantitative reverse transcriptase PCR. The levels of the marker genes were similar between the two types of culture; although some variability was observed, there were no fold differences in expression. NSPCs adhered to the PVA gel as clusters and grew without differentiating into neurons and glia. The proliferation rate of cells grown on the soft PVA gel [3.75–7.5% (w/v) PVA] was approximately 70% of that of neurospheres in suspension. We conclude that gamma-crosslinked PVA hydrogels can function as a novel scaffold for maintaining adherent NSPCs in an undifferentiated state.