Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture

Abstract

Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do invivo because the culture conditions are different from invivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce invivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an invivo-like environment invitro to produce cell responses similar to those invivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an invivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an invivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-μm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing.