Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix
Figure 5
Effect of the coverslip and PuraMatrix on the neuronal ratio of primary neurons.
Bars: 100 µm. Neurons from E18 rat hippocampi were plated at 3×104 cells/mL in basic medium #0 and coverslipped 3 h after plating (+), or not (−). Before plating, 25% PuraMatrix was already prepared in a well (A–C) or the wells were not treated (D–F). The neurons attached to PuraMatrix (A–C) or the polystyrene, tissue culture-treated bottom (D–F) and grew. The neurons were plated into five multiplates simultaneously, and the neuronal ratio in each plate was only examined once at DIV 4, 7, 21, 42, or 69. Neurons and astrocytes were double-stained with anti-MAP2 for neurons (green) and anti-GFAP for astrocytes (red), with DAPI as a nuclear counterstain (blue). Neurons and astrocytes were counted in six areas in each well: three areas in the center and three areas at the edge, and their total numbers were used for analysis. The neuronal ratio was consistently higher in the coverslip+ compared with the coverslip– condition at DIV 4–69 (A, D). (A) Our combined sandwich culture technique using both a coverslip and PuraMatrix (coverslip+) achieved a high neuronal ratio of nearly 100%. Fluorescence images at DIV 42 at the edge of the coverslip+ (B) and coverslip− (C) wells. (B) Neurons (green) preceded astrocytes (red). (C) Astrocytes proliferated locally, mainly at the edge of the well. (D) Sandwich culture using coverslip only (coverslip+) also yielded a high neuronal ratio of nearly 100%. Fluorescence images at DIV 42 at the edge of the coverslip+ (E) and in the center of the coverslip− (F) wells. (E) Neurons (green) preceded astrocytes (red). The image is unfocused because of the influence of intrinsic fluorescence of the polystyrene well itself, especially at the edge. (F) Astrocytes proliferated locally, mainly in the center of the well, where many cells aggregated.