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.

Ai Kaneko,Yoshiyuki Sankai,Kari Hoyt

doi:10.1371/journal.pone.0102703

Abstract

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×104 cells/mL (8.9×103 cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Highlights

The primary culture of neuronal cells plays an important role in neuroscience, especially in studies of their differentiation, nutritional requirements, and synapse formation
When plated at low density (#104 cells/cm2), rat primary neurons from hippocampi or other brain regions typically die within days, suggesting that neuronal survival is critically dependent on their density [5,6,7,8,9,4,10]
When plated on PuraMatrix, PC12 cells adhered within several hours, extended neurites by the day, and sometimes those neurites extended . 1,000 mm

Summary

Introduction

The primary culture of neuronal cells plays an important role in neuroscience, especially in studies of their differentiation, nutritional requirements, and synapse formation. For research on individual neurons or subcellular components, neurons should be plated at low density and maintained with a chemically defined medium because undefined components, such as serum, make it difficult to evaluate what factors are influencing neuronal growth. There has long been a need for methods enabling the long-term culture of primary neurons at low density in defined, serum-free, medium [1,2,3,4]. The lower the cell density, the more difficult it is to maintain the cultures of primary neurons in serum-free medium. When plated at low density (#104 cells/cm2), rat primary neurons from hippocampi or other brain regions typically die within days, suggesting that neuronal survival is critically dependent on their density (around 104 cells/cm2) [5,6,7,8,9,4,10]

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Conclusion