Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Gaurav Das; Varsha Gupta; Juhee Khan; Deepshikha Mukherjee; Surajit Ghosh

doi:10.3791/59800

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-...

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JoVE Journal Neuroscience

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

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12:22 min

August 31, 2019

DOI: 10.3791/59800-v

Gaurav Das^1,2, Varsha Gupta¹, Juhee Khan¹, Deepshikha Mukherjee¹, Surajit Ghosh^1,2

¹Organic and Medicinal Chemistry Division,CSIR-Indian Institute of Chemical Biology, ²Academy of Scientific and Innovative Research (AcSIR)

Overview

This study presents a protocol for generating neurospheres from high-density plated neurons while also facilitating prolonged cultures of primary rat neurons at lower densities. Using primary hippocampal and cortical neurons from E14-E16 Sprague Dawley rat embryos, the research aims to provide a low-cost platform for studying neurotherapeutics.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neurotherapeutics

Background

Brain research often utilizes in vitro experiments for studying neuronal functions.
Commercial neural lineage-derived cell lines can exhibit variability.
Primary neuron cultures are preferred for consistent results.
This protocol highlights the density-dependent growth of neurons.

Purpose of Study

To develop a robust and economical approach for culturing primary neurons.
To investigate the effects of cell density on neuronal culture outcomes.
To utilize this platform for screening neurotherapeutic leads.

Methods Used

This study employs a cell culture platform with primary neurons from rat embryos.
The biological model consists of primary hippocampal and cortical neurons.
Key steps include plating neurons at different densities and maintaining culture conditions.
Cell viability and morphology were assessed through imaging and MTT assays.

Main Results

Neurons in low-density cultures can be maintained for up to 30 days with high viability.
High-density cultures spontaneously form neurospheres within 24 hours.
Healthy neuronal morphology was observed in both culture conditions.
Immunostaining confirmed the purity of neuron cultures.

Conclusions

This study demonstrates a dual-density protocol that offers insights into neuronal growth and therapeutic screening.
The method's adaptability allows for robust cultures suitable for various applications.
Results contribute to understanding neuronal mechanisms and future studies in neurotherapeutics.

Frequently Asked Questions

What are the advantages of this neuron culture protocol?

This protocol allows for versatility in culturing primary neurons, supporting both prolonged cultures and spontaneous neurosphere generation. It is also cost-effective and easy to implement.

How is the primary neuronal model implemented?

Primary hippocampal and cortical neurons from E14-E16 rat embryos are isolated and plated at varying densities to study their growth and viability.

What types of data or outcomes are obtained from this study?

Key outcomes include neuronal viability, morphology, and the ability to form neurospheres, assessed through imaging and viability assays.

How can the method be applied or adapted for other studies?

The protocol can be adapted for different types of neurotherapeutic screenings and for studying various neuronal responses by adjusting cell densities.

Are there any limitations or considerations for using this protocol?

Maintenance of cell cultures requires careful monitoring and medium changes to ensure high viability and prevent contamination.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

The overall goal of this procedure is to demonstrate the development of neurospheres from mixed primary hippocampal and cortical neurons isolated from E14-E16 Sprague Dawley rat embryo, in order to offer a robust and low-cost platform for studying the effect of various neurotherapeutic leads. As you know, that brain is a complex network of neurons associated with large number of supporting cells. To understand the brain function, often most of the research is re-initiated from in vitro experiments, which involves commercially available neural lineage derived cell lines.

However, this cell lines are transformed cell lines that suffer from genotypic and phenotypic variations. To address this issues, primary neuron culture is the suitable approach. Here, we illustrated a simple and cost-effective protocol to culture primary neurons and built a robust screening platform for various neural therapeutics.

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