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

Summary

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.

Abstract

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Introduction

The brain is an intricate circuitry of neuronal and non-neuronal cells. For years, scientists have been trying to gain insight into this complex machinery. To do so, neuroscientists initially resorted to various transformed nerve-based cell lines for investigations. However, the inability of these clonal cell lines to form strong synaptic connections and proper axons or dendrites have shifted scientific interest to primary neuron cultures^1,2. The most exciting aspect of primary neuron culture is that it creates an opportunity to observe and manipulate living neurons³. Moreover, it is le....

Protocol

All experimental procedures involving animal were approved by the Institutional Animal Ethics Committee of CSIR-Indian Institute of Chemical Biology (IICB/AEC/Meeting/Apr/2018/1).

1. Reagent and media preparation

1. Poly-D-lysine (PDL) solution: prepare PDL solutions at concentrations of 0.1 mg/mL in deionized water and store in 4 °C until use.
2. Dissociation medium: To 1 L of sterile, filtered deionized water, combine the following components in the respective concentrat.......

Discussion

This protocol describes that how by altering the cell plating densities of primary neurons, two variable neuronal platforms are obtained. Though this is a simple method, each step must be meticulously performed to achieve the desired results. Other previous methods have either reported long-term primary neuron cultures or neurosphere cultures. Most primary neuron culture protocols have involved the culturing of hippocampal neurons for 3-5 weeks, but most have failed, as the neurons die and wither away due to lo.......

Materials

Name	Company	Catalog Number	Comments
Anti-GFAP	Abcam	AB7260
Anti- Nestin	Abcam	AB92391
Anti-O4	Millipore	MAB345
Anti-Tau	Abcam	AB76128
Anti-Tuj1	Millipore	MAB1637
B27 Serum Free Supplement	Gibco	17504-044
Cell Counter	Life technologies	Countess II FL
CO2 Incubator	Eppendorf	Galaxy 170 R
D-glucose	SDFCL	38450-K05
Ethanol	Merck Millipore	100983
Fluorescence Microscope	Olympus	IX83 Model
Formaldehyde	Sigma Aldrich	47608
GlutaMax-I Supplement	Gibco	35050-061
GtXMs IgG Fluor	Millipore	AP1814
GtXMs IgG (H+L)	Millipore	AP124C
HEPES	SRL	16826
Hoechst 33258	Calbiochem	382061
Horse Serum	HiMedia	RM10674
Hydrochloric Acid	Rankem	H0100
Laminar Hood	BioBase	BBS-V1800
MEM Eagle’s with Earle’s BSS	Sigma Aldrich	M-2279
Microscope	Dewinter	Victory Model
Neurobasal Medium	Gibco	21103-049
Plasticware (24 well plate, cell strainers, and low adherence plates)	BD Falcon	353047, 352350 and 3471
90 mm Petridishes	Himedia	PW001
Penicillin/Streptomycin	Gibco	15140-122
Poly-D-Lysine	Millipore	A.003.E
Potassium Chloride	Fisher Scientific	BP366-500
Potassium Phosphate Monobasic	Merck	MI6M562401
Sodium Chloride	Qualigem	15918
Sodium Phosphate Dibasic	Merck	MI6M562328
Stereomicrosope	Dewinter	Zoomstar Model
Triton-X 100	SRL	2020130
Trypan Blue Solution	Gibco	15250-061
0.25 % Trypsin-EDTA	Gibco	25200-072