Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Summary

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Abstract

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 10⁶ neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

Introduction

Understanding spinal cord pathology requires the use of various models, both on the macroscopic and microscopic levels. Large and small animal models^1,2,3 are used for in vivo investigations of spinal cord disease and injury. While studying these issues in vivo has its merits, analysis of the spinal cord is limited to whole spinal cord homogenate or to tissue sections⁴. This creates some ambiguity when trying to isolate specific responses and targets in the spinal cord among its resident neurons and surrounding glia. The increasing av....

Protocol

The care and treatment of animals in this procedure were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Colorado.

1. Preparing Solutions

1. Prepare and store all solutions at appropriate temperatures, as shown in Table 1.

2. Coating Wells and Slides

NOTE: Neurons do not adhere well to plastic or glass surfaces.

1. One day prior to the isolation of the neurons, coat the wells of a sterile 24-well culture plate with 0.5 mL of Poly-D-Lysine (PDL; Table 2) and lea....

Results

Using this technique, a single litter (4-10 pups) allows for the isolation of 1-2.5 10⁶ neurons suitable for seeding onto culture plates. Typically, 4-8 wells are seeded at the concentration mentioned above (i.e. 300,000 cells/mL). Figure 3 demonstrates the appearance of neurons at this concentration after a week in culture at low- (a) and high- (b) magnification light microscopy. However, we have also bee.......

Discussion

This technique allows for the reliable culture of spinal cord neurons. Once proficiency in the technique is achieved, it takes approximately 3.5 h to complete. We have been able to carry out the isolation of neurons from 2 separate litters (16 mice total) in approximately 4 h. The key step in feasibility is being able to proficiently extract the spinal cords from the mice. The yield allows for plating several wells and for the ability to test the neurons under various conditions. We have been able to treat the neurons af.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hibernate A Medium - 500 mL	Thermo-Fisher	A1247501	https://www.thermofisher.com/order/catalog/product/A1247501
Hibernate A Minus Calcium - 500 mL	Brainbits	HA-Ca	http://www.brainbitsllc.com/hibernate-a-minus-calcium/
Glutamax 100X - 100 mL	Thermo-Fisher	35050061	https://www.thermofisher.com/order/catalog/product/35050079
B27 Supplement 50X - 10 mL	Thermo-Fisher	17504044	https://www.thermofisher.com/order/catalog/product/17504044
Papain, Lyophilized - 100 mg	Worthington	LS003119	http://www.worthington-biochem.com/pap/cat.html
Neurobasal A Medium - 500 mL	Thermo-Fisher	10888022	https://www.thermofisher.com/order/catalog/product/10888022
Penicillin-Streptomycin (10,000 U/mL)	Thermo-Fisher	15140122	https://www.thermofisher.com/order/catalog/product/15140122
Poly-D-Lysine (PDL) hydrobromide - 5 mg	Sigma-Aldrich	P6407-5MG	http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en®ion=US
Mouse Laminin - 1 mg	Thermo-Fisher	23017015	https://www.thermofisher.com/order/catalog/product/23017015
Trypan Blue - 20 mL	Sigma-Aldrich	T8154-20ML	http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en®ion=US
OptiPrep Density Gradient Medium - 250 mL	Sigma-Aldrich	D1556-250ML	http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en®ion=US
Dichlorodimethylsilane (DMDCS, Sigma Silicoat)	Sigma-Aldrich	440272-100ML	http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en®ion=US
Chloroform	Sigma-Aldrich	288306-1L	http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en®ion=US
Glass Pippette - 9"	Sigma-Aldrich	13-678-20C	http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en®ion=US
Pipette bulb - 5 mL	Sigma-Aldrich	Z186678-3EA	http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1
BRAND® Petri dish, glass - 60 x 15 mm	Sigma-Aldrich	BR455717-10EA	http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en®ion=US
Sterile 24-Well Cell Culture Plate	Sigma-Aldrich	M8812-100EA	http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en®ion=US
Hausser Hemacytometer (glass counting chamber)	Fischer Scientific	02-671-6	https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716
Glass Slides - 12 mm sterile cover glass - uncoated	Neuvitro	GG-12-1.5-Pre	http://www.neuvitro.com/german-coverslip-12mm-diameter.htm
NeuN Rabbit Monoclonal Antibody - 100 µL	Abcam	ab177487	After fixing in paraformaldehyde (PFA) and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:200 for 18 h in 4 °C
MAP-2 Mouse Monoclonal Antibody - 50 µL	Abcam	ab11267	After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:500 for 18 h in 4 °C