Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Summary

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Abstract

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.

Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.

This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Introduction

Cultures of dissociated neural cells, in this case hippocampal cells, are a valuable experimental model for studying neural growth and function by providing high cell isolation and accessibility^1,2,3. This type of culture is frequently used in neuroscience, drug development, and tissue engineering due to the large amount of information that can be collected, such as rates of growth and viability, neurotoxicity, neurite outgrowth and networking, synaptic connectivity and plasticity, morphological modifications, neurites organization and wiring, etc.¹

Protocol

The use of animals in this protocol was approved by the National Animal Care and Use Committee.

NOTE: Calcium carbonated coral skeletons should be used in the crystalline form of aragonite. The coral types tested so far for neural cultures are Porites Lutea, Stylophora Pistillata, and Trachyphyllia Geoffroyi. The skeletons can be purchased whole or ground.

1. Cleaning the coral skeleton pieces

CAUTION: The following steps should be performed in a chemical hood at room temperature, because the solutions described below are hazardous and may cause burns and irritations.

Discussion

The technique presented here describes a way to improve the maintenance and functionality of neural cells in culture. This is achieved by adhering the cells to a matrix made of coral skeleton grains that nurtures the cells and promotes their growth and activity. Using this technique increases the capacity of the neural culture model to mimic the cells' environment in the brain.

The introduction of the matrix as a culture substrate has several advantages over other substrates used in classi.......

Materials

Name	Company	Catalog Number	Comments
24-well plates	Greiner	#60-662160
B-27	Gibco	#17504-044
Bovine Serum Albumin (BSA)	Sigma	#A4503
D – glucose	Sigma	#G8769
Dulbecco's Minimal Essential Eagle (DMEM)	Sigma	#D5796
Electrical sieve	Ari Levy	#3700
Fetal Bovine Serun (FBS)	Biological Industries	#04-007-1A
First Day Medium			85.1% Minimum Essential Eagle’s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.
Flasks	Greiner	#60-690160	25cm^2, Tissue culture treated
Fluoro-deoxy-uridine	Sigma	#F0503
Glass Coverslips	Menzel-Glaser	#BNCB00120RA1
H2O2	Romical	#007130-72-19	Hazardous
Ham's F-12 Nutrient Mixture	Sigma	#N4888
HANK'S solution	Sigma	#H6648
Kynurenic acid	Sigma	#K3375
L - glutamine	Sigma	#G7513
Manual strainer (40µm)	VWR	#10199-654
Minimun Essential Eagle (MEM)	Sigma	#M2279
Mortar and pestle	De-Groot	4-P090
NaClO (Sodium Hypochlorite)	Sigma	#425044	Hazardous
NaOH	Sigma	#S8045	Hazardous
Neuronal Growth Medium			45% MEM, 40% Dulbecco's modified eagle's medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.
Petri dish	Greiner	#60-628160, #60-627160	60mm, 35mm, respectively.
Poly D – Lysine	Sigma	#P7280
Smart Dentin Grinder	KometaBio	#GR101
Trypsin	Gibco	#15-090-046
Uridine	Sigma	#U3750