Neuron and glial cell culture protocols
(Curr Protoc Neurosci)
1: Curr Protoc Neurosci. 2006 Aug;Chapter 3:Unit 3.19.
Rodent sensory neuron culture and analysis.
Case Western Reserve University, Cleveland, Ohio, USA.
Sensory neurons have proven very useful for analysis of neuronal differentiation
in vivo and in vitro. Their utility for in vitro work is based on the fact that
sensory neurons are relatively easy to isolate in large numbers and are amenable
to manipulations in culture. Lumbar ganglia are usually used because their
location in the caudal nervous system means they are the least differentiated at
any developmental stage, allowing the analysis of relatively undifferentiated
cells. Rodent sensory ganglia from embryonic to adult stages can be dissected
effectively and maintained in serum-free medium or in coculture with other cells
or factors. This unit describes generation of embryonic rat lumbar dorsal root
ganglia (DRG) cultures, which form an important model system for investigating
the cellular and molecular mechanisms that regulate neuronal differentiation.
Adult DRG can also be successfully cultured, with a few modifications of the
PMID: 18428634 [PubMed - in process]
2: Curr Protoc Neurosci. 2004 May;Chapter 3:Unit 3.2.
Long-term culture of hippocampal neurons.
Centro de Investigaciones Biológicas, Censejo Superior de Investigaciones
Cientificas, Madrid, Spain.
In culture, hippocampal cells can develop to express neuronal antigens and
acquire mature neuronal morphologies, including axons, complex dendritic trees,
and synapses that are electrophysiologically active. This system is suitable for
studying neuronal differentiation and other events, such as synaptogenesis. It is
also a valuable model for investigating synaptic plasticity and exploring the
mechanisms of neuronal degeneration. This unit provides a protocol for culturing
neurons prepared from embryonic (E-18) rat or mouse hippocampus, but could also
be used to grow neurons from embryonic cortex, olfactory bulb, striatum, or
spinal cord. A second method is included for preparing neuronal cultures from
embryos with different genotypes, such as those from transgenic mice. Also
described is the preparation of polyornithine- and fibronectin-coated coverslips,
which are highly adhesive and promote neurite outgrowth, for use in the culture
PMID: 18428599 [PubMed - in process]
3: Curr Protoc Neurosci. 2001 May;Chapter 6:Unit 6.5.
Acute isolation of neurons from the mature mammalian central nervous system.
Kay AR, Krupa DJ.
University of Iowa, Iowa City, Iowa, USA.
The acute dissociation procedure provides a simple means of isolating neurons
from the mature mammalian central nervous system. The method was primarily
devised to isolate neurons for patch-clamp electrophysiology. It may also prove
useful for single-cell PCR, immunocytochemistry, sorting of fluorescently labeled
cells, or long-term tissue culture of mature neurons. Dissociation is brought
about by a combination of proteolysis and an ionic environment that encourages
breakdown of the tissue. The method allows the isolation of neurons free of glial
ensheathments in as little as 45 min after the sacrifice of the animal. Neurons
so isolated lose fine dendritic branches, although the structure proximal to the
cell body is often maintained, allowing identification of the morphological type
of the neuron. The preparation has the following advantages: (1) the neurons are
fully differentiated; (2) the cells are electronically compact, which improves
the fidelity of the voltage clamp; (3) the cells are removed from the influence
of surrounding cells; and (4) neurons can be isolated from small, circumscribed
loci within the adult central nervous system.
PMID: 18428515 [PubMed - in process]
4: Curr Protoc Neurosci. 2001 May;Chapter 6:Unit 6.11.
Preparation and maintenance of organotypic slice cultures of CNS tissue.
Gähwiler BH, Thompson SM, Muller D.
University of Zurich, Zurich, Switzerland.
Organotypic slice cultures are the in vitro method of choice for applications
requiring long-term survival of the preparation and a high degree of cellular
differentiation and organization resembling that of the original tissue.
Long-term survival is achieved by culturing slices at the air/liquid interface,
either by continuously rotating the preparation (roller-tube cultures) or by
culturing them on semiporous membranes (stationary interface cultures). Both
culture techniques yield nerve cells which are highly differentiated in terms of
their morphological and physiological characteristics. Because most of these
cultures are prepared from 1-week-old postnatal animals, in which the cellular
and tissue organization is already relatively advanced, the original
cytoarchitecture is often remarkably well maintained. Moreover, the presence of a
full complement of glial and nerve cells is thought to provide a microenvironment
facilitating differentiation of neurons. Slice culture also offers unique
advantages for recording from pairs of cells, as a consequence of the high degree
of connectivity between nerve cells. Recently, new applications have emerged such
as the cultivation of slices from knock-out animals with limited postnatal
survival time or alteration of gene expression by viral vectors.
PMID: 18428510 [PubMed - in process]
5: Curr Protoc Neurosci. 2001 May;Chapter 3:Unit 3.7.
Immortalizing central nervous system cells.
University of Miami School of Medicine, Miami, Florida, USA.
This unit presents methods for isolating clonal, neural-derived cell lines. One
approach for isolating such neural cell lines involves a replication-deficient
retrovirus encoding a specific oncogene and a selectable marker which are used to
infect dissociated CNS cells dissected at a developmental stage at which the cell
population of interest has not undergone its terminal mitotic division. Also
presented is a method for cloning by limiting dilution, which may be necessary to
obtain a pure population of cells. Following growth under appropriate selection
conditions, clones are isolated and tested for their ability to differentiate
with the desired phenotypic properties. A method is also provided for coating
tissue culture dishes, which is necessary for successful culture of CNS neurons.
PMID: 18428471 [PubMed - in process]
6: Curr Protoc Neurosci. 2001 May;Chapter 3:Unit 3.3.
Culture of substantia nigra neurons.
National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
Primary cultures of nigral tissue are widely used as a model system to assay
effects of trophic and toxic agents on dopaminergic neurons. Cultured
dopaminergic neurons have been successfully transplanted in animals and led to
behavioral improvement in animal models of Parkinson's disease. Cell cultures
have also been used to study the development of substantia nigra, allowing
investigators to identify early inductive events important for nigral development
and to study dopaminergic differentiation and target innervation. This unit
provides simple and reliable culture protocols for these applications. The first
approach presented is the preparation of dissociated nigral cell cultures, the
later steps of which can be used as a simple and efficient assay for testing
growth factors. A second approach is the preparation of free-floating roller tube
cultures, which may be used as a tool for neural transplantation and to study
more complex developmental events. A third approach is the production of
organotypic cultures using chicken plasma as a matrix. Organotypic cultures can
maintain the in vivo cytoarchitecture of a host region in vitro.
PMID: 18428467 [PubMed - in process]
How to grow primary neurons (Neuroscience forum)