Protocols for Neural Cell Culture

284 Pages·1997·10.943 MB·English

by  Valérie Castellani, Jürgen Bolz (auth.), Sergey Fedoroff, Arleen Richardson (eds.)

§b~ @~ @g @= ~ ~ @~ = n y ~"' = = = =
[P[f@1l@~@~~
~@lr ~~rmlrCil~ ((:~~~
((:rm~llrm[f~
edited by
and
Sergey Fedoroff Arleen Richardson
Department of Anatomy and Cell Biology, University
of Saskatchewan, Saskatoon, Saskatchewan, Canada
Springer Science+Business Media, LLC
ISBN 978-0-89603-454-9 ISBN 978-1-4757-2586-5 (eBook)
DOI 10.1007/978-1-4757-2586-5
© 1997 Springer Science+Business Media New York
Originally published by Humana Press Inc. in 1997
For additional copies, pricing for bulk purchases, and lor information about Humana titles, contact Humana at the above address
or at any of the following numbers:
All rights reserved.
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic,
mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher.
All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily
reflect the views of the publisher.
0
This publication is printed on acid-free paper.
ANSI Z39.48-1984 (American National Standards Institute
Permanence of Paper for Printed Library Materials
Cover illustration: Figure 4C in Chapter 2, "Microexplant Cultures of the Cerebellum," by Bernard Rogister and Gustave Moonen.
Cover design by Patricia F. Cleary.
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by
Humana Press Inc., provided that the base fee of US$5.00 per copy, plus US$00.25 per page, is paid directly to the Copyright
Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy
license from the CCc, a separate system of payment has been arranged and is acceptable to Springer Science+Business Media, LLC.
The fee code for users of the Transactional Reporting Service is [0-89603-454-2/97 $5.00 + $00.251.
Library of Congress Cataloging-in-Publication Data
Protocols for Neural Cell Culture / edited by Sergey Fedoroff and Arleen Richardson-2nd ed.
p. cm.
Includes bibliographical references and index.
1. Nerve tissue-Cultures and culture media-Laboratory manuals. 2. Neurons-Growth-Laboratory manuals. I. Fedoroff,
Sergey. II. Richardson, Arleen.
[DNLM: 1. Nerve Tissue-cytology-Iaboratory manuals. 2. Tissue Culture-methods-Iaboratory manuals. 3. Neurons-cy
tology-Iaboratory manuals.WL 25 P9671
QP356.25.P765 1992
611' .0188-dc20
DNLM/DLC
for Library of Congress 92-1532
CIP
In recent years tissue culture has become one of the major methodologies
of biomedical research. It has applications in clinical diagnosis, toxicology, and
industrial biotechnology. In the future, its usefulness will undoubtedly expand
into gene and cell therapies.
Every worker in tissue culture has favorite methods for doing things. The
procedures in this book by no means represent the only ways of achieving desired
results. However, certain principles prevail. Only proven protocols that are used
routinely in well-established laboratories have been selected for the present vol
ume. Most of the protocols have been used successfully for many years in teach
ing tissue culture at the University of Saskatchewan. Four chapters deal with
culturing neurons from the central and peripheral nervous systems and include a
protocol for the study of myelination by oligodendrocytes and Schwann cells. Three
chapters cover procedures for isolating and growing glia cells, including astro
cytes, oligodendrocytes, and microglia from mice, rats, and humans. Protocols for
quantification of cells in culture and growth assays, as well as protocols for bio
logical assays for neuroactive agents, are included. Procedures are described for
the application of immunocytochemistry to the study of neural cells, the identifi
cation of antigens, the preparation of cultures for electron microscopic analysis,
and the care and use of dissecting instruments.
To assist users of this volume, we have presented protocols as they are used
in the laboratories of origin, even though this has resulted in some duplication
and differences in details of similar procedures from chapter to chapter. We
believe that good sterile techniques are much more important as preventive mea
sures against contamination than the routine use of antibiotics; therefore, we
have avoided inclusion of antibiotics as part of the culture media. We have
included general discussion, where appropriate, based on experience gained in
our tissue culture laboratory during more than forty years of research and teaching.
v
VI Preface
This volume is designed to be kept close at hand as a ready reference and a
guide to laboratory procedures. It is based on tissue culture manuals used for a
number of years at international courses on tissue culture at the University of
Saskatchewan, made possible by the generous support of the Canadian Council
of Animal Care and the Medical Research Council of Canada.
Sergey Fedoroff
Arleen Richardson
The second edition of Protocols for Neural Cell Culture adheres to the prin
ciples enunciated in the first edition, but the content has been extensively
revised and expanded. Two new chapters have been added to reflect the increased
interest in the development and differentiation of the nervous system and in the
reconstruction of its circuitry in tissue culture. One chapter deals with slice cul
tures in which the organization of the nervous system is preserved. When slice
cultures are combined with explant cultures, afferent and efferent projections
can be reconstructed. The other chapter deals with aggregating neural cell cul
tures, in which "minibrains" can form. Theses are small, uniformly sized spheres
of nervous tissue, usually having nerve cells in the center and astrocytes, oligo
dendrocytes, and microglia in the periphery. Such cultures can be used to study
neutral cell interactions in an organized milieu and for qualitative as well as
quantitative studies at biochemical and molecular levels.
A new development is the isolation and propagation of progenitor cells that
can be stimulated to differentiate into either neurons or various types of glial
cells. Two chapters deal with the isolation of neopallial cells and the stimulation
of their development into astroglia, microglia, or oligodendroglia. Such cultures
can easily be scaled-up and made highly enriched in one cell type.
Highly enriched "pure" cultures of one cell type are often required for the
study of a particular cell type or for co culturing with other cell types in order to
discern neural cell interactions. We have added a new chapter that reviews a
number of procedures for elimination of unwanted (contaminating) neutral or
nonneural cells. We have included a chapter on procedures for transfection of
cells in culture with nontoxic mutant herpes simplex virus because of the rapid
advances in gene therapy as applied to the nervous system, and because nontoxic
mutant herpes simplex virus can be used as a vector for transfer of genes to non
dividing neurons.
The practical tips gathered together in a new chapter should be useful to
those operating a tissue culture laboratory. They will be especially helpful to
vii
viii Preface to the Second Edition
beginners in the field and serve as reminders to every experienced tissue culturist.
As with the first edition, this manual is intended to be a useful, practical compan
ion in the neural cell culture laboratory, and in its improved and expanded ver
sion, should be an even more valuable resource.
Sergey Fedoroff
Arleen Richardson
The brain, spinal cord, and peripheral ganglia are the epitome of heteroge
neous tissues. They contain unique neuronal and glial cells as well as vascular
and connective tissue and other miscellaneous cell elements. Glial cells comprise
astroglial, oligo dendroglial , and microglial cell subsets in the central nervous
system or Schwann cells in the peripheral nervous system. Each glial cell subset
can be further subdivided based on in situ location and phenotypic expression.
The categorization of neuronal cell subsets is based on in situ criteria, such as
anatomical location, developmental age, electrophysiological properties, and
molecular expression repertoire. There are perhaps thousands of different neu
ronal subsets, each with their own particular array of properties and intercellular
associations. Any particular property expressed by a neural cell will be controlled
to a large extent by molecular signals within the immediate environment. Since
environmental signals often change during the life of a cell, the expressed cellu
lar characteristics may also change. The incredible cellular complexity of nervous
tissue presents unique problems to the neuroscientist attempting to understand
how individual neural cells or cell communities contribute to the proper function
ing of the nervous system. Despite the complexities, a driving force in neuro
science research is the belief that a detailed understanding of neural cells will
lead to the development of rationales for treating the unacceptable number of
developmental abnormalities, pathologies, and traumatic injuries imposed on the
human nervous system.
The in vitro approach to the study of the nervous system attempts to reduce
cellular complexity and to characterize extrinsic influences. When neural tissue
or tissue derivatives are transferred to a culture vessel, the tissue loses to vari
ous extents:
1. Contributions to its humoral environment from the remaining organ and
organism;
2. Its physiochemical connections with other neuronal, glial, peripheral, and
target cells; and
3. The anchorage that enabled it to maintain three-dimensional structure
within the tissue.
IX
x Introduction
In vitro, the culture medium and gas phase provide a new humoral environ
ment and artificial growth surfaces provide reanchorage. Although there can be
no completely compensatory contribution to the loss of specific intercellular
interactions, cultures can be set up to retain or re-establish connections similar
to the original tissue.
The culture medium and substratum can be defined to decided extents. How
ever, medium and substratum will become less defined after the cellular elements
are introduced, since neural cells will remove certain components and contribute
others. A common way to delay and minimize cellular modifications of the in vitro
environment is to provide the medium and substratum in large excess relative to
the number of neural cells.
The two major experimental advantages of using neural cultures to charac
terize the nervous system are:
1. Reduced cellular complexity; and
2. Ability to manipulate the cellular environment.
The major disadvantage, promulgated for all in vitro studies, is the uncer
tainty of physiologically relevant phenotypic expression. However, there is over
whelming evidence that neural cells often express the same properties in vivo
and in vitro. Some few examples include astroglial cell production of vimentin
and glial fibrillary acidic protein, oligodendroglial and Schwann cell elaboration
of myelin, neuronal postmitosis, neuronal expression of action potentials,
neurofilaments, growth cone formation, and axonal growth. Neuroscientists gen
erally agree that most, if not all, in vitro phenotypic expressions of neural cells
are responses to the extrinsic regulation imposed by the specific culture environ
ment and that, as such, they represent potential properties of the cells in vivo.
The advantages of reducing neural tissue complexity and manipulation of
the cellular environment in vitro have permitted scientists to predict and regu
late the behavior of specific neural cells in vivo. The purification, characteriza
tion, and cloning of neurotrophic factors (NTFs) depended almost entirely on the
use of in vitro techniques. NTFs enhance the survival and growth of specific sets
of cultured neuronal cells. By administering NTFs or anti-NTF antibodies in situ,
investigators have demonstrated that specific neurons require NTFs for their
survival, maturation, and maintenance.
In practical terms, the following considerations are important in using neu
ral cultures:
1. Source of neural tissue: animal species, animal age, anatomical site of tissue;
2. Type of culture: organ, slice, explant (fragment), disaggregate, reaggregate,
cell line;