Cell Culture

427 Pages·1990·14.805 MB·English

by  P. Michael Conn (Eds.)

Methods in Neurosciences
Edited by
P. Michael Conn
Department of Pharmacology
The University of Iowa
College of Medicine
Iowa City, Iowa
Volume 2
Cell Culture
ACADEMIC PRESS, INC.
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Front cover photograph (paperback edition only) courtesy of Sean Murphy,
Department of Pharmacology, The University of Iowa College of Medicine,
Iowa City, Iowa.
This book is printed on acid-free paper. @
Copyright © 1990 by Academic Press, Inc.
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90 91 92 93 9 8 7 6 5 4 3 2 1
Contributors to Volume 2
Article numbers are in parentheses following the names of contributors. Affiliations listed are
current.
RUBEN ADLER (10), Retinal Degenerations Research Center, Retinal Degen­
erations Laboratories, Wilmer Eye Institute, The Johns Hopkins University,
School of Medicine, Baltimore, Maryland 21205
EFRAIN C. AZMITIA (17), Department of Biology, New York University,
New York, New York 10003
BARBARA A. BENNETT (21), Department of Physiology and Pharmacology,
The Bowman Gray School of Medicine, Wake Forest University, Winston-
Salem, North Carolina 27103
MICHAEL E. BERENS (16), Brain Tumor Research Center, Department of
Neurosurgery, School of Medicine, University of California, San Francisco,
San Francisco, California 94143
ROLF BJERKVIG (15), Department of Pathology, The Gade Institute, Univer­
sity of Bergen, Haukeland Hospital, N-5021 Bergen, Norway
IRA B. BLACK (1), Division of Developmental Neurology, Department of
Neurology and Neuroscience, Cornell University Medical College and The
New York Hospital, New York, New York 10021
LIANE BOLOGA (11), Institute of Anatomy, University of Fribourg, CH-1700
Fribourg, Switzerland
E. PETER BOSCH (4), Division of Neurochemistry and Neurobiology, De­
partment of Neurology, The University of Iowa, College of Medicine, Iowa
City, Iowa 52242
JANE E. BOTTENSTEIN (5), Marine Biomedicai Institute and Department of
Human Biological Chemistry & Genetics, The University of Texas Medical
Branch at Galveston, Galveston, Texas 77550
P. MICHAEL CONN (13), Department of Pharmacology, The University of
Iowa, College of Medicine, Iowa City, Iowa 52242
STANLEY M. CRAIN (6), Departments of Neuroscience and of Physiology
and Biophysics, Albert Einstein College of Medicine of Yeshiva University,
Bronx, New York 10461
IX
X CONTRIBUTORS TO VOLUME 2
CHERYL F. DREYFUS (1), Division of Developmental Neurology, Depart­
ment of Neurology and Neuroscience, Cornell University Medical College
and The New York Hospital, New York, New York 10021
GARY R. DUTTON (7), Department of Pharmacology, The University of
Iowa, College of Medicine, Iowa City, Iowa 52242
LAURA FACCI (2), Department of CNS Research, Fidia Research Laborato­
ries, 35031 Abano Terme, Italy
ANNIE FAIVRE-BAUMAN (23), Groupe de Neuroendocrinologie Cellulaire et
Moléculaire URA 1115, Collège de France, Centre National de la Recherche
Scientifique (CNRS), 75231 Paris Cedex 05, France
MUHAMMAD FAROOQ (12), Department of Neurology, Albert Einstein Col­
lege of Medicine of Yeshiva University, Bronx, New York 10461
PAUL C. GOLDSMITH (16), Reproductive Endocrinology Center, Depart­
ment of Obstetrics, Gynecology and Reproductive Sciences, School of Med­
icine, University of California, San Francisco, San Francisco, California
94143
JAMES R. HANSEN (13), Department of Pediatrics, The University of Iowa,
College of Medicine, Iowa City, Iowa 52242
NORBERT HERSCHKOWITZ (11), Department of Pediatrics, University of
Bern, CH-3010 Bern, Switzerland
SAMUEL F. HUNTER (5), Department of Pharmacology & Toxicology, The
University of Texas Medical Branch at Galveston, Galveston, Texas
77550
MARCUS JACOBSON (25), Department of Anatomy, University of Utah Medi­
cal School, Salt Lake City, Utah 84132
RICHARD M. KRIS (22), Rorer Biotechnology Incorporated, King of Prussia,
Pennsylvania 19406
KIN Y A KURIYAMA (8), Department of Pharmacology, Kyoto Prefectural
University of Medicine, Kawaramachi-Hirokoij, Kamikyo-Ku, Kyoto 602,
Japan
OLE DIDRIK LAERUM (15), Department of Pathology, The Gade Institute,
University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway
MEI LEE (22), Department of Biochemistry, The George Washington Uni­
versity Medical Center, Washington, D.C. 20037
ALBERTA LEON (2), Department of CNS Research, Fidia Research Labora­
tories, 35031 Abano Terme, Italy
CONTRIBUTORS TO VOLUME 2 XI
JON E. LEVINE (20), Section of Biological Sciences, Department of Neurobi-
ology and Physiology, Northwestern University, Evanston, Illinois 60208
RAMON LIM (4), Division of Neurochemistry and Neurobiology, Depart­
ment of Neurology, The University of Iowa, College of Medicine, Iowa
City, Iowa 52242
DARIA MILANI (2), Department of CNS Research, Fidia Research Laborato­
ries, 35031 Abano Terme, Italy
TERRY W. MOODY (22), Department of Biochemistry, The George Washing­
ton University Medical Center, Washington, D.C. 20037
MARIANA MORRIS (21), Department of Physiology and Pharmacology, The
Bowman Gray School of Medicine, Wake Forest University, Winston-
Salem, North Carolina 27103
SEAN MURPHY (3), Department of Pharmacology, The University of Iowa,
College of Medicine, Iowa City, Iowa 52242
WILLIAM T. NORTON (12), Department of Neurology, Albert Einstein Col­
lege of Medicine of Yeshiva University, Bronx, New York 10461
SEITARO OHKUMA (8), Department of Pharmacology, Kyoto Prefectural
University of Medicine, Kawaramachi-Hirokoij, Kamikyo-Ku, Kyoto 602,
Japan
JOHN S. RAMSDELL (19), Division of Molecular and Cellular Endocrinology,
Department of Anatomy and Cell Biology, Medical University of South
Carolina, Charleston, South Carolina 29425
RUSSELL P. SANETO (9), Department of Cell Biology and Anatomy, Oregon
Health Sciences University, Portland, Oregon 97201
ABRAHAM SHAHAR (14), Section of Electron Microscopy, Department of
Virology, Israel Institute for Biological Research (IIBR), 70450 Ness-Ziona,
Israel
STEPHEN D. SKAPER (2), Department of CNS Research, Fidia Research
Laboratories, 35031 Abano Terme, Italy
JULIE STALE Y (22), Department of Biochemistry, The George Washington
University Medical Center, Washington, D.C. 20037
GÜNTHER S. STENT (24), Department of Molecular & Cell Biology, Univer­
sity of California, Berkeley, Berkeley, California 94720
FRANK J. STROBL (20), Section of Biological Sciences, Department of Neu­
robiology and Physiology, Northwestern University, Evanston, Illinois
60208
XÜ CONTRIBUTORS TO VOLUME 2
DUNCAN K. STUART (24), Department of Molecular & Cell Biology, Univer­
sity of California, Berkeley, Berkeley, California 94720
DAVID K. SUNDBERG (21), Department of Physiology and Pharmacology,
The Bowman Gray School of Medicine, Wake Forest University, Winston-
Salem, North Carolina 27103
ANDRÉE TIXIER-VIDAL (23), Groupe de Neuroendocrinologie Cellulaire et
Moléculaire URA 1115, Collège de France, Centre National de la Recherche
Scientifique (CNRS), 75231 Paris Cedex 05, France
GINO TOFFANO (2), Fidia Research Laboratories, 35031 Abano Terme, Italy
C. DOMINIQUE TORAN-ALLERAND (18), Department of Anatomy and Cell
Biology and Center for Reproductive Sciences, College of Physicians and
Surgeons of Columbia University, New York, New York 10032
STEVEN A. TORRENCE (24), Department of Molecular & Cell Biology, Uni­
versity of California, Berkeley, Berkeley, California 94720
It is appropriate that an early volume in the Methods in Neurosciences series
deal with cell culture techniques. The ability to assess the response of neural
cells (and targets of neurally derived substances) is an important goal of
studies in the neurosciences. The means to measure responses in a chemi­
cally defined medium (and away from the influences of other tissues) has
been a positive feature of the use of cell cultures and is the major reason that
it is so central to this field.
In describing methods, the authors have been encouraged to identify
shortcuts not included in earlier publications and to include comparisons
among similar techniques in order that the reader be able to make appropri­
ate decisions when selecting methods. Much of the material presented is
published for the first time.
General techniques (Section I: General Approaches to Preparation of Cell
Cultures) that can be adapted to many different systems as well as means for
the purification of cells for culture (Section II: Purification of Cell Types)
have been included as have procedures appropriate for large-scale isolation
(Section III: Bulk Isolation), for functionally esoteric uses (Section IV: Spe­
cial Approaches) which may be easily adapted to other systems, for assess­
ing differentiated function (Section V: Functional Assessment), and for trac­
ing lineage (Section VI: Cell Lineage).
Although some topics could not be covered due to space limitations or
prior commitments of some of the invited authors, the sampling offered is
broad and sufficiently referenced to enable the reader to identify methods
that will be readily adaptable to many systems.
Particular thanks go to the authors for making time in their schedules to
participate, for meeting deadlines, and for maintaining high standards of
quality. Also, thanks are due to the staff of Academic Press for their help
and for the timely publication of this material.
P. MICHAEL CONN
Xlll
[1] Multiple Approaches to Brain Culture
Cheryl F. Dreyfus and Ira B. Black
Introduction
It may appear paradoxical to employ culture approaches in the brain sci­
ences, which, after all, are ultimately concerned with the genesis of menta­
tion and behavior. How can in vitro models, singularly lacking in behavioral
output, inform study of the brain? The mammalian central nervous system
(CNS), composed of heterogeneous cells and systems, and exhibiting multi­
ple levels of complexity and organization, would seem to defy such simplis­
tic, reductionistic avenues. In fact, however, the profound complexity and
heterogeneity demand the simplifying strategies that typify cell and tissue
culture. The judicious, parallel use of in vivo and in vitro techniques has
provided insights unobtainable through either method alone. The resolution
provided by culture models is now beginning to indicate how cell and molec­
ular biology underlies systems and behavioral function.
Culture approaches are particularly powerful in this regard, since different
systems may be developed to approximate an enormous range of complexi­
ties. Preparations vary from extremely complex, interacting systems of neu­
rons to the culture of single, identified cells. Indeed, a prime problem facing
the neuroscientist concerns the choice of an optimal model to address the
particular problem under investigation. To help the uninitiated, as well as the
experienced neuroscientist in the choice of in vitro systems, we describe
both complex and relatively simple culture systems. However, the field of
cell culture is so vast that we can hardly provide even a cursory survey of all
available approaches. Rather, we have adopted a different strategy in the
present brief review.
We describe exemplars of the most complex, heterogeneous, "life-like"
cultures, and those representing the most simplified and highly defined. Our
goal is to present the spectrum, allowing the reader to choose the most
appropriate system. Initially, we characterize complex expiant cultures that
largely preserve cytoarchitectonic, synaptic, and gross anatomic relations.
We proceed to indicate how complex systems can then be dissected into
component parts, allowing rigorous characterization of individual elements.
Finally, we describe reconstitution methods that allow investigation of the
interaction of defined cellular and molecular components. This treatment
tacitly acknowledges that each particular method has both strengths and
limitations, and that no single method is wholly adequate.
Methods in Neurosciences, Volume 2
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 3
4 I GENERAL APPROACHES TO PREPARATION OF CELL CULTURES
This seemingly self-evident truism bears special emphasis in the area of
neural culture. The more simplified the culture system, the further from the
in vivo CNS situation, the greater the potential for irrelevance, and the
greater the interpretative difficulty. The more complex the culture system,
the greater the approximation of the in vivo situation, and the more difficult
the control of cellular and molecular variables. The tension between control­
lability and relevance and simplicity and complexity should not be con­
founded with issues of rigor. Different systems are simply suited to address
different questions, and no one system addresses them all. It is apparent that
a number of approaches have to be employed simultaneously to study most
problems in neurobiology.
Nevertheless, this brief review must, of necessity, omit description of
entire areas of neural culture. Space restrictions do not permit description of
neoplastic cell lines, transformed lines, various reaggregate techniques and
the whole area of in vivo culture, or grafting, which are important examples
of approaches that have provided critical insights. The reader is referred to a
number of reviews of these areas (1-10).
Organotypic Expiant Culture
One of the earliest techniques developed is also one of the most complex,
the culture of intact fragments of neural tissue. Maximow used the term
"organotypic" in 1925, which emphasizes the degree of preservation of
normal, in vivo, cellular, and systems relations. In many instances, synaptic
interactions are maintained, allowing analysis of interactions of very differ­
ent cellular elements that are simply inaccessible in vivo. Moreover, ex-
plants are dynamic, growing areas of brain that undergo synaptogenesis and
myelinogenesis, and that generate spontaneous action potentials (11-17).
These properties result in the development of neural systems that exhibit
appropriate synaptic specificity ultrastructurally, and normal chemical and
pharmacological characteristics. These astounding observations attest to the
power of the technique, and simultaneously constitute a remarkable set of
scientific insights. Simply stated, many aspects of specificity, selectivity,
and sensitivity, the hallmarks of neural function, can be reproduced in cul­
ture. Explantation provides accessibility for molecular, cellular, and sys­
tems analysis. Several specific examples may help illustrate the potential for
examination of diverse areas of the nervous system.
Cerebellar expiants develop normal architectural arrangements of cortical
laminae and deep nuclei (13, 18-20), permitting detailed characterization of
interactions in this critically important model of motor control and mapping
functions. Expiants of hypothalamus, the governor of vegetative life, exhibit
[1] MULTIPLE APPROACHES TO BRAIN CULTURE 5
the development of appropriate peptidergic neurons (21-23), allowing de­
tailed, chronic biochemical (23, 24) and electrophysiological (25) ap­
proaches. The locus coeruleus (26-33), which appears to subserve arousal
and attention, and the substantia nigra (34-36), which degenerates in Parkin­
son's disease, express catecholaminergic phenotypic traits in culture, per­
mitting analysis of underlying molecular genetic mechanisms. In one final
example, the hippocampus, which has been implicated in memory mecha­
nisms, displays functional, synaptic networks in culture (11) and pyramidal
and granule cells develop normal dendritic arborizations (13). Explantation
of virtually any area of the CNS allows definition of the cell and systems
biology of a variety of specific neural functions.
It is apparent that expiant culture is particularly well-suited for study of
interacting systems and nuclei within the local environment, but divorced
from the confounding variables of the whole organism. The preservation of
normal cellular relationships approximates aspects of in vivo conditions in
an accessible, simplified form. Mechanisms underlying systems develop­
ment and function can be conveniently examined in the expiant environ­
ment, and extensive investigation indicates that expiants faithfully reflect
growth in vivo. Nevertheless, fidelity to the in vivo situation is purchased at
the expense of complexity: frequently, elucidation of specific cellular and
molecular mechanisms requires the use of simplified, dissociated cell cul­
ture, as detailed below.
Growth and Maintenance: General Considerations
A number of specialized techniques have been developed to grow and main­
tain organotypic cultures. One approach that we have successfully em­
ployed involves modification of the original methods of Maximow (37). This
technique permits the growth and differentiation of selected brain regions for
extended periods of at least 4-5 weeks. Prolonged viability allows observa­
tion of the lengthy processes of development and maintenance of neural
systems. For example, we have used this approach to document the devel­
opment of neurons from areas as diverse as the substantia nigra (36), basal
forebrain (38), and striatum (39). In particular, we have defined the differen­
tiation of catecholaminergic neurons of the locus coeruleus (28, 32). In or­
ganotypic culture the neurons express an array of noradrenergic phenotypic
traits, including the high-affinity norepinephrine uptake system, the biosyn-
thetic enzymes tyrosine monooxygenase and dopamine ß-monooxygenase,
and the transmitter itself, norepinephrine. Moreover, the culture preparation
has permitted manipulations that have begun to define the molecular mecha­
nisms governing the development of normal phenotypic expression. The