Cell Culture

639 Pages·1979·11.082 MB·English

by  Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Ira H. Pastan

Preface
Many of the problems that have caught the interest and imagination of
biochemists are studied best with cultured cells. We offer in this volume,
in a format familiar to investigators in biochemistry, the general tech-
niques necessary for working with cells in culture and illustrate such
general methods with specific examples from the large variety of cells that
have been cultivated.
The tools and methods for cell culture are presented in Part I. Part II
provides a group of specialized techniques that are useful for many of the
applications that biochemists and other investigators with their widely
different approaches may require. Part III is concerned with specific
methods for specific cell types that have been chosen to represent the
wide range of cells that may now be prepared.
There is some duplication in the presentations. For example, portions
of certain methods are repeated in one or another form both in Part I and
Part III. We believe that this repetition is necessary to convey faithfully to
the reader a complete method of proven effectiveness. Additionally, we
hope that a heuristic effect will be achieved that will enable investigators
unfamiliar with cell culture to assess what is available and to predict what
might be most suitable for their own purposes.
MAILLIW B. YBOKAJ
ARI H. NATSAP
iiix
Contributors to Volume LVIII
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
DLANOR T. ACTON (17, 18), Department of TREBOR B. TONEPMAC (25), Department of
Microbiology and the Diabetes Research Neurobiology, Harvard Medical School,
and Training Center, University of Boston, Massachusetts 51120
Alabama in Birmingham, University Sta- WILLIAM ELSILRAC (54), Salk ehTInstitute,
tion, Birmingham, Alabama 49253 P.O. Box 1809, San Diego, California
DOLPH O. ADAMS (43), Department of 21129
Pathology, Duke Medical Center, P. COFF1NO (19), Departments of Medicine
Durham, North Carolina 01772 and Microbiology, University of -rofila C
W. FRENCH NOSREDNA (44), Laboratory of nia, San Francisco, California 34149
Molecular Hematology, National Heart, LEwis L. CORIELL (3), Institute for Medical
Lung, and Blood Institutes, National In- Research, Copewood Street, Camden,
stitutes of Health, Bethesda, Maryland New Jersey 30180
41002 TREBOR T. DELL'ORco (1), Biochemical -iD
ASAKUST ASHIHARA (20), Department of vision, ehT S. R. Noble Foundation,
Pathology, Shiga Medical College, Route ,1 Ardmore, Oklahoma 10437
Moriyama-cho, Moriyama City, Shiga OREIPMAIG DI ACROYAM (24), Department
524, Japan of Microbiology, University of Illinois at
W. EMMETT BARKLEY (4), Building ,31 the Medical Center, Chicago, Illinois
Room 2E47, National Institutes of 08606
Health, Bethesda, Maryland 41002 WILLIAM H. J. SALGUOD (1,10), Department
PAUL A. DATSRAB (17), Department of Mi- of Anatomy, Tufts School University of
crobiology and Diabetes Research the and Medicine, Boston, Massachusetts 51120
Center, Training University of Alabama in ENIREHTAC DUFF (27), Genetics Depart-
Birmingham, University Station, Birming- ment and Research Institute, ehT Hospi-
ham, Alabama 49253 tal for Sick Children, Toronto, Ontario,
RENATO BASERGA (20), Department of adanaC
Pathology and Fels Research Institute, TREBOR M. FRIEDMAN (23), Laboratory of
Temple University School of Medicine, Experimental Pathology, National Insti-
Philadelphia, Pennsylvania 04191 tute of Arthritis, Metabolism, and Diges-
MARK M. BASHOR (9), Letterman Army tive Diseases, National Institutes of
Institute of Research, Presidio of San Health, Bethesda, Maryland 41002
Francisco, San : Francisco, California T. V. NANHSlRKALAPOG (44), Laboratory of
92149 Molecular Hematology, National Heart,
YBLEHS L. BERGER (42), Section of Cellular Lung, and Blood Institutes, National In-
and Molecular Physiology, Laboratory of stitutes of Health, Bethesda, Maryland
Pathophysiology, National Cancer Insti- 41002
tute, National Institutes of Health, J. W. GRAY (19), Biomedical Sciences,
Bethesda, Maryland 41002 Lawrence Livermore Laboratory, Univer-
JANE NIETSNETTOB (6), Department of -iB sity of California, P. .O Box 808, Liver-
ology, University of California, San -iD more, California 34149
ego, La Jolla, California 39029 ECIRUAM GREEN (36), Institute for Molecu-
NOEL BOUCK (24), Department of -iM ral Virology, St. Louis University
crobiology, University of Illinois at the School of Medicine, St. Louis, Missouri
Medical Center, Chicago, Illinois 08606 01136
ix
X CONTRIBUTORS TO VOLUME LVIII
JEFFREY GRUBB (38), Department of IRWIN R. GREBSGINOK (45), Department of
Pediatrics, Division of Medical Genetics, Biology, University of Virginia, Char-
Washington University School of Medi- lottesville, Virginia 10922
cine, St. Louis, Missouri 01136 CHING-Ju LAI (34), Laboratory of DNA
P. M. GULLINO (14), Laboratory of National Institute, Viruses, Tumor Cancer
Pathophysiology, National Cancer Insti- National Institutes of Health, Bethesda,
tute, National Institutes of Health, Maryland 41002
Bethesda, Maryland 41002 H. L. LEFFERT (47), ehT Salklnstitute, Post
DRAHCIR G. HAM (5), Department of Mo- Office Box 1809, San Diego, California
lecular, Cellular, and Developmental Bi- 21129
ology, University of Colorado, Boulder, DAVID W. LEVINE (15), ehT Cell Culture
Colorado 90308 Center, Massachusetts Institute of -hceT
EDWARD HAWROT (53), Department of nology, Cambridge, Massachusetts 93120
Neurobiology, Harvard Medical School, JAMES A. MCATEER (10), .W Alton Jones
Boston, Massachusetts 51120 Science Cell Center, dlO Barn Road, Lake
HAYASHI IZUMI (6), Department of Biology, Placid, New York 64921
University of California, San Diego, La NOD B: McCLURE (6), Department of -loiB
Jolla, California 39029 ogy, University of California, San Diego,
W. FRED HINt (39), Department of En- La Jolla, California 39029
tomology, Ohio State University, Colum- DLAREG J. MCGARRITV (2, 37), Institute for
bus, Ohio 01234 Medical Research, Copewood Street,
ITARAHB HUKKU (13), The Child Research Camden, New Jersey 30180
Center of Michigan, Children's Hospital ECALLAW L. MCKEEHAN (5), Department
of Michigan, Detroit, Michigan 10284 of Molecular, Cellular, and Develop-
ERIC HUNTER (32), Department of Mi- mental Biology, University of Colorado,
crobiology, ehT Medical Center, Univer- Boulder, Colorado 90308
sity of Alabama ni Birmingham, Birm- WILLIAM F. McLIMANS (16), Roswell Park
ingham, Alabama 49253 Memorial Cancer Institute, New York
NORAHS HUTCHINGS (6), Department of Bi- State Department of Health, Buffalo,
ology, University of California, San -iD New York 36241
ego, La Jolla, California 39029 HIDEO MASUI (6), Department of Biology,
ROGER H. KENNETT (28), Department of University of California, San Diego, La
Human Genetics, The Human Genetics Jolla, California 39029
Cell Center, University of Pennsylvania JENNIE MATHER (6), Department of Biol-
School of Medicine, Philadelphia, ogy, University of California, San Diego,
Pennsylvania 40191 La Jolla, California 39029
EGROEG KHOURY (34), Laboratory of DNA T. MORAN (47), ehT Salk Institute, Post
Viruses, National Institute, Tumor Cancer Office Box 1809, San Diego, California
National Institutes of Health, Bethesda, 21129
Maryland 41002 TOSHIO MURASHIGE (41), Department of
MICHAEL NURSSGALK (50), Departments of Plant Science, University of California,
Research Surgical and Biological Chemis- Riverside, California 12529
try, Children's Hospital Medical Center, IKUYAGUS OHASA (6), Department of -loiB
Harvard Medical School, Boston, Massa- ogy, University of California, San Diego,
chusetts 51120 La Jolla, California 39029
R. A. KNAZEK (14), Laboratory of ARI NATSAP (30), Room 37,Na- Building B27,
Pathophysiology, National Cancer Insti- tional Institutes of Health, Bethesda,
tute, National Institutes of Health, Maryland 41002
Bethesda, Maryland 41002 MANFORD K. PATTERSON, JR. (11), ehT
K. S. KOCH (47), ehT Salk Institute, Post Samuel Roberts Noble Foundation, Route
Office Box 1809, San Diego, California One, Ardmore, Oklahoma 10437
21129 PAUL H. NOSRETTAP (53), Department of
CONTRIBUTORS TO VOLUME LVIII xi
Neurobiology, Harvard Medical School, tute, National Institutes of Health,
Boston, Massachusetts 51120 Bethesda, Maryland 41002
JOHN PAWELEK (51), Department of Der- SEUNG-IL SHIN (31), Department of Genet-
matology, Yale University, School of ics, Albert Einstein College of Medicine,
Medicine, New Haven, Connecticut Bronx, New York 16401
01560 WILLIAM F. SIMPSON (13), The Child Re-
D. NAMLREP (7), School of Pharmacy, Uni- search Center of Michigan, Children's
versity of Wisconsin, Madison, Wisconsin Hospital of Michigan, Detroit, Michigan
60735 10284
WARD D. ,NOSRETEP JR. (13), ehT ChildRe- MAILLIW S. YLS (38), Department of
search Center of Michigan, Children's Pediatrics, Division of Medical Genetics,
Hospital of Michigan, Detroit, Michigan Washington University School of Medi-
10284 cine, St. Louis, Missouri 01136
LOLA C. M. REID (12, 21), Department of RALPH E. SMIT(H3 3), Departments of Mi-
Molecular Pharmacology and Liver Re- crobiology and Immunology, Duke Uni-
search Center, Departments of Medicine versity Medical Center, Durham, North
and Biochemistry, Albert Einstein College Carolina 01772
of Medicine, Bronx, New York 16401 NEHCTERG H. STEIN (22), Department of
NHOJ F. REYNOLDS (41), Department of Molecular, Cellular, and Developmental
Plant Science, University of California, Biology, University of Colorado, Boulder,
Riverside, Riverside, California Colorado 90308
ANGIE RIZZlNO (6), Department of Biology, NEMRA H. ,NAIJHSAT JR. (46), Laboratory of
University of California, San Diego, La Toxicology, Harvard School of Public
Jolla, California 39029 Health, and Department of Pharmacology,
SOCRAM ROJKIND (21), Department of Mo- Harvard Medical School, Boston, Massa-
lecular Pharmacology and Liver Re- chusetts 51120
search Center, Departments of Medi- YRAM TAUB (49), ehT Department of Biol-
cine and Biochemistry, Albert Einstein ogy, The John Muir College, University of
College of Medicine, Bronx, New York California, San Diego, La Jolla, Cali-
16401 fornia 39029
ALBERT W. RUESINK (29), Department of WILLIAM G. THILLY (15), Genetic Tox-
Biology, Indiana University, Jordan Hall icology Group, Department of Nutrition
,831 Bloomington, Indiana 10474 and Food Science, Massachusetts Insti-
MILTON H. JR. SAIER, (49), ehT Department tute of Technology, Cambridge, Massa-
of Biology, ehT John Muir College, Uni- chusetts 93120
versity of California, San Diego, La E. BRAD THOMPSON (48), Laboratory of
Jolla, California 39029 Biochemistry, National Cancer Institute,
NODROG SA(T6O) , Department of Biology, National Institutes of Health, Bethesda,
University of California, San Diego, La Maryland 41002
Jolla, California 39029 YRRAL I. NOSPMOHT (26), Biomedical Sci-
DRANREB P. SCHIMMER (52), Banting and ences Division L-452, Lawrence Liver-
Best Department of Medical Research, more Laboratory, University of -rofilaC
University of Toronto, Toronto, Ontario nia, Livermore, California 05549
M5G IL6, Canada EGROEG J. TODARO (35), Laboratory of
DAVID SCHUBERT (54), ehT Salk Institute, Viral Carcinogenesis, National Cancer
P.O. Box 1809, San Diego, California Institute, National Institutes of Health,
21129 Bethesda, Maryland 41002
GINETTE ORERRES (6), Department of -i B M, WILLIAMS (47), ehT Salk Institute, Post
ology, University of California, San -iD O~ce Box 1809, San Diego, California
ego, La Jolla, California 39029 21129
SELRAHC J. SHERR (35), Laboratory of lariV KIM S. WISE (18), Department of -iM
Carcinogenesis, National Cancer Insti- Research and Diabetes the and crobiology,
xii CONTRIBUTORS TO VOLUME LVIII
Center, University Training of Alabama ni partment and Research Institute, ehT
Birmingham, Station, University Birming- Hospital for Sick Children, Toronto, -nO
ham, Alabam 49a25 3 tario, Canada
WILLIAM S. M. WOLD (36), Institute for REEN WO (6), Department of Biology, Uni-
Molecular Virology, St. Louis Univer- versity of California, San Diego, La Jolla,
sity School of Medicine, St. Louis, Mis- California 39029
souri 01136 DNILASOR YKSVEHSINAY (22), Department
KEN WOLF (8, 40), National Fisheries of Molecular, Cellular and Develop-
Center-Leetown, Fish and Wildlife Ser- mental Biology, University of Colorado,
vice, Department of the Interior, Route Boulder, Colorado 90308
3, Box 41, Kearneyville, West Virginia TREBOR K. ZWERNER (17, 18), Department
03452 of Microbiology and the Diabetes Re-
DRAHCIR WOLFE (6), Department of Biol- search and Center, Training University of
ogy, University of California, San Diego, Alabama in Birmingham, University Sta-
La Jolla, California 39029 tion, Birmingham, Alaba 4m92a5 3
RONALD G. WORTON (27), Genetics De-
3
[1] ASPECTS OF A TISSUE CULTURE LABORATORY
[1 ] Physical Aspects of a Tissue Culture Laboratory
yB WILLIAM H. J. SALGUOD and TREBOR T. DELL'ORCO
I. Introduction
The material included in this section is intended to present the basic
requirements necessary for the introduction of cell and tissue culture
techniques into a biochemistry laboratory. The information will be pre-
sented as the space and equipment needs for performing the routine oper-
ations that are necessary for cell culture production regardless of the size
of the proposed facility. These will be considered under four headings:
.1 Cleaning and sterilization facilities
2. Media preparation and storage facilities
3. Work area for aseptic manipulation of cell cultures
4. Equipment for routine cell maintenance
These four topics are generally applicable to any type of proposed cell
culture; however, this presentation deals exclusively with the culture of
mammalian cells. While it will offer a suitable starting point, certain
modifications will be necessary for the cultivation of cells from other
sources, such as invertebrates and plants. More detailed information on
the requirements for these systems can be obtained in recently published
reviews. 2,1
Regardless of the cell system to be employed, the scope of the labora-
tory facilities will depend largely upon the role planned for cell culture
procedures in the individual investigative program. When only a minor
role is planned, a minimum of space will be dedicated to cell production
and support facilities. When a more active role is anticipated, however,
space requirements will be increased and more elaborate facilities may be
deemed necessary. Thus, the facilities could all be compressed into one
laboratory or separated into individual laboratories each performing only
one function. Whether or not a major involvement is planned, another
factor to be considered in overall space and equipmentr equirements is the
type of investigations that will be done. For example, cells grown for the
harvest of a biological product, such as a hormone, or for the purification
of a particular enzyme would require large quantities of cells and the
necessary space and equipment requirements for mass culture
o. L. grobmaG dna L. .R ,retteW "Plant eussiT erutluC Methods." eiriarP .geR Lab., .taN
.seR .cnuoC Can., ,nootaksaS .5791 eeS this also emulov .]14[
2 .K ,hcsoromaraM ed., "Invertebrate eussiT :erutluC hcraeseR ".snoitacilppA cimedacA
,sserP weN York, .6791 eeS this also emulov .]93[
Copyright ~ 1979 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. LVIII All rights of reproduction in any form reserved.
ISBN 0-12-181958-2
4 BASIC METHODS [1]
capabilities. In contrast, most routine biochemical procedures, such as
enzyme assays, can be performed with relatively little cellular material
and proportionately less space and equipment. Therefore, the types of
specialized equipment that are necessary for particular programs can be
predetermined with some degree of accuracy.
Although some of the facilities needed for an adequate cell culture
laboratory can be used for nothing else, much of the required facilities and
equipment need not be dedicated exclusively to this purpose. By sharing
equipment through collaborative efforts, the initial investment necessary
to begin a tissue culture laboratory can be reduced. Central services and
already available resources such as sterility testing, glassware washing,
and animal handling areas should be utilized whenever possible. Also, the
shared use of ancillary equipment, e.g., microscopes, pH meters, cen-
trifuges, etc., within the same laboratory or with other laboratories should
be considered. However, certain precautions, to be detailed later, must be
taken when the sharing of facilities is contemplated.
It is hoped that this article will cover most of the main requirements
for setting up and running a functional cell culture laboratory. Additional
information concerning not only laboratory set-up but also detailed cell
culture techniques can be found in several well-written books, a-6 These
texts should be referred to before introducing cell and tissue culture
technology into any laboratory.
II. Cleaning and Sterilization Facilities
Although this area of a cell culture laboratory remains critically impor-
tant, some of the impact of poor laboratory practices has been lost in
recent years due to the ready availability of sterile, disposable labware
and commercially prepared media and reagents. With the exception of
very small operations in which it is financially feasible to purchase all
materials in a prepackaged, disposable form, at least some cleaning and
sterilization of glassware is necessary in almost every laboratory. Be-
cause of this and because cells in culture can be nutritionally fastidious,
investigators should be aware of the care that must be taken in the proper
handling of glassware which not only comes into direct contact with the
z .p .F and Jr. Kruse, .M Patterson, K. Jr., Methods Culture: 'Tissue eds., ' dna Applications."
Press, Academic New York, .3791
4 j. Paul, "Cell Culture," Tissue and ht5 London, and Edinburgh ed. Churchill-Livingstone,
.5791
5 ..RC Parker, "Methods of Culture," Tissue dr3 ed. York, (Hoeber), New Harper .1691
and Penso G. .D in Cultures "Tissue Balducci, lacigoloiB Amsterdam, Research." Elsevier,
.3691
[1] ASPECTS OF A TISSUE CULTURE LABORATORY 5
cells but also with such things as pipettes which are used to transfer
culture media. This is of particular importance in connection with toxic
substances introduced into glassware by normal processing. While
everyone is aware of the problems associated with microbial contamina-
tion, little thought may be given to toxic organic products introduced
during the manufacture of certain items, inorganic residues from deter-
gent washes, or contamination by metal ions sloughed from pipes. There-
fore, proper procedures for cleaning and sterilization of glassware should
be carefully followed; several such procedures are available in the litera-
ture. 7-~''3 Also it is recommended that glassware used in cell culture pro-
cedures be employed exclusively for this purpose and not mixed with
glassware used for other purposes. This includes not only the culture
vessels themselves but flasks, pipettes, and other miscellaneous items.
This precaution insures that all material used in culturing techniques has
been subjected to the same vigorous cleaning and that diffficult-to-remove
reagents do not contaminate the culture systems.
A complete separation of the cleaning facilities from the preparation
and the aseptic areas is the ideal situation; however, because of space
limitations, the preparation area can be combined with the cleaning area if
the glassware is not routinely contaminated with viruses or bacteria. If at
all possible, the aseptic areas should be maintained in a location isolated
from the cleaning area. The general size of the cleanup area is largely
dependent upon the quantity of material to be handled, but a laboratory of
001 to 051 ft 2 will accommodate the maximum amount of equipment that
would be needed. In laboratories where acid cleaning is to be employed,5.7
a fume hood with sufficient ventilation and safety features should be in-
corporated into the overall design.
In general the layout of the laboratory will be determined by the loca-
tion of the sink. The sources of hot and cold tap water will dictate the
placement of the washing equipment, i.e., decontamination and soaking
buckets, water purification system, and pipette washer. If the volume of
glassware is sufficiently large, a built-in glass washer would be advantage-
ous. Several commercially available models are acceptable; however, an
adequate supply of purified water is necessary for the final rinses. Water
of suitable purity for these final rinses can be obtained by a single glass
distillation, demineralization, or reverse osmosis. The choice of purifica-
tion method depends on such factors as the condition of the untreated
source water, the quantity of water needed, and the amount of space
available for the necessary equipment. It may be practical to employ a
single water purification system for all laboratory needs if the system is
r F. M. Price and K. K. Sanford, Tissue Cult. Assoc. Man. 2, 379 (1976).
6 BASIC METHODS [1]
capable of producing ultrapure, reagent-grade water. Such systems are
discussed in greater detail in Section III which deals with the media prep-
aration area of the laboratory.
After final rinsing, glassware is ready for drying and prepared for
sterilization. Large, bulky items may be drained and dried at room tem-
perature on a drying rack. Most glassware is dried at elevated temper-
atures in a drying oven. It is also convenient to use a specially designed
dryer for pipettes since large numbers of pipettes are frequently used in
cell culture procedures. While the drying ovens and the sterilizing equip-
ment may be located in a separate room or area, we have found it more
convenient for the drying apparatus to be located close to the washing
area. After drying, all glass vessels should be covered with paper or
aluminum foil and stored in a covered area to prevent dust accumulation.
Pipettes should be plugged with cotton and immediately stored in draw-
ers. For larger operations an apparatus for automatically plugging pipettes
is available. Adequate bench and storage space needs to be allotted for
the handling of glassware; a -01 to 15-foot bench area with overhead and
under-counter storage is sufficient for most medium-size laboratories.
Since glassware is seldom being washed and prepared for sterilization at
the same time, this bench area can be used for both functions.
Sterilization of glassware and other materials can be accomplished by
either dry heat with a sterilizing oven or with moist heat by autoclaving.
Both pieces of equipment are necessary, and their size and subsequent
location in the laboratory design depends upon the projected amount of
use. Because of obvious problems with heat and ventilation, it would be
better to locate this equipment in a separate room which is readily acces-
sible to the cleanup area. If smaller units are suitable for the intended
traffic, they can be placed in the same room as the washing facilities. All
sterilizing units should be equipped with temperature-recording charts to
maintain a complete record of sterilizing time and temperature. Because
of loading and/or air circulation problems within any unit, certain articles
in any one load may not reach the desired temperature. It is, therefore, a
good practice to label each article with a heat-sensitive indicator tape
which changes when the proper temperature has been reached and main-
tained for the proper time.
Almost all glassware, except that containing rubber tubing connec-
tions, may be sterilized by dry heat. The method also is used for material
such as silicone grease which cannot be effectively sterilized by moist
heat. However, dry heat sterilization is time-consuming and more difficult
to control even when a forced-air circulation system maintains uniform
conditions within the oven. Because of this, most sterilization procedures
are carded out with moist heat by autoclaving.
[1] ASPECTS OF A TISSUE CULTURE LABORATORY 7
Since autoclaving is the most commonly used method, a word of cau-
tion is necessary about the quality of steam used to supply the autoclave.
When the steam is heavily contaminated with impurities, they settle on
the surfaces during autoclaving and the advantages of careful washing and
rinsing procedures are lost. This may be a major problem where larger,
shared facilities are supplied by house steam. Whatever the situation,
however, it is recommended that any autoclave using house steam be
equipped with a filtering device to remove contaminating material. A
better solution to the problem is to use an autoclave that has provisions
for its own steam generation. The water for such a unit can be obtained
from a purified source thereby eliminating the contaminants at the source.
In addition to the major items of equipment mentioned in this section,
several minor ones have proven useful and should be considered when
outfitting the cleaning and sterilizing facilities. These include carts to
facilitate the transfer of articles between the different areas of the labora-
tory. These are almost a necessity when the different functional units are
very widely separated. Also, provisions should be made for disposal con-
tainers in the cleanup area. Ideally, these should be closed containers
which would serve as receptacles for used disposable labware, wrappings
from sterilized items, and the like. Other items are pipette jars for soaking
pipettes before washing, a liquid detergent dispenser, and an ultrasonic
cleaning bath for hard-to-clean glassware.
III. Media Preparation and Storage Facilities
As with the other functional units described in this article, it would be
ideal if a separate area were set aside exclusively for media and reagent
preparation. If laboratory space is available, a 001 to 051 ft 2 room would
be adequate to handle the equipment and to provide the bench space for
the necessary operations. However, as noted in the previous section, this
area can be conveniently combined with that designated for cleanup and
sterilization. Although media and other reagents can be purchased as
sterile, ready-to-use material, most investigators formulate at least part of
what they use. The operations involved in preparing any reagent for use in
cell culture are extremely critical, and several things, including a suitable
water source, high-quality chemicals, good filtration equipment, and
proper storage facilities, are essential for the successful maintenance of
cell populations in vitro.
The major component of media and other reagents for the propagation
of cells in culture is water. Although completely chemically defined media
are not yet possible, it is necessary to know within reasonable limits what