Culture of Animal Cells: A Manual of Basic Technique 5th Edition

566 Pages·2005·8.28 MB·English

by  R. Ian Freshney

Contents

Introduction 1
Trainng Programs 11
Biology of Cultures Cells 31
Laboratory design and Layout 43
Equipment 55
Aseptic Technique 73
Safety, Bioethics, and Validation 87
Culture Vessels and Substrates 105
Defined Media and Supplements 115
Serum-Free Media 129
Preparation and Sterilization 145
Primary Culture 175
Subculture and Cell Lines 199
Cloning and Selection 217
Cell Separation 237
Characterization 247
Differentiation 281
Transformation and 291
Immortalization
Contamination 307
Cryopreservation 321
Quantitation 335
Cytotoxicity 359
Culture of Specific Cell Types 375
Culture of Tumor Cells 421
Organotypic Culture 435
Scale-Up 451
Specialized Techniques 467
Problem Solving 503
In Conclusion 515
Appendix
CHAPTER 1
Introduction
1.1 HISTORICALBACKGROUND continuoushumancellline,HeLa[Geyetal.,1952];thiswas
subsequentlyclonedbyPuck[PuckandMarcus,1955]when
the concept of an X-irradiated feeder layer was introduced
Tissue culture was first devised at the beginning of the
into cloning. Tissue culture became more widely used at
twentiethcentury[Harrison,1907;Carrel,1912](Table1.1)
this time because of the introduction of antibiotics, which
as a method for studying the behavior of animal cells free
facilitated long-term cell line propagation although many
of systemic variations that might arise invivo both during
peoplewere already warning againstcontinuoususe andthe
normal homeostasis and under the stress of an experiment.
associated risk of harboring cryptic, or antibiotic-resistant,
As the name implies, the technique was elaborated first
contaminations[Parker,1961].The1950swerealsotheyears
with undisaggregated fragments of tissue, and growth was
of the development of defined media [Morgan etal., 1950;
restricted to the migration of cells from the tissue fragment,
Parker etal., 1954; Eagle, 1955, 1959; Waymouth, 1959],
withoccasionalmitosesintheoutgrowth.Ascultureofcells
whichledultimatelytothedevelopmentofserum-freemedia
from such primary explants of tissue dominated the field for
[Ham,1963,1965](seeSection10.6).
more than 50years [Fischer, 1925; Parker, 1961], it is not
surprisingthatthename‘‘tissueculture’’hasremainedinuse
as a generic term despite the fact that most of the explosive
expansion in this area in the second half of the twentieth 40000
century (Fig.1.1) was made possible by the use of dispersed 35000
cellcultures.
30000
Disaggregation of explanted cells and subsequent plating
out of the dispersed cells was first demonstrated by Rous hits 25000
[Rous and Jones, 1916], although passage was more often of 20000
o.
by surgical subdivision of the culture [Fischer, Carrel, and N 15000
others] to generate what were then termed cell strains. 10000
L929 was the first cloned cell strain, isolated by capillary
5000
cloningfrommouseL-cells[Sanfordetal.,1948].Itwasnot
0
until the 1950s that trypsin became more generally used for
1960 1970 1980 1990 2000
subculture, following procedures described by Dulbecco to Cumulative total Publication year
obtain passaged monolayer cultures for viral plaque assays [Fischer, 1925]
[Dulbecco, 1952], and the generation of a single cell Fig.1.1. Growth of Tissue Culture. Number of hits in PubMed
suspension by trypsinization, which facilitated the further for‘‘cellculture’’from1965. Thepre-1960figureisderivedfrom
development of single cell cloning. Gey established the first thebibliographyofFischer[1925].
CultureofAnimalCells:AManualofBasicTechnique,FifthEdition,byR.IanFreshney
Copyright2005JohnWiley&Sons,Inc.
1
2 CULTUREOFANIMALCELLS
TABLE1.1. KeyEventsintheDevelopmentofCellandTissueCulture
Date Event Reference
1907 Frogembryonervefiberoutgrowthinvitro Harrison,1907
1912 Explantsofchickconnectivetissue;heartmusclecontractilefor Carrel,1912;Burrows,1912
2–3months
1916 Trypsinizationandsubcultureofexplants Rous&Jones,1916
1920s/30s Subcultureoffibroblasticcelllines Carrel&Ebeling,1923
1925–1926 Differentiationinvitroinorganculture Strangeways&Fell,1925,1926
1940s Introductionofuseofantibioticsintissueculture Keilova,1948;Cruikshank&Lowbury,1952
1943 EstablishmentoftheL-cellmousefibroblast;firstcontinuouscellline Earleetal.,1943
1948 CloningoftheL-cell Sanfordetal.,1948
1949 Growthofvirusincellculture Endersetal.,1949
1952 Useoftrypsinforgenerationofreplicatesubcultures Dulbecco,1952
Virusplaqueassay Dulbecco,1952
1952–1955 Establishmentthefirsthumancellline,HeLa,fromacervical Geyetal.,1952
carcinoma,
1952 Nucleartransplantation seeBriggs&King,1960
1954 Fibroblastcontactinhibitionofcellmotility Abercrombie&Heaysman,1954
Salkpoliovaccinegrowninmonkeykidneycells seeGriffiths,1991
1955 CloningofHeLaonahomologousfeederlayer Puck&Marcus,1955
Developmentofdefinedmedia Eagle,1955,1959
Requirementofdefinedmediaforserumgrowthfactors Sanfordetal.,1955;Harris,1959
Late1950s Realizationofimportanceofmycoplasma(PPLO)infection Corielletal.,1958;Rothblat&Morton,
1959;Nelson,1960
1961 Definitionoffinitelifespanofnormalhumancells Hayflick&Moorhead,1961
Cellfusion–somaticcellhybridization Sorieul&Ephrussi,1961
1962 EstablishmentandtransformationofBHK21 Macpherson&Stoker,1962
Maintenanceofdifferentiation(pituitary&adrenaltumors) Buonassisietal.,1962;Yasamuraetal.,
1966;Sato&Yasumura,1966
1963 3T3cells&spontaneoustransformation Todaro&Green,1963
1964 Pluripotencyofembryonalstemcells Kleinsmith&Pierce,1964
Selectionoftransformedcellsinagar Macpherson&Montagnier,1964
1964–1969 Rabies,RubellavaccinesinWI-38humanlungfibroblasts Wiktoretal.,1964;Andzaparidze,1968
1965 Serum-freecloningofChinesehamstercells Ham,1965
Heterokaryons—man–mousehybrids Harris&Watkins,1965
1966 Nervegrowthfactor Levi-Montalcini,1966
Differentiationinrathepatomas Thompsonetal.,1966
1967 Epidermalgrowthfactor Hoober&Cohen,1967
HeLacellcross-contamination Gartler,1967
Densitylimitationofcellproliferation Stoker&Rubin,1967
Lymphoblastoidcelllines Mooreetal.,1967;Gerperetal.,1969;
Milleretal.,1971
1968 Retentionofdifferentiationinculturednormalmyoblasts Yaffe,1968
Anchorage-independentcellproliferation Stokeretal.,1968
1969 Colonyformationinhematopoieticcells Metcalf,1969;seealsoMetcalf,1990
1970s Developmentoflaminar-flowcabinets seeKruseetal.,1991;Collins&Kennedy,
1999
1973 DNAtransfer,calciumphosphate Graham&VanderEb,1973
1975 Fibroblastgrowthfactor Gospodarowiczetal.,1975
Hybridomas—monoclonalantibodies Kohler&Milstein,1975
1976 Totipotencyofembryonalstemcells Illmensee&Mintz,1976
Growthfactor-supplementedserum-freemedia Hayashi&Sato,1976
1977 ConfirmationofHeLacellcross-contaminationofmanycelllines Nelson-Rees&Flandermeyer,1977
3T3feederlayerandskinculture Rheinwald&Green,1975
1978 MCDB-selective,serum-freemedia Ham&McKeehan,1978
Matrixinteractions Gospodarowiczetal.,1978b;Reid&
Rojkind,1979
Cellshapeandgrowthcontrol Folkman&Moscona,1978
CHAPTER1 INTRODUCTION 3
TABLE1.1. KeyEventsintheDevelopmentofCellandTissueCulture(Continued)
Date Event Reference
1980s Regulationofgeneexpression see,e.g.,Darnell,1982
Oncogenes,malignancy,andtransformation seeWeinberg,1989
1980 MatrixfromEHSsarcoma(laterMatrigel) Hasselletal.,1980
1983 Regulationofcellcycle Evansetal.,1983;seealsoNurse,1990
ImmortalizationbySV40 Huschtscha&Holliday,1983
1980–1987 Developmentofmanyspecializedcelllines Peehl&Ham,1980;Hammondetal.,1984;
Knedler&Ham,1987
1983 Reconstitutedskincultures Belletal.,1983
1984 Productionofrecombinanttissue-typeplasminogenactivatorin Collenetal.,1984
mammaliancells
1990s Industrial-scalecultureoftransfectedcellsforproductionof Butler,1991
biopharmaceuticals
1991 Cultureofhumanadultmesenchymalstemcells Caplan,1991
1998 Tissue-engineeredcartilage Aigneretal.,1998
1998 Cultureofhumanembryonicstemcells Thomsonetal.,1998
2000+ HumanGenomeProject:genomics,proteomics,genetic Dennisetal.,2001
deficienciesandexpressionerrors
Exploitationoftissueengineering Atala&Lanza,2002;Vunjak-Novakovic&
Freshney,2004
SeealsoPollack,1981.
Throughout this book, the term tissue culture is used as made the embryonated hen’s egg a favorite choice; but the
a generic term to include organ culture and cell culture. developmentof experimental animal husbandry, particularly
The term organ culture will alwaysimply a three-dimensional withgeneticallypurestrainsofrodents,broughtmammalsto
cultureofundisaggregatedtissue retainingsomeorall ofthe theforefrontasthefavoritematerial.Althoughchickembryo
histological features of the tissue invivo. Cell culture refers to tissuecouldprovideadiversityofcelltypesinprimaryculture,
a culture derived from dispersed cells taken from original rodenttissuehadtheadvantageofproducingcontinuouscell
tissue,fromaprimaryculture,orfromacelllineorcellstrain lines [Earle etal., 1943] and a considerable repertoire of
by enzymatic, mechanical, or chemical disaggregation. The transplantabletumors.Thedevelopmentoftransgenicmouse
termhistotypiccultureimpliesthatcellshavebeenreaggregated technology [Beddington, 1992; Peat etal., 1992], together
or grown to re-create a three-dimensional structure with with the well-established genetic background of the mouse,
tissuelikecelldensity,e.g.,bycultivationathighdensityina hasaddedfurtherimpetustotheselectionofthisanimalasa
filterwell,perfusionandovergrowthofamonolayerinaflask favoritespecies.
or dish, reaggregation in suspension over agar or in real or The demonstration that human tumors could also give
simulated zero gravity, or infiltration of a three-dimensional rise to continuous cell lines [e.g., HeLa; Gey etal., 1952]
matrix such as collagen gel. Organotypic implies the same encouraged interest in human tissue, helped later by the
procedures but recombining cells of different lineages, e.g., classic studies of Leonard Hayflick on the finite life span
epidermal keratinocytes in combined culture with dermal of cells in culture [Hayflick & Moorhead, 1961] and the
fibroblasts,inanattempttogenerateatissueequivalent. requirementofvirologistsandmoleculargeneticists to work
Harrison [1907] chose the frog as his source of tissue, withhumanmaterial.Thecultivationofhumancellsreceived
presumably because it was a cold-blooded animal, and a further stimulus when a number of different serum-free
consequently, incubation was not required. Furthermore, selectivemediaweredevelopedforspecificcelltypes,suchas
because tissue regeneration is more common in lower epidermal keratinocytes, bronchial epithelium, and vascular
vertebrates, he perhaps felt that growth was more likely to endothelium (see Section10.2.1). These formulations are
occur than with mammalian tissue. Although his technique now available commercially,although the cost remains high
initiated a new wave of interest in the cultivation of tissue relativetothecostofregularmedia.
invitro, few later workers were to follow his example in For many years, the lower vertebrates and the
the selection of species. The stimulus from medical science invertebrates were largely ignored, although unique aspects
carried future interest into warm-bloodedanimals,in which of their development (tissue regeneration in amphibians,
both normal development and pathological development metamorphosis in insects) make them attractive systems for
are closer to that found in humans. The accessibility of the study of the molecular basis of development. More
different tissues, many of which grew well in culture, recently, the needs of agriculture and pest control have
4 CULTUREOFANIMALCELLS
encouraged toxicity and virological studies in insects, and neoplasia.Thestandardizationofconditionsandcelllinesfor
developmentsin gene technologyhave suggested that insect the production and assay of viruses undoubtedly provided
cell lines with baculovirus and other vectors may be useful much impetus to the development of modern tissue culture
producer cell lines because of the possibility of inserting technology, particularly the production of large numbers
larger genomic sequences in the viral DNA and a reduced of cells suitable for biochemical analysis. This and other
risk ofpropagating humanpathogenic viruses. Furthermore, technical improvements made possible by the commercial
the economic importance of fish farming and the role of supplyofreliablemediaandseraandbythegreatercontrolof
freshwaterandmarinepollutionhavestimulatedmorestudies contaminationwithantibioticsandclean-airequipmenthave
ofnormaldevelopmentandpathogenesisinfish.Procedures madetissuecultureaccessibletoawiderangeofinterests.
forhandlingnonmammaliancellshavetendedtofollowthose An additional force of increasing weight from public
developed for mammalian cell culture, although a limited opinionhasbeentheexpressionofconcernbymanyanimal-
numberofspecializedmediaarenowcommerciallyavailable rights groups over the unnecessary use of experimental
forfishandinsectcells(seeSections27.7.1,27.7.2). animals. Although most accept the idea that some
The types of investigation that lend themselves requirement for animals will continue for preclinical trials
particularly to tissue culture are summarized in Fig.1.2: of new pharmaceuticals, there is widespread concern that
(1)intracellularactivity,e.g.,thereplicationandtranscription extensive use of animals for cosmetics development and
of deoxyribonucleic acid (DNA), protein synthesis, energy similar activities may not be morally justifiable. Hence,
metabolism,anddrugmetabolism;(2)intracellularflux,e.g., there is an ever-increasing lobby for more invitro assays,
RNA, the translocation of hormone receptor complexes the adoption of which, however, still requires their proper
and resultant signal transduction processes, and membrane validation and general acceptance. Although this seemed a
trafficking; (3)environmental interaction, e.g., nutrition, distant prospect some years ago, the introduction of more
infection, cytotoxicity, carcinogenesis, drug action, and sensitive andmore readily performed invitro assays, together
ligand–receptor interactions; (4)cell–cell interaction, e.g., withaveryrealprospectofassayingforinflammationinvitro,
morphogenesis, paracrine control, cell proliferation kinetics, has promoted an unprecedented expansion in invitro testing
metabolic cooperation, cell adhesion and motility, matrix (seeSection22.4).
interaction, and organotypic models for medical prostheses In addition to cancer research and virology, other areas
and invasion; (5)genetics, including genome analysis in of research have come to depend heavily on tissue culture
normal and pathological conditions, genetic manipulation, techniques. The introduction of cell fusion techniques (see
transformation, and immortalization; and (6)cell products Section27.9)andgeneticmanipulation[Maniatisetal.,1978;
and secretion, biotechnology, bioreactor design, product Sambrook etal., 1989; Ausubel etal., 1996] established
harvesting,anddownstreamprocessing. somatic cell genetics as a major component in the genetic
The development of cell culture owed much to the analysis of higher animals, including humans. A wide range
needs of two major branches of medical research: the oftechniquesforgeneticrecombinationnowincludesDNA
production of antiviral vaccines and the understanding of transfer[Ravid&Freshney,1998],monochromsomaltransfer
CELL PRODUCTS:
Proteomics, secretion,
biotechnology, biorector design,
product harvesting, down-
stream processing
INTRACELLULAR ACTIVITY: IMMUNOLOGY: Cell surface epitopes,
DNA transcription, protein synthesis, hybridomas, cytokines and signaling,
energy metabolism, drug metabolism, inflammation
cell cycle , differentiation, apoptosis
GENOMICS: Genetic analysis,
INTRACELLULAR transfection, infection,
FLUX: RNA processing, transformation, immortalization,
hormone receptors, senescence
metabolite flux, calcium
mobilization, signal
TISSUE ENGINEERING: Tissue
transduction, membrane
constructs, matrices and
trafficking
scaffolds, stem cell sources,
propagation, differentiation
PHARMACOLOGY: Drug
action, ligand receptor
interactions, drug metabolism, TOXICOLOGY: Infection,
drug resistance CELL-CELL INTERACTION: cytotoxicity, mutagenesis,
Morphogenesis, paracrine control, cell carcinogenesis, irritation,
proliferation kinetics, metabolic inflammation
cooperation, cell adhesion and motility,
matrix interaction, invasion
Fig.1.2. TissueCultureApplications.
CHAPTER1 INTRODUCTION 5
[Newbold & Cuthbert, 1998], and nuclear transfer [Kono, Furtherdevelopmentsintheapplicationoftissuecultureto
1997], which have been added to somatic hybridization medicalproblemshavefollowedfromthedemonstrationthat
as tools for genetic analysis and gene manipulation. DNA cultures of epidermal cells form functionally differentiated
transfer itself has spawned many techniques for the transfer sheets [Green etal., 1979] and endothelial cells may
of DNA into cultured cells, including calcium phosphate form capillaries [Folkman & Haudenschild, 1980], offering
coprecipitation, lipofection, electroporation, and retroviral possibilitiesinhomograftingandreconstructivesurgeryusing
infection(seeSection27.11). an individual’s own cells [Tuszynski etal., 1996; Gustafson
In particular, human genetics has progressed under the etal., 1998; Limat etal., 1996], particularly for severe burns
stimulus of the Human Genome Project [Baltimore, 2001], [Gobet etal., 1997; Wright etal., 1998; Vunjak-Novakovic
andthedatageneratedtherefromhaverecentlymadefeasible & Freshney, 2005] (see also Section25.3.8). With the ability
the introduction of multigene array expression analysis [Iyer to transfect normal genes into genetically deficient cells, it
etal.,1999]. hasbecomepossibletograftsuch‘‘corrected’’cellsbackinto
Tissueculturehascontributedgreatly,viathemonoclonal the patient. Transfected cultures of rat bronchial epithelium
antibody technique, to the study of immunology, already carryingtheβ-galreportergenehavebeenshowntobecome
dependent on cell culture for assay techniques and the pro- incorporated into the rat’s bronchial lining when they are
duction of hematopoietic cell lines. The insight into the introducedasanaerosolintotherespiratorytract[Rosenfeld
mechanism of action of antibodies and the reciprocal infor- etal., 1992]. Similarly, cultured satellite cells have been
mationthatthisprovidedaboutthestructureoftheepitope, shownto be incorporated intowoundedrat skeletalmuscle,
derived from monoclonal antibody techniques [Kohler & with nuclei from grafted cells appearing in mature, syncytial
Milstein, 1975], was, like the technique of cell fusion itself, myotubes[Morganetal.,1992].
aprologuetoawholenewfieldofstudiesingeneticmanip- The prospects for implanting normal cells from adult
ulation. This field has supplied much basic information on or fetal tissue-matched donors or implanting genetically
the control of gene transcription, and a vast new technol- reconstituted cells from the same patient have generated
ogy and a multibillion-dollar industry have grown out of a whole new branch of culture, that of tissue engineering
the ability to insert exploitable genes into prokaryotic and [Atala and Lanza, 2002; Vunjak-Novakovic and Freshney,
eukaryotic cells. Cell products such as human growth hor- 2005], encompassing the generation of tissue equivalents
mone, insulin, and interferon are now produced routinely by organotypic culture (see Section25.3.8), isolation and
by transfected cells, although the absence of posttranscrip- differentiationofhumanembryonalstem(ES)cellsandadult
tionalmodifications,suchasglycosylation,inbacteriasuggests totipotentstemcellssuchasmesenchymalstemcells(MSCs),
that mammalian cells may provide more suitable vehicles gene transfer, materials science, utilization of bioreactors,
[Grampp etal., 1992], particularly in light of developments and transplantation technology. The technical barriers are
inimmortalizationtechnology(seeSection18.4). steadily being overcome, bringing the ethical questions to
Other areas of major interest include the study of cell the fore. The technical feasibility of implanting normal
interactions and intracellular control mechanisms in cell fetal neurons into patients with Parkinson disease has been
differentiation and development [Jessell and Melton, 1992; demonstrated; society must now decide to what extent fetal
Ohmichi etal., 1998; Balkovetz & Lipschutz, 1999] and materialmaybeusedforthispurpose.Whereapatient’sown
attempts to analyze nervous function [Richard etal., 1998; cells can be grown and subjected to genetic reconstitution
Dunn etal., 1998; Haynes, 1999]. Progress in neurological by transfection of the normal gene—e.g., transfecting the
research has not had the benefit, however, of working with normalinsulingeneintoβ-isletcellsculturedfromdiabetics,
propagated cell linesfrom normal brain or nervoustissue, as or even transfecting other cell types such as skeletal muscle
the propagation of neurons invitro has not been possible, progenitors [Morgan etal., 1992]—it would allow the
until now, without resorting to the use of transformed cells to be incorporated into a low-turnover compartment
cells(seeSection18.4).However,developmentswithhuman and, potentially, give a long-lasting physiological benefit.
embryonalstemcellcultures[Thomsonetal.,1998;Rathjen Although the ethics of this type of approach seem less
etal., 1998; Wolf etal., 1998; Webber & Minger, 2004] contentious, the technical limitations of this approach are
suggest that this approach may provide replicating cultures stillapparent.
thatwilldifferentiateintoneurons. Invitro fertilization (IVF), developed from early
Tissueculturetechnologyhasalsobeenadoptedintomany experiments in embryo culture [see review by Edwards,
routineapplicationsinmedicineandindustry.Chromosomal 1996], is now widely used [see, e.g., Gardner and Lane,
analysis of cells derived from the womb by amniocentesis 2003] and has been accepted legally and ethically in many
(see Section27.6) can reveal genetic disorders in the unborn countries. However, another area of development raising
child, the quality of drinking water can be determined, and significantethicaldebateisthe generationofgametesinvitro
the toxic effectsofpharmaceuticalcompoundsandpotential fromthe cultureofprimordial germcellsisolatedfromtestis
environmentalpollutantscanbemeasuredincolony-forming and ovary [Dennis, 2003] or from ES cells. Oocytes have
andotherinvitroassays(seeSections22.3.1,22.3.2,22.4). been cultured from embryonic mouse ovary and implanted,
6 CULTUREOFANIMALCELLS
generating normal mice [Eppig, 1996; Obata etal., 2002], TABLE1.3. LimitationsofTissueCulture
andspermatidshavebeenculturedfromnewbornbulltestes
and co-cultured with Sertoli cells [Lee etal., 2001]. Similar Category Examples
workwithmousetestesgeneratedspermatidsthatwereused
Necessaryexpertise Sterilehandling
tofertilizemouseeggs,whichdevelopedintomature,fertile
Chemicalcontamination
adults[Marh,etal.,2003].
Microbialcontamination
Cross-contamination
Environmentalcontrol Workplace
1.2 ADVANTAGESOFTISSUECULTURE
Incubation,pHcontrol
Containmentanddisposalof
1.2.1 ControloftheEnvironment
biohazards
The two major advantages of tissue culture (Table1.2) Quantityandcost Capitalequipmentforscale-up
are control of the physiochemical environment (pH, Medium,serum
temperature, osmotic pressure, and O and CO tension), Disposableplastics
2 2
whichmaybecontrolledveryprecisely,andthephysiological Geneticinstability Heterogeneity,variability
conditions, which may be kept relatively constant, but Phenotypicinstability Dedifferentiation
Adaptation
cannot always be defined. Most cell lines still require
Selectiveovergrowth
supplementation of the medium with serum or other
Identificationofcelltype Markersnotalwaysexpressed
poorly defined constituents. These supplements are prone
Histologydifficulttorecreateand
to batch variation and contain undefined elements such as
atypical
hormonesandotherregulatorysubstances.Theidentification
Geometryandmicroenvironment
ofsomeoftheessentialcomponentsofserum(seeTable9.5),
changecytology
togetherwithabetterunderstandingoffactorsregulatingcell
proliferation (see Table10.3), has made the replacement of
serumwithdefinedconstituentsfeasible(seeSection10.4).As
not always precisely defined, yet it can be regulated and,
laboratoriesseektoexpressthenormalphenotypicproperties
as cloned matrix constituents become available, may still be
of cells invitro, the role of the extracellular matrix becomes
fullydefined.
increasingly important. Currently, that role is similar to the
use of serum—that is, the matrix is often necessary, but
1.2.2 CharacterizationandHomogeneity
ofSample
TABLE1.2. AdvantagesofTissueCulture Tissue samples are invariably heterogeneous. Repli-
cates—even from one tissue—vary in their constituent cell
Category Advantages types. After one or two passages, cultured cell lines assume
a homogeneous (or at least uniform) constitution, as the
Physico-chemical ControlofpH,temperature,
cells are randomly mixed at each transfer and the selective
environment osmolality,dissolvedgases
pressureofthecultureconditionstendstoproduceahomo-
Physiologicalconditions Controlofhormoneandnutrient
geneous culture of the most vigorous cell type. Hence, at
concentrations
Microenvironment Regulationofmatrix,cell–cell eachsubculture,replicatesamplesareidenticaltoeachother,
interaction,gaseousdiffusion and the characteristics of the line may be perpetuated over
Celllinehomogeneity Availabilityofselectivemedia, several generations, or even indefinitely if the cell line is
cloning storedinliquidnitrogen.Becauseexperimentalreplicatesare
Characterization Cytologyandimmunostainingare virtually identical, the need for statistical analysis of variance
easilyperformed isreduced.
Preservation Canbestoredinliquidnitrogen
The availability of stringent tests for cell line identity
Validation& Origin,history,puritycanbe
(Chapter15) and contamination (Chapter18) means that
accreditation authenticatedandrecorded
preserved stocks may be validated for future research and
Replicatesand Quantitationiseasy
commercialuse.
variability
Reagentsaving Reducedvolumes,directaccess
tocells,lowercost 1.2.3 Economy,Scale,andMechanization
ControlofC×T Abilitytodefinedose, Cultures may be exposed directly to a reagent at a lower,
concentration(C),andtime(T) and defined, concentration and with direct access to the
Mechanization Availablewithmicrotitrationand cell.Consequently,lessreagentisrequiredthanforinjection
robotics invivo, where 90% is lost by excretion and distribution to
Reductionofanimaluse Cytotoxicityandscreeningof
tissues other than those under study. Screening tests with
pharmaceutics,cosmetics,etc.
many variables and replicates are cheaper, and the legal,
CHAPTER1 INTRODUCTION 7
moral, and ethical questions of animal experimentation are of the phenotypic characteristics typical of the tissue from
avoided.Newdevelopmentsinmultiwellplatesandrobotics which the cells had been isolated. This effect was blamed
alsohaveintroducedsignificanteconomiesintimeandscale. on dedifferentiation, a process assumed to be the reversal of
differentiation, but later shown to be largely due to the
1.2.4 InVitroModelingofInVivoConditions overgrowthofundifferentiatedcellsofthesameoradifferent
Perfusion techniques allow the delivery of specific lineage. The development of serum-free selective media
experimental compounds to be regulated in concentration, (see Section10.2.1) has now made the isolation of specific
duration of exposure (see Table1.2), and metabolic state. lineagesquitepossible,anditcanbeseenthat,undertheright
The developmentof histotypic and organotypic modelsalso conditions,manyofthedifferentiatedpropertiesofthesecells
increasestheaccuracyofinvivomodeling. mayberestored(seeSection17.7).
1.3.4 OriginofCells
1.3 LIMITATIONS
If differentiated properties are lost, for whatever reason, it
is difficult to relate the cultured cells to functional cells in
1.3.1 Expertise
the tissue from which they were derived. Stable markersare
Culture techniques must be carried out under strict aseptic
requiredforcharacterizationofthecells(seeSection16.1);in
conditions, because animal cells grow much less rapidly
addition,thecultureconditionsmayneedtobemodifiedso
than many of the common contaminants, such as bacteria,
thatthesemarkersareexpressed(seeSections3.4.1,17.7).
molds,andyeasts.Furthermore,unlikemicroorganisms,cells
from multicellularanimalsdo not normallyexist in isolation
1.3.5 Instability
and, consequently, are not able to sustain an independent
Instability is a major problem with many continuous cell
existence without the provision of a complex environment
lines, resulting from their unstable aneuploid chromosomal
simulatingbloodplasmaorinterstitialfluid.Theseconditions
constitution.Evenwithshort-termculturesofuntransformed
imply a level of skill and understanding on the part of the
cells, heterogeneity in growth rate and the capacity to
operatorinordertoappreciatetherequirementsofthesystem
differentiate within the population can produce variability
and to diagnose problems as they arise (Table1.3; see also
fromonepassagetothenext(seeSection18.3).
Chapter28).Also,caremustbetakentoavoidtherecurrent
problem of cross-contamination and to authenticate stocks.
Hence, tissue culture should not be undertaken casually to
1.4 MAJORDIFFERENCESINVITRO
runoneortwoexperiments.
1.3.2 Quantity Many of the differences in cell behavior between cultured
Amajorlimitationofcellcultureistheexpenditureofeffort cellsandtheircounterpartsinvivostemfromthedissociation
and materials that goes into the production of relatively of cells from a three-dimensional geometry and their
little tissue. A realistic maximum per batch for most small propagation on a two-dimensional substrate. Specific cell
laboratories (with two or three people doing tissue culture) interactions characteristic of the histology of the tissue are
might be 1–10g of cells. With a little more effort and lost, and, as the cells spread out, become mobile, and, in
the facilities of a larger laboratory, 10–100g is possible; many cases, start to proliferate, so the growth fraction of
above100gimpliesindustrialpilot-plantscale,alevelthatis the cell populationincreases. When a cell line forms, it may
beyondthereachofmostlaboratoriesbutisnotimpossibleif represent only one or two cell types, and many heterotypic
special facilities are provided, when kilogram quantities can cell–cellinteractionsarelost.
begenerated. The culture environment also lacks the several systemic
The cost of producing cells in culture is about 10 times components involved in homeostatic regulation invivo,
thatofusinganimaltissue.Consequently,iflargeamountsof principally those of the nervous and endocrine systems.
tissue(>10g)arerequired,thereasonsforprovidingthemby Without this control, cellular metabolism may be more
culturemustbeverycompelling.Forsmalleramountsoftissue constant invitro than invivo, but may not be truly
(∼10g), the costs are more readily absorbed into routine representativeofthetissuefromwhichthecellswerederived.
expenditure,butitisalwaysworthconsideringwhetherassays Recognitionofthisfacthasledtotheinclusionofanumber
or preparative procedures can be scaled down. Semimicro- of different hormones in culture media (see Sections10.4.2,
ormicroscaleassayscanoftenbequicker,becauseofreduced 10.4.3),anditseemslikelythatthistrendwillcontinue.
manipulation times, volumes, centrifuge times, etc., and are Energy metabolism invitro occurs largely by glycolysis,
frequentlymorereadilyautomated(seeSections21.8,22.3.5). andalthoughthecitric acidcycleisstill functional,itplaysa
lesserrole.
1.3.3 DedifferentiationandSelection It is not difficult to find many more differences between
When the first major advancesin cell line propagation were theenvironmentalconditionsofacellinvitroandinvivo(see
achieved in the 1950s, many workers observed the loss Section22.2),andthisdisparityhasoftenledtotissueculture
8 CULTUREOFANIMALCELLS
being regarded in a rather skeptical light. Still, although favors the retention of a spherical or three-dimensional
the existence of such differences cannot be denied, many shape. (2)In primary explant culture, a fragment of tissue is
specialized functions are expressed in culture, and as long as placed at a glass (or plastic)–liquid interface, where, after
the limits of the model are appreciated, tissue culture can attachment, migration is promoted in the plane of the solid
becomeaveryvaluabletool. substrate (see Section12.3.1). (3)Cell culture implies that the
tissue, or outgrowth from the primary explant, is dispersed
(mechanicallyorenzymatically)intoacellsuspension,which
1.5 TYPESOFTISSUECULTURE may then be cultured as an adherent monolayer on a solid
substrate or as a suspension in the culture medium (see
There are three main methods of initiating a culture Sections12.3,13.7).
[Schaeffer, 1990; see Appendix IV, Fig.1.3, and Table1.4): Because of the retention of cell interactions found in the
(1)Organcultureimpliesthatthearchitecturecharacteristicof tissuefromwhichtheculturewasderived,organculturestend
the tissue invivo is retained, at least in part, in the culture to retain the differentiated properties of that tissue. They do
(see Section25.2). Toward this end, the tissue is cultured notgrowrapidly(cellproliferationislimitedtotheperiphery
at the liquid–gas interface (on a raft, grid, or gel), which of the explant and is restricted mainly to embryonic tissue)
ORGAN EXPLANT DISSOCIATED ORGANOTYPIC
CULTURE CULTURE CELL CULTURE CULTURE
Tissue at gas-liquid Tissue at solid-liquid Disaggregated tissue; Different cells co-cultured with
interface; histological interface; cells migrate cells form monolayer or without matrix; organotypic
structure maintained to form outgrowth at solid-liquid interface structure recreated
Fig.1.3. TypesofTissueCulture.
TABLE1.4. PropertiesofDifferentTypesofCulture
Category Organculture Explant Cellculture
Source Embryonicorgans,adulttissue Tissuefragments Disaggregatedtissue,primary
fragments culture,propagatedcellline
Effort High Moderate Low
Characterization Easy,histology Cytologyandmarkers Biochemical,molecular,
immunological,andcytological
assays
Histology Informative Difficult Notapplicable
Biochemicaldifferentiation Possible Heterogeneous Lost,butmaybereinduced
Propagation Notpossible Possiblefromoutgrowth Standardprocedure
Replicatesampling, Highintersamplevariation Highintersamplevariation Lowintersamplevariation
reproducibility,
homogeneity
Quantitation Difficult Difficult Easy;manytechniquesavailable