Contents · 1916 Trypsinization and subculture of explants Rous & Jones, 1916 1920s/30s Subculture of ﬁbroblastic cell lines Carrel & Ebeling, 1923 1925–1926 Differentiation in

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 Immortalization

291

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 HISTORICAL BACKGROUND

Tissue culture was first devised at the beginning of thetwentieth century [Harrison, 1907; Carrel, 1912] (Table 1.1)as a method for studying the behavior of animal cells freeof systemic variations that might arise in vivo both duringnormal homeostasis and under the stress of an experiment.As the name implies, the technique was elaborated firstwith undisaggregated fragments of tissue, and growth wasrestricted to the migration of cells from the tissue fragment,with occasional mitoses in the outgrowth. As culture of cellsfrom such primary explants of tissue dominated the field formore than 50 years [Fischer, 1925; Parker, 1961], it is notsurprising that the name ‘‘tissue culture’’ has remained in useas a generic term despite the fact that most of the explosiveexpansion in this area in the second half of the twentiethcentury (Fig. 1.1) was made possible by the use of dispersedcell cultures.

Disaggregation of explanted cells and subsequent platingout of the dispersed cells was first demonstrated by Rous[Rous and Jones, 1916], although passage was more oftenby surgical subdivision of the culture [Fischer, Carrel, andothers] to generate what were then termed cell strains.L929 was the first cloned cell strain, isolated by capillarycloning from mouse L-cells [Sanford et al., 1948]. It was notuntil the 1950s that trypsin became more generally used forsubculture, following procedures described by Dulbecco toobtain passaged monolayer cultures for viral plaque assays[Dulbecco, 1952], and the generation of a single cellsuspension by trypsinization, which facilitated the furtherdevelopment of single cell cloning. Gey established the first

continuous human cell line, HeLa [Gey et al., 1952]; this wassubsequently cloned by Puck [Puck and Marcus, 1955] whenthe concept of an X-irradiated feeder layer was introducedinto cloning. Tissue culture became more widely used atthis time because of the introduction of antibiotics, whichfacilitated long-term cell line propagation although manypeople were already warning against continuous use and theassociated risk of harboring cryptic, or antibiotic-resistant,contaminations [Parker, 1961]. The 1950s were also the yearsof the development of defined media [Morgan et al., 1950;Parker et al., 1954; Eagle, 1955, 1959; Waymouth, 1959],which led ultimately to the development of serum-free media[Ham, 1963, 1965] (see Section 10.6).

1960

Cumulative total[Fischer, 1925]

1970 1980

Publication year

1990 20000

5000

10000

15000

20000

25000

30000

35000

40000

No.

of h

its

Fig. 1.1. Growth of Tissue Culture. Number of hits in PubMedfor ‘‘cell culture’’ from 1965. The pre-1960 figure is derived fromthe bibliography of Fischer [1925].

Culture of Animal Cells: A Manual of Basic Technique, Fifth Edition, by R. Ian FreshneyCopyright 2005 John Wiley & Sons, Inc.

1

2 CULTURE OF ANIMAL CELLS

TABLE 1.1. Key Events in the Development of Cell and Tissue Culture

Date Event Reference

1907 Frog embryo nerve fiber outgrowth in vitro Harrison, 19071912 Explants of chick connective tissue; heart muscle contractile for

2–3 monthsCarrel, 1912; Burrows, 1912

1916 Trypsinization and subculture of explants Rous & Jones, 19161920s/30s Subculture of fibroblastic cell lines Carrel & Ebeling, 19231925–1926 Differentiation in vitro in organ culture Strangeways & Fell, 1925, 19261940s Introduction of use of antibiotics in tissue culture Keilova, 1948; Cruikshank & Lowbury, 19521943 Establishment of the L-cell mouse fibroblast; first continuous cell line Earle et al., 19431948 Cloning of the L-cell Sanford et al., 19481949 Growth of virus in cell culture Enders et al., 19491952 Use of trypsin for generation of replicate subcultures Dulbecco, 1952

Virus plaque assay Dulbecco, 19521952–1955 Establishment the first human cell line, HeLa, from a cervical

carcinoma,Gey et al., 1952

1952 Nuclear transplantation see Briggs & King, 19601954 Fibroblast contact inhibition of cell motility Abercrombie & Heaysman, 1954

Salk polio vaccine grown in monkey kidney cells see Griffiths, 19911955 Cloning of HeLa on a homologous feeder layer Puck & Marcus, 1955

Development of defined media Eagle, 1955, 1959Requirement of defined media for serum growth factors Sanford et al., 1955; Harris, 1959

Late 1950s Realization of importance of mycoplasma (PPLO) infection Coriell et al., 1958; Rothblat & Morton,1959; Nelson, 1960

1961 Definition of finite life span of normal human cells Hayflick & Moorhead, 1961Cell fusion–somatic cell hybridization Sorieul & Ephrussi, 1961

1962 Establishment and transformation of BHK21 Macpherson & Stoker, 1962Maintenance of differentiation (pituitary & adrenal tumors) Buonassisi et al., 1962; Yasamura et al.,

1966; Sato & Yasumura, 19661963 3T3 cells & spontaneous transformation Todaro & Green, 19631964 Pluripotency of embryonal stem cells Kleinsmith & Pierce, 1964

Selection of transformed cells in agar Macpherson & Montagnier, 19641964–1969 Rabies, Rubella vaccines in WI-38 human lung fibroblasts Wiktor et al., 1964; Andzaparidze, 19681965 Serum-free cloning of Chinese hamster cells Ham, 1965

Heterokaryons—man–mouse hybrids Harris & Watkins, 19651966 Nerve growth factor Levi-Montalcini, 1966

Differentiation in rat hepatomas Thompson et al., 19661967 Epidermal growth factor Hoober & Cohen, 1967

HeLa cell cross-contamination Gartler, 1967Density limitation of cell proliferation Stoker & Rubin, 1967Lymphoblastoid cell lines Moore et al., 1967; Gerper et al., 1969;

Miller et al., 19711968 Retention of differentiation in cultured normal myoblasts Yaffe, 1968

Anchorage-independent cell proliferation Stoker et al., 19681969 Colony formation in hematopoietic cells Metcalf, 1969; see also Metcalf, 19901970s Development of laminar-flow cabinets see Kruse et al., 1991; Collins & Kennedy,

19991973 DNA transfer, calcium phosphate Graham & Van der Eb, 19731975 Fibroblast growth factor Gospodarowicz et al., 1975

Hybridomas—monoclonal antibodies Kohler & Milstein, 19751976 Totipotency of embryonal stem cells Illmensee & Mintz, 1976

Growth factor-supplemented serum-free media Hayashi & Sato, 19761977 Confirmation of HeLa cell cross-contamination of many cell lines Nelson-Rees & Flandermeyer, 1977

3T3 feeder layer and skin culture Rheinwald & Green, 19751978 MCDB-selective, serum-free media Ham & McKeehan, 1978

Matrix interactions Gospodarowicz et al., 1978b; Reid &Rojkind, 1979

Cell shape and growth control Folkman & Moscona, 1978

CHAPTER 1 INTRODUCTION 3

TABLE 1.1. Key Events in the Development of Cell and Tissue Culture (Continued)

Date Event Reference

1980s Regulation of gene expression see, e.g., Darnell, 1982Oncogenes, malignancy, and transformation see Weinberg, 1989

1980 Matrix from EHS sarcoma (later Matrigel) Hassell et al., 19801983 Regulation of cell cycle Evans et al., 1983; see also Nurse, 1990

Immortalization by SV40 Huschtscha & Holliday, 19831980–1987 Development of many specialized cell lines Peehl & Ham, 1980; Hammond et al., 1984;

Knedler & Ham, 19871983 Reconstituted skin cultures Bell et al., 19831984 Production of recombinant tissue-type plasminogen activator in

mammalian cellsCollen et al., 1984

1990s Industrial-scale culture of transfected cells for production ofbiopharmaceuticals

Butler, 1991

1991 Culture of human adult mesenchymal stem cells Caplan, 19911998 Tissue-engineered cartilage Aigner et al., 19981998 Culture of human embryonic stem cells Thomson et al., 19982000+ Human Genome Project: genomics, proteomics, genetic

deficiencies and expression errorsDennis et al., 2001

Exploitation of tissue engineering Atala & Lanza, 2002; Vunjak-Novakovic &Freshney, 2004

See also Pollack, 1981.

Throughout this book, the term tissue culture is used asa generic term to include organ culture and cell culture.The term organ culture will always imply a three-dimensionalculture of undisaggregated tissue retaining some or all of thehistological features of the tissue in vivo. Cell culture refers toa culture derived from dispersed cells taken from originaltissue, from a primary culture, or from a cell line or cell strainby enzymatic, mechanical, or chemical disaggregation. Theterm histotypic culture implies that cells have been reaggregatedor grown to re-create a three-dimensional structure withtissuelike cell density, e.g., by cultivation at high density in afilter well, perfusion and overgrowth of a monolayer in a flaskor dish, reaggregation in suspension over agar or in real orsimulated zero gravity, or infiltration of a three-dimensionalmatrix such as collagen gel. Organotypic implies the sameprocedures but recombining cells of different lineages, e.g.,epidermal keratinocytes in combined culture with dermalfibroblasts, in an attempt to generate a tissue equivalent.

Harrison [1907] chose the frog as his source of tissue,presumably because it was a cold-blooded animal, andconsequently, incubation was not required. Furthermore,because tissue regeneration is more common in lowervertebrates, he perhaps felt that growth was more likely tooccur than with mammalian tissue. Although his techniqueinitiated a new wave of interest in the cultivation of tissuein vitro, few later workers were to follow his example inthe selection of species. The stimulus from medical sciencecarried future interest into warm-blooded animals, in whichboth normal development and pathological developmentare closer to that found in humans. The accessibility ofdifferent tissues, many of which grew well in culture,

made the embryonated hen’s egg a favorite choice; but thedevelopment of experimental animal husbandry, particularlywith genetically pure strains of rodents, brought mammals tothe forefront as the favorite material. Although chick embryotissue could provide a diversity of cell types in primary culture,rodent tissue had the advantage of producing continuous celllines [Earle et al., 1943] and a considerable repertoire oftransplantable tumors. The development of transgenic mousetechnology [Beddington, 1992; Peat et al., 1992], togetherwith the well-established genetic background of the mouse,has added further impetus to the selection of this animal as afavorite species.

The demonstration that human tumors could also giverise to continuous cell lines [e.g., HeLa; Gey et al., 1952]encouraged interest in human tissue, helped later by theclassic studies of Leonard Hayflick on the finite life spanof cells in culture [Hayflick & Moorhead, 1961] and therequirement of virologists and molecular geneticists to workwith human material. The cultivation of human cells receiveda further stimulus when a number of different serum-freeselective media were developed for specific cell types, such asepidermal keratinocytes, bronchial epithelium, and vascularendothelium (see Section 10.2.1). These formulations arenow available commercially, although the cost remains highrelative to the cost of regular media.

For many years, the lower vertebrates and theinvertebrates were largely ignored, although unique aspectsof their development (tissue regeneration in amphibians,metamorphosis in insects) make them attractive systems forthe study of the molecular basis of development. Morerecently, the needs of agriculture and pest control have


encouraged toxicity and virological studies in insects, anddevelopments in gene technology have suggested that insectcell lines with baculovirus and other vectors may be usefulproducer cell lines because of the possibility of insertinglarger genomic sequences in the viral DNA and a reducedrisk of propagating human pathogenic viruses. Furthermore,the economic importance of fish farming and the role offreshwater and marine pollution have stimulated more studiesof normal development and pathogenesis in fish. Proceduresfor handling nonmammalian cells have tended to follow thosedeveloped for mammalian cell culture, although a limitednumber of specialized media are now commercially availablefor fish and insect cells (see Sections 27.7.1, 27.7.2).

The types of investigation that lend themselvesparticularly to tissue culture are summarized in Fig. 1.2:(1) intracellular activity, e.g., the replication and transcriptionof deoxyribonucleic acid (DNA), protein synthesis, energymetabolism, and drug metabolism; (2) intracellular flux, e.g.,RNA, the translocation of hormone receptor complexesand resultant signal transduction processes, and membranetrafficking; (3) environmental interaction, e.g., nutrition,infection, cytotoxicity, carcinogenesis, drug action, andligand–receptor interactions; (4) cell–cell interaction, e.g.,morphogenesis, paracrine control, cell proliferation kinetics,metabolic cooperation, cell adhesion and motility, matrixinteraction, and organotypic models for medical prosthesesand invasion; (5) genetics, including genome analysis innormal and pathological conditions, genetic manipulation,transformation, and immortalization; and (6) cell productsand secretion, biotechnology, bioreactor design, productharvesting, and downstream processing.

The development of cell culture owed much to theneeds of two major branches of medical research: theproduction of antiviral vaccines and the understanding of

neoplasia. The standardization of conditions and cell lines forthe production and assay of viruses undoubtedly providedmuch impetus to the development of modern tissue culturetechnology, particularly the production of large numbersof cells suitable for biochemical analysis. This and othertechnical improvements made possible by the commercialsupply of reliable media and sera and by the greater control ofcontamination with antibiotics and clean-air equipment havemade tissue culture accessible to a wide range of interests.

An additional force of increasing weight from publicopinion has been the expression of concern by many animal-rights groups over the unnecessary use of experimentalanimals. Although most accept the idea that somerequirement for animals will continue for preclinical trialsof new pharmaceuticals, there is widespread concern thatextensive use of animals for cosmetics development andsimilar activities may not be morally justifiable. Hence,there is an ever-increasing lobby for more in vitro assays,the adoption of which, however, still requires their propervalidation and general acceptance. Although this seemed adistant prospect some years ago, the introduction of moresensitive and more readily performed in vitro assays, togetherwith a very real prospect of assaying for inflammation in vitro,has promoted an unprecedented expansion in in vitro testing(see Section 22.4).

In addition to cancer research and virology, other areasof research have come to depend heavily on tissue culturetechniques. The introduction of cell fusion techniques (seeSection 27.9) and genetic manipulation [Maniatis et al., 1978;Sambrook et al., 1989; Ausubel et al., 1996] establishedsomatic cell genetics as a major component in the geneticanalysis of higher animals, including humans. A wide rangeof techniques for genetic recombination now includes DNAtransfer [Ravid & Freshney, 1998], monochromsomal transfer

INTRACELLULAR ACTIVITY:DNA transcription, protein synthesis, energy metabolism, drug metabolism, cell cycle, differentiation, apoptosis

INTRACELLULAR FLUX: RNA processing, hormone receptors, metabolite flux, calcium mobilization, signal transduction, membrane trafficking

PHARMACOLOGY: Drug action, ligand receptor interactions, drug metabolism, drug resistance CELL-CELL INTERACTION:

Morphogenesis, paracrine control, cell proliferation kinetics, metabolic cooperation, cell adhesion and motility, matrix interaction, invasion

GENOMICS: Genetic analysis, transfection, infection, transformation, immortalization, senescence

CELL PRODUCTS: Proteomics, secretion, biotechnology, biorector design, product harvesting, down-stream processing

TISSUE ENGINEERING: Tissue constructs, matrices and scaffolds, stem cell sources, propagation, differentiation

IMMUNOLOGY: Cell surface epitopes, hybridomas, cytokines and signaling, inflammation

TOXICOLOGY: Infection, cytotoxicity, mutagenesis, carcinogenesis, irritation, inflammation

Fig. 1.2. Tissue Culture Applications.


[Newbold & Cuthbert, 1998], and nuclear transfer [Kono,1997], which have been added to somatic hybridizationas tools for genetic analysis and gene manipulation. DNAtransfer itself has spawned many techniques for the transferof DNA into cultured cells, including calcium phosphatecoprecipitation, lipofection, electroporation, and retroviralinfection (see Section 27.11).

In particular, human genetics has progressed under thestimulus of the Human Genome Project [Baltimore, 2001],and the data generated therefrom have recently made feasiblethe introduction of multigene array expression analysis [Iyeret al., 1999].

Tissue culture has contributed greatly, via the monoclonalantibody technique, to the study of immunology, alreadydependent on cell culture for assay techniques and the pro-duction of hematopoietic cell lines. The insight into themechanism of action of antibodies and the reciprocal infor-mation that this provided about the structure of the epitope,derived from monoclonal antibody techniques [Kohler &Milstein, 1975], was, like the technique of cell fusion itself,a prologue to a whole new field of studies in genetic manip-ulation. This field has supplied much basic information onthe control of gene transcription, and a vast new technol-ogy and a multibillion-dollar industry have grown out ofthe ability to insert exploitable genes into prokaryotic andeukaryotic cells. Cell products such as human growth hor-mone, insulin, and interferon are now produced routinelyby transfected cells, although the absence of posttranscrip-tional modifications, such as glycosylation, in bacteria suggeststhat mammalian cells may provide more suitable vehicles[Grampp et al., 1992], particularly in light of developmentsin immortalization technology (see Section 18.4).

Other areas of major interest include the study of cellinteractions and intracellular control mechanisms in celldifferentiation and development [Jessell and Melton, 1992;Ohmichi et al., 1998; Balkovetz & Lipschutz, 1999] andattempts to analyze nervous function [Richard et al., 1998;Dunn et al., 1998; Haynes, 1999]. Progress in neurologicalresearch has not had the benefit, however, of working withpropagated cell lines from normal brain or nervous tissue, asthe propagation of neurons in vitro has not been possible,until now, without resorting to the use of transformedcells (see Section 18.4). However, developments with humanembryonal stem cell cultures [Thomson et al., 1998; Rathjenet al., 1998; Wolf et al., 1998; Webber & Minger, 2004]suggest that this approach may provide replicating culturesthat will differentiate into neurons.

Tissue culture technology has also been adopted into manyroutine applications in medicine and industry. Chromosomalanalysis of cells derived from the womb by amniocentesis(see Section 27.6) can reveal genetic disorders in the unbornchild, the quality of drinking water can be determined, andthe toxic effects of pharmaceutical compounds and potentialenvironmental pollutants can be measured in colony-formingand other in vitro assays (see Sections 22.3.1, 22.3.2, 22.4).

Further developments in the application of tissue culture tomedical problems have followed from the demonstration thatcultures of epidermal cells form functionally differentiatedsheets [Green et al., 1979] and endothelial cells mayform capillaries [Folkman & Haudenschild, 1980], offeringpossibilities in homografting and reconstructive surgery usingan individual’s own cells [Tuszynski et al., 1996; Gustafsonet al., 1998; Limat et al., 1996], particularly for severe burns[Gobet et al., 1997; Wright et al., 1998; Vunjak-Novakovic& Freshney, 2005] (see also Section 25.3.8). With the abilityto transfect normal genes into genetically deficient cells, ithas become possible to graft such ‘‘corrected’’ cells back intothe patient. Transfected cultures of rat bronchial epitheliumcarrying the β-gal reporter gene have been shown to becomeincorporated into the rat’s bronchial lining when they areintroduced as an aerosol into the respiratory tract [Rosenfeldet al., 1992]. Similarly, cultured satellite cells have beenshown to be incorporated into wounded rat skeletal muscle,with nuclei from grafted cells appearing in mature, syncytialmyotubes [Morgan et al., 1992].

The prospects for implanting normal cells from adultor fetal tissue-matched donors or implanting geneticallyreconstituted cells from the same patient have generateda whole new branch of culture, that of tissue engineering[Atala and Lanza, 2002; Vunjak-Novakovic and Freshney,2005], encompassing the generation of tissue equivalentsby organotypic culture (see Section 25.3.8), isolation anddifferentiation of human embryonal stem (ES) cells and adulttotipotent stem cells such as mesenchymal stem cells (MSCs),gene transfer, materials science, utilization of bioreactors,and transplantation technology. The technical barriers aresteadily being overcome, bringing the ethical questions tothe fore. The technical feasibility of implanting normalfetal neurons into patients with Parkinson disease has beendemonstrated; society must now decide to what extent fetalmaterial may be used for this purpose. Where a patient’s owncells can be grown and subjected to genetic reconstitutionby transfection of the normal gene—e.g., transfecting thenormal insulin gene into β-islet cells cultured from diabetics,or even transfecting other cell types such as skeletal muscleprogenitors [Morgan et al., 1992]—it would allow thecells to be incorporated into a low-turnover compartmentand, potentially, give a long-lasting physiological benefit.Although the ethics of this type of approach seem lesscontentious, the technical limitations of this approach arestill apparent.

In vitro fertilization (IVF), developed from earlyexperiments in embryo culture [see review by Edwards,1996], is now widely used [see, e.g., Gardner and Lane,2003] and has been accepted legally and ethically in manycountries. However, another area of development raisingsignificant ethical debate is the generation of gametes in vitrofrom the culture of primordial germ cells isolated from testisand ovary [Dennis, 2003] or from ES cells. Oocytes havebeen cultured from embryonic mouse ovary and implanted,


generating normal mice [Eppig, 1996; Obata et al., 2002],and spermatids have been cultured from newborn bull testesand co-cultured with Sertoli cells [Lee et al., 2001]. Similarwork with mouse testes generated spermatids that were usedto fertilize mouse eggs, which developed into mature, fertileadults [Marh, et al., 2003].

1.2 ADVANTAGES OF TISSUE CULTURE

1.2.1 Control of the EnvironmentThe two major advantages of tissue culture (Table 1.2)are control of the physiochemical environment (pH,temperature, osmotic pressure, and O2 and CO2 tension),which may be controlled very precisely, and the physiologicalconditions, which may be kept relatively constant, butcannot always be defined. Most cell lines still requiresupplementation of the medium with serum or otherpoorly defined constituents. These supplements are proneto batch variation and contain undefined elements such ashormones and other regulatory substances. The identificationof some of the essential components of serum (see Table 9.5),together with a better understanding of factors regulating cellproliferation (see Table 10.3), has made the replacement ofserum with defined constituents feasible (see Section 10.4). Aslaboratories seek to express the normal phenotypic propertiesof cells in vitro, the role of the extracellular matrix becomesincreasingly important. Currently, that role is similar to theuse of serum—that is, the matrix is often necessary, but

TABLE 1.2. Advantages of Tissue Culture

Category Advantages

Physico-chemicalenvironment

Control of pH, temperature,osmolality, dissolved gases

Physiological conditions Control of hormone and nutrientconcentrations

Microenvironment Regulation of matrix, cell–cellinteraction, gaseous diffusion

Cell line homogeneity Availability of selective media,cloning

Characterization Cytology and immunostaining areeasily performed

Preservation Can be stored in liquid nitrogenValidation &

accreditationOrigin, history, purity can be

authenticated and recordedReplicates and

variabilityQuantitation is easy

Reagent saving Reduced volumes, direct accessto cells, lower cost

Control of C × T Ability to define dose,concentration (C), and time (T)

Mechanization Available with microtitration androbotics

Reduction of animal use Cytotoxicity and screening ofpharmaceutics, cosmetics, etc.

TABLE 1.3. Limitations of Tissue Culture

Category Examples

Necessary expertise Sterile handlingChemical contaminationMicrobial contaminationCross-contamination

Environmental control WorkplaceIncubation, pH controlContainment and disposal of

biohazardsQuantity and cost Capital equipment for scale-up

Medium, serumDisposable plastics

Genetic instability Heterogeneity, variabilityPhenotypic instability Dedifferentiation

AdaptationSelective overgrowth

Identification of cell type Markers not always expressedHistology difficult to recreate and

atypicalGeometry and microenvironment

change cytology

not always precisely defined, yet it can be regulated and,as cloned matrix constituents become available, may still befully defined.

1.2.2 Characterization and Homogeneityof SampleTissue samples are invariably heterogeneous. Repli-cates—even from one tissue—vary in their constituent celltypes. After one or two passages, cultured cell lines assumea homogeneous (or at least uniform) constitution, as thecells are randomly mixed at each transfer and the selectivepressure of the culture conditions tends to produce a homo-geneous culture of the most vigorous cell type. Hence, ateach subculture, replicate samples are identical to each other,and the characteristics of the line may be perpetuated overseveral generations, or even indefinitely if the cell line isstored in liquid nitrogen. Because experimental replicates arevirtually identical, the need for statistical analysis of varianceis reduced.

The availability of stringent tests for cell line identity(Chapter 15) and contamination (Chapter 18) means thatpreserved stocks may be validated for future research andcommercial use.

1.2.3 Economy, Scale, and MechanizationCultures may be exposed directly to a reagent at a lower,and defined, concentration and with direct access to thecell. Consequently, less reagent is required than for injectionin vivo, where 90% is lost by excretion and distribution totissues other than those under study. Screening tests withmany variables and replicates are cheaper, and the legal,


moral, and ethical questions of animal experimentation areavoided. New developments in multiwell plates and roboticsalso have introduced significant economies in time and scale.

1.2.4 In Vitro Modeling of In Vivo ConditionsPerfusion techniques allow the delivery of specificexperimental compounds to be regulated in concentration,duration of exposure (see Table 1.2), and metabolic state.The development of histotypic and organotypic models alsoincreases the accuracy of in vivo modeling.

1.3 LIMITATIONS

1.3.1 ExpertiseCulture techniques must be carried out under strict asepticconditions, because animal cells grow much less rapidlythan many of the common contaminants, such as bacteria,molds, and yeasts. Furthermore, unlike microorganisms, cellsfrom multicellular animals do not normally exist in isolationand, consequently, are not able to sustain an independentexistence without the provision of a complex environmentsimulating blood plasma or interstitial fluid. These conditionsimply a level of skill and understanding on the part of theoperator in order to appreciate the requirements of the systemand to diagnose problems as they arise (Table 1.3; see alsoChapter 28). Also, care must be taken to avoid the recurrentproblem of cross-contamination and to authenticate stocks.Hence, tissue culture should not be undertaken casually torun one or two experiments.

1.3.2 QuantityA major limitation of cell culture is the expenditure of effortand materials that goes into the production of relativelylittle tissue. A realistic maximum per batch for most smalllaboratories (with two or three people doing tissue culture)might be 1–10 g of cells. With a little more effort andthe facilities of a larger laboratory, 10–100 g is possible;above 100 g implies industrial pilot-plant scale, a level that isbeyond the reach of most laboratories but is not impossible ifspecial facilities are provided, when kilogram quantities canbe generated.

The cost of producing cells in culture is about 10 timesthat of using animal tissue. Consequently, if large amounts oftissue (>10 g) are required, the reasons for providing them byculture must be very compelling. For smaller amounts of tissue(∼10 g), the costs are more readily absorbed into routineexpenditure, but it is always worth considering whether assaysor preparative procedures can be scaled down. Semimicro-or microscale assays can often be quicker, because of reducedmanipulation times, volumes, centrifuge times, etc., and arefrequently more readily automated (see Sections 21.8, 22.3.5).

1.3.3 Dedifferentiation and SelectionWhen the first major advances in cell line propagation wereachieved in the 1950s, many workers observed the loss

of the phenotypic characteristics typical of the tissue fromwhich the cells had been isolated. This effect was blamedon dedifferentiation, a process assumed to be the reversal ofdifferentiation, but later shown to be largely due to theovergrowth of undifferentiated cells of the same or a differentlineage. The development of serum-free selective media(see Section 10.2.1) has now made the isolation of specificlineages quite possible, and it can be seen that, under the rightconditions, many of the differentiated properties of these cellsmay be restored (see Section 17.7).

1.3.4 Origin of CellsIf differentiated properties are lost, for whatever reason, itis difficult to relate the cultured cells to functional cells inthe tissue from which they were derived. Stable markers arerequired for characterization of the cells (see Section 16.1); inaddition, the culture conditions may need to be modified sothat these markers are expressed (see Sections 3.4.1, 17.7).

1.3.5 InstabilityInstability is a major problem with many continuous celllines, resulting from their unstable aneuploid chromosomalconstitution. Even with short-term cultures of untransformedcells, heterogeneity in growth rate and the capacity todifferentiate within the population can produce variabilityfrom one passage to the next (see Section 18.3).

1.4 MAJOR DIFFERENCES IN VITRO

Many of the differences in cell behavior between culturedcells and their counterparts in vivo stem from the dissociationof cells from a three-dimensional geometry and theirpropagation on a two-dimensional substrate. Specific cellinteractions characteristic of the histology of the tissue arelost, and, as the cells spread out, become mobile, and, inmany cases, start to proliferate, so the growth fraction ofthe cell population increases. When a cell line forms, it mayrepresent only one or two cell types, and many heterotypiccell–cell interactions are lost.

The culture environment also lacks the several systemiccomponents involved in homeostatic regulation in vivo,principally those of the nervous and endocrine systems.Without this control, cellular metabolism may be moreconstant in vitro than in vivo, but may not be trulyrepresentative of the tissue from which the cells were derived.Recognition of this fact has led to the inclusion of a numberof different hormones in culture media (see Sections 10.4.2,10.4.3), and it seems likely that this trend will continue.

Energy metabolism in vitro occurs largely by glycolysis,and although the citric acid cycle is still functional, it plays alesser role.

It is not difficult to find many more differences betweenthe environmental conditions of a cell in vitro and in vivo (seeSection 22.2), and this disparity has often led to tissue culture


being regarded in a rather skeptical light. Still, althoughthe existence of such differences cannot be denied, manyspecialized functions are expressed in culture, and as long asthe limits of the model are appreciated, tissue culture canbecome a very valuable tool.

1.5 TYPES OF TISSUE CULTURE

There are three main methods of initiating a culture[Schaeffer, 1990; see Appendix IV, Fig. 1.3, and Table 1.4):(1) Organ culture implies that the architecture characteristic ofthe tissue in vivo is retained, at least in part, in the culture(see Section 25.2). Toward this end, the tissue is culturedat the liquid–gas interface (on a raft, grid, or gel), which

favors the retention of a spherical or three-dimensionalshape. (2) In primary explant culture, a fragment of tissue isplaced at a glass (or plastic)–liquid interface, where, afterattachment, migration is promoted in the plane of the solidsubstrate (see Section 12.3.1). (3) Cell culture implies that thetissue, or outgrowth from the primary explant, is dispersed(mechanically or enzymatically) into a cell suspension, whichmay then be cultured as an adherent monolayer on a solidsubstrate or as a suspension in the culture medium (seeSections 12.3, 13.7).

Because of the retention of cell interactions found in thetissue from which the culture was derived, organ cultures tendto retain the differentiated properties of that tissue. They donot grow rapidly (cell proliferation is limited to the peripheryof the explant and is restricted mainly to embryonic tissue)

EXPLANT CULTURE

DISSOCIATED CELL CULTURE

ORGANOTYPIC CULTURE

ORGAN CULTURE

Tissue at gas-liquid interface; histologicalstructure maintained

Tissue at solid-liquidinterface; cells migrate

to form outgrowth

Disaggregated tissue;cells form monolayer at solid-liquid interface

Different cells co-cultured with or without matrix; organotypic

structure recreated

Fig. 1.3. Types of Tissue Culture.

TABLE 1.4. Properties of Different Types of Culture

Category Organ culture Explant Cell culture

Source Embryonic organs, adult tissuefragments

Tissue fragments Disaggregated tissue, primaryculture, propagated cell line

Effort High Moderate LowCharacterization Easy, histology Cytology and markers Biochemical, molecular,

immunological, and cytologicalassays

Histology Informative Difficult Not applicableBiochemical differentiation Possible Heterogeneous Lost, but may be reinducedPropagation Not possible Possible from outgrowth Standard procedureReplicate sampling,

reproducibility,homogeneity

High intersample variation High intersample variation Low intersample variation

Quantitation Difficult Difficult Easy; many techniques available


TABLE 1.5. Subculture

Advantages Disadvantages

Propagation Trauma of enzymatic ormechanical disaggregation

More cells Selection of cells adapted toculture

Possibility of cloning Overgrowth of unspecializedor stromal cells

Increased homogeneity Genetic instabilityCharacterization of

replicate samplesLoss of differentiated properties

(may be inducible)Frozen storage Increased risk of

misidentification orcross-contamination

and hence cannot be propagated; each experiment requiresfresh explantations, which implies greater effort and poorerreproducibility of the sample than is achieved with cellculture. Quantitation is, therefore, more difficult, and theamount of material that may be cultured is limited by thedimensions of the explant (∼1 mm3) and the effort requiredfor dissection and setting up the culture. However, organcultures do retain specific histological interactions withoutwhich it may be difficult to reproduce the characteristics ofthe tissue.

Cell cultures may be derived from primary explantsor dispersed cell suspensions. Because cell proliferation isoften found in such cultures, the propagation of cell linesbecomes feasible. A monolayer or cell suspension with asignificant growth fraction (see Section 21.11.1) may bedispersed by enzymatic treatment or simple dilution andreseeded, or subcultured, into fresh vessels (Table 1.5; seealso Sections 13.1, 13.7). This constitutes a passage, and thedaughter cultures so formed are the beginnings of a cell line.

The formation of a cell line from a primary culture implies(1) an increase in the total number of cells over severalgenerations and (2) the ultimate predominance of cells or celllineages with the capacity for high growth, resulting in (3) adegree of uniformity in the cell population (see Table 1.5).The line may be characterized, and the characteristics willapply for most of its finite life span. The derivation ofcontinuous (or ‘‘established,’’ as they were once known) celllines usually implies a phenotypic change, or transformation(see Sections 3.8, 18.2).

When cells are selected from a culture, by cloning or bysome other method, the subline is known as a cell strain.A detailed characterization is then implied. Cell lines orcell strains may be propagated as an adherent monolayer

or in suspension. Monolayer culture signifies that, giventhe opportunity, the cells will attach to the substrate andthat normally the cells will be propagated in this mode.Anchorage dependence means that attachment to (and usually,some degree of spreading onto) the substrate is a prerequisitefor cell proliferation. Monolayer culture is the mode ofculture common to most normal cells, with the exception ofhematopoietic cells. Suspension cultures are derived from cellsthat can survive and proliferate without attachment (anchorageindependent); this ability is restricted to hematopoietic cells,transformed cell lines, and cells from malignant tumors. It canbe shown, however, that a small proportion of cells that arecapable of proliferation in suspension exists in many normaltissues (see Section 18.5.1). The identity of these cells remainsunclear, but a relationship to the stem cell or uncommittedprecursor cell compartment has been postulated. This conceptimplies that some cultured cells represent precursor poolswithin the tissue of origin (see Section 3.10). Cultured celllines are more representative of precursor cell compartmentsin vivo than of fully differentiated cells, as, normally, mostdifferentiated cells do not divide.

Because they may be propagated as a uniform cellsuspension or monolayer, cell cultures have many advantages,in quantitation, characterization, and replicate sampling, butlack the potential for cell–cell interaction and cell–matrixinteraction afforded by organ cultures. For this reason, manyworkers have attempted to reconstitute three-dimensionalcellular structures by using aggregates in cell suspension(see Section 25.3.3) or perfused high-density cultures onmicrocapillary bundles or membranes (see Section 25.3.2).Such developments have required the introduction, or at leastredefinition, of certain terms. Histotypic or histotypic culture,or histoculture (I use histotypic culture), has come to meanthe high-density, or ‘‘tissuelike,’’ culture of one cell type,whereas organotypic culture implies the presence of more thanone cell type interacting as they might in the organ of origin(or a simulation of such interaction). Organotypic culture hasgiven new prospects for the study of cell interaction amongdiscrete, defined populations of homogeneous and potentiallygenetically and phenotypically defined cells.

In many ways, some of the most exciting developmentsin tissue culture arise from recognizing the necessity ofspecific cell interaction in homogeneous or heterogeneouscell populations in culture. This recognition may mark thetransition from an era of fundamental molecular biology, inwhich many of the regulatory processes have been workedout at the cellular level, to an era of cell or tissue biology, inwhich that understanding is applied to integrated populationsof cells and to a more precise elaboration of the signalstransmitted among cells.

CHAPTER 2

Training Programs

2.1 OBJECTIVES

This book has been designed, primarily, as a source ofinformation on procedures in tissue culture, with additionalbackground material provided to place the practical protocolsin context and explain the rationale behind some of theprocedures used. There is a need, however, to assist thosewho are engaged in the training of others in tissue culturetechnique. Whereas an independent worker will access thoseparts of the book most relevant to his or her requirements, astudent or trainee technician with limited practical experiencemay need to be given a recommended training program,based on his/her previous experience and the requirementsof his/her supervisor. This chapter is intended to provideprograms at basic and advanced levels for an instructor to useor modify in the training of new personnel.

The programs are presented as a series of exercises ina standard format with cross-referencing to the appropriateprotocols and background text. Standard protocol instructionsare not repeated in the exercises, as they are provided indetail in later chapters, but suggestions are made for possibleexperimental modifications to the standard protocol to makeeach exercise more interesting and to generate data that thetrainee can then analyze. Most are described with a minimalnumber of samples to save manipulation time and complexity,so the trainee should be made aware of the need for a greaternumber of replicates in a standard experimental situation. Theexercises are presented in a sequence, starting from the mostbasic and progressing toward the more complex, in terms oftechnical manipulation. They are summarized in Table 2.1,with those that are regarded as indispensable presented in bold

type. The basic and advanced exercises are assumed to be ofgeneral application and good general background althoughavailable time and current laboratory practices may dictate adegree of selection.

Where more than one protocol is required, the protocolnumbers are separated by a semicolon; where there is achoice, the numbers are separated by ‘‘or’’, and the instructorcan decide which is more relevant or best suited to the workof the laboratory. It is recommended that all the basic andadvanced exercises in Table 2.1 are attempted, and those inbold type are regarded as essential. The instructor may chooseto be more selective in the specialized section.

Additional ancillary or related protocols are listed withineach exercise. These do not form a part of the exercise butcan be included if they are likely to be of particular interestto the laboratory or the student/trainee.

2.2 BASIC EXERCISES

These are the exercises that a trainee or student shouldattempt first. Most are simple and straightforward to perform,and the protocols and variations on these protocols to makethem into interesting experiments are presented in detail inthe cross-referenced text. Amounts specified in the Materialssections for each protocol are for the procedure described,but can be scaled up or down as required. Exercises presentedin bold font in Table 2.1 are regarded as essential.

A tour of the tissue culture facilities is an essentialintroduction; this lets the trainee meet other staff, determinetheir roles and responsibilities, and see the level of preparation

Culture of Animal Cells: A Manual of Basic Technique, Fifth Edition, by R. Ian FreshneyCopyright 2005 John Wiley & Sons, Inc.

11


TABLE 2.1. Training Programs

Exerciseno. Procedure Training objectives Protocol

Basic:

1 Pipetting and transfer of fluids. Familiarization. Handling and accuracy skills. In exercise2 Observation of cultured cells. Use of inverted microscope. Appreciation

differences in cell morphology within and amongcell lines. Use of camera and preparation ofreference photographs.

16.116.6

3 Aseptic technique: preparingmedium for use.

Aseptic handling. Skill in handling sterile reagentsand flasks without contamination. Addingsupplements to medium.

6.1; 11.7

4 Feeding a culture. Assessing a culture. Changing medium. 13.15 Washing and sterilizing glassware. Familiarization with support services. Appreciation

of need for clean and nontoxic glass containers.11.1

6 Preparation and sterilization ofwater.

Appreciation of need for purity and sterility.Applications and limitations. Sterilization byautoclaving.

11.5

7 Preparation of PBS. Constitution of salt solutions. Osmolality. Bufferingand pH control. Sterilization of heat-stablesolutions by autoclaving.

11.6

8 Preparation of pH standards. Familiarization with use of phenol red as a pHindicator.

9.1

9 Preparation of stock medium andsterilization by filtration.

Technique of filtration and appreciation of range ofoptions.

11.11, 11.12,11.13, 11.14

10 Preparation of complete mediumfrom powder or 10× stock.

Aseptic handling. Constitution of medium. Controlof pH.

11.8, 11.9

11 Counting cells by hemocytometerand an electronic counter.

Quantitative skill. Counting cells and assessment ofviability. Evaluation of relative merits of twomethods.

21.1, 21.2

12 Subculture of continuous cell linegrowing in suspension.

Assessing a culture. Aseptic handling. Cell countingand viability. Selecting reseeding concentration.

13.3

13 Subculture of continuous cell linegrowing in monolayer.

Assessing a culture. Aseptic handling. How todisaggregate cells. Technique of trypsinization.

13.2

14 Staining a monolayer cell culturewith Giemsa.

Cytology of cells. Phase-contrast microscopy.Fixation and staining. Photography.

16.2, 16.6

15 Construction and analysis of growthcurve.

Replicate subcultures in multiwell plates. Cellcounting. Selecting reseeding concentration.

21.8, 21.1,21.2

Advanced:16 Cell line characterization. Confirmation of cell line identity. Increase

awareness of overgrowth, misidentification, andcross-contamination.

16.7 or 16.8 or16.9 or16.10

17 Detection of mycoplasma. Awareness of importance of mycoplasma screening.Experience in fluorescence method or PCR forroutine screening of cell lines for mycoplasmacontamination.

19.2 or 19.3

18 Cryopreservation. How to freeze cells, prepare cell line and freezerinventory records, stock control.

20.1; 20.2

19 Primary culture. Origin and diversity of cultured cells. Variations inprimary culture methodology.

12.2; 12.6 or12.7

20 Cloning of monolayer cells. Technique of dilution cloning. Determination ofplating efficiency. Clonal isolation.

14.1, 21.10,14.6

Specialized:21 Cloning in suspension. Technique of dilution cloning in suspension.

Isolation of suspension clones.14.4 or 14.5;

14.822 Selective media. Demonstration of selective growth of specific cell

types.23.1 or 23.2

CHAPTER 2 TRAINING PROGRAMS 13

TABLE 2.1. Training Programs (Continued)

ExerciseNo. Procedure Training objectives Protocol

23 Cell separation. Isolation of cell type with desired phenotype by oneof several separation methods.

15.1 or 15.2

24 Preparation of feeder layers How to improve cloning efficiency. Selective effectsof feeder layers.

14.3

25 Histotypic culture in filter wellinserts.

Familiarization with high-density culture. Potentialfor differentiation, nutrient transport, and invasionassay.

25.4

26 Cytotoxicity assay. Familiarization with high-throughput screeningmethods. Positive and negative effects.

22.4

27 Survival assay. (Can be run as acomponent of Exercise 20 or as aseparate exercise.)

Use of clonal growth to identify positive andnegative effects on cell survival and proliferation.

22.3

that is required. The principles of storage should also beexplained and attention drawn to the distinctions in locationand packaging between sterile and nonsterile stocks, tissueculture grade and non-tissue culture grade plastics, usingstocks and backup storage, fluids stored at room temperatureversus those stored at 4◦C or −20◦C. The trainee shouldknow about replacement of stocks: what the shelf life isfor various stocks, where replacements are obtained, who toinform if backup stocks are close to running out, and how torotate stocks so that the oldest is used first.

Exercise 1 Aseptic Technique I: Pipetting andTransfer of Fluids

Purpose of ProcedureTo transfer liquid quickly and accurately from one containerto another.

Training ObjectivesSkill in handling pipettes; appreciation of level of speed,accuracy, and precision required.

Supervision: Continuous initially, then leave trainee to repeatexercise and record accuracy.

Time: 30 min −1 h.

Background InformationSterile liquid handling (see Section 5.2.7); handling bottlesand flasks (see Section 6.3.4); pipetting (see Section 6.3.5).

Demonstration material or operations: Instructor should demon-strate handling pipette, inserting in pipetting aid, andfluid transfer and give some guidance on the compromiserequired between speed and accuracy. Instructor should alsodemonstrate fluid withdrawal by vacuum pump (if used inlaboratory) and explain the mechanism and safety constraints.

ExerciseSummary of ProcedureTransfer of liquid by pipette from one vessel to another.

Standard ProtocolAseptic Technique in Vertical Laminar Flow (see Protocol6.1) or Working on the Open Bench (see Protocol 6.2).Before embarking on the full protocol, it is useful to havethe trainee practice simple manipulations by simply pipettingfrom a bottle of water into a waste beaker. This gives somefamiliarization with the manipulations before undertakingaseptic work.

Experimental Variations1) This exercise is aimed at improving manual dexterity and

handling of pipettes and bottles in an aseptic environment.The following additional steps are suggested to add interestand to monitor how the trainee performs:

2) Preweigh the flasks used as receiving vessels.a) Add 5 ml to each of 5 flasks.b) Using a 5-mL pipette.

3) Using a 25-mL pipette.4) Record the time taken to complete the pipetting.5) Weigh the flasks again.6) Incubate the flasks to see whether any are contaminated.

Data1) Calculate the mean weight of liquid in each flask.2) Note the range and

a) Calculate as a percentage of the volume dispensed, orb) Calculate the mean and standard deviation:

i) Key values into Excel.ii) Place the cursor in the cell below the column

of figures.iii) Press arrow to right of � button on standard toolbar,

and select average.


iv) Select column of figures, if not already selected,and enter. This will give the average or mean ofyour data.

v) Place cursor in next cell.vi) Press arrow to right of � button on standard toolbar,

and select other functions and then select STDEV.vii) Select column of figures, if not already selected, and

enter. This will give the standard deviation of yourdata, which you can then calculate as a percentageof the mean to give you an idea of how accurateyour pipetting has been.

Analysis1) Compare the results obtained with each pipette and

comment on the differences:a) In accuracyb) In time

2) When would it be appropriate to use each pipette?3) What is an acceptable level of error in the precision

of pipetting?4) Which is more important: absolute accuracy or consistency?

Exercise 2 Introduction to Cell Cultures

Purpose of ProcedureCritical examination of cell cultures.

ApplicationsChecking consistency during routine maintenance; evalu-ation of status of cultures before feeding, subculture, orcryopreservation; assessment of response to new or experi-mental conditions; detection of overt contamination.

Training ObjectivesFamiliarization with appearance of cell cultures of differenttypes and at different densities; use of digital or film camera;distinction between sterile and contaminated, and healthy andunhealthy cultures; assessment of growth phase of culture.

Supervision: Continuous during observation, then intermittentduring photography.

Time: 30 min.

Background InformationMorphology, photography (see Section 16.4.5).

Demonstration material or operations: Photo examples of cellmorphology, phase contrast, fixed and stained, immuno-stained; types of culture vessel suitable for morphologicalstudies, e.g., Petri dishes (see Fig. 8.3), coverslip tubes,chamber slides (see Fig. 16.3); cytocentrifuge for suspensioncultures (see Fig. 16.4).

Safety: No special safety requirements.

ExerciseSummary of ProcedureExamine and photograph a range of cell lines at differentcell densities.

Equipment and Materials� Range of flask or Petri dish cell cultures at different densities,

preferably with normal and transformed variants of the samecell (e.g., 3T3 and SV3T3, or BHK21-C13 and BHK21-PyY) at densities including mid-log phase (∼50% confluentwith evidence of mitoses), confluent (100% of growth areacovered cells packed but not piling up), and postconfluent(cells multilayering and piling up if transformed). Includesuspension cell cultures and low and high concentrationsif available.

� If possible, include examples of contaminated cultures(preferably not Petri dishes to avoid risk of spread) andunhealthy cultures, e.g., cultures that have gone too longwithout feeding.

� Inverted microscope with 4×, 10×, and 20× phase-contrastobjectives and condenser.

� Automatic camera, preferably digital with monitor, or filmcamera with photo-eyepiece.

Standard Protocol1) Set up microscope and adjust lighting (see Protocol 16.1).2) Bring cultures from incubator. It is best to examine a few

flasks at a time, rather than have too many out of theincubator for a prolonged period. Choose a pair, e.g.,the same cells at low or high density, or a normal andtransformed version of the same cell type.

3) Examine each culture by eye, looking for turbidity of themedium, a fall in pH, or detached cells. Also try to identifymonolayer of cells and look for signs of patterning. This canbe normal, e.g., swirling patterns of fibroblasts at confluence(see Fig 16.2b,h and Plate 5b).

4) Examine at low power (4× objective) by phase-contrastmicroscopy on inverted microscope, and check cell densityand any sign of cell–cell interaction.

5) Examine at medium (10× objective) and high (20×objective) power and check for the healthy status of thecells (see Fig. 13.1), signs of rounding up, contraction of themonolayer, or detachment.

6) Check for any sign of contamination (see Fig. 19.1a–c).7) Look for mitoses and estimate, roughly, their frequency.8) Photograph each culture (see Protocol 16.6), noting the

culture details (cell type, date form last passage) and celldensity and any particular feature that interests you.

9) Return cultures to incubator and repeat with a new set.

Ancillary Protocols: Staining (see Protocols 16.2, 16.3; Cyto-centrifuge (see Protocol 16.4); Indirect Immunofluorescence(see Protocol 16.11).

Experimental Variations1) Look for differences in growth pattern, cell density, and

morphology in related cultures.


2) Assess health status of cells.3) Is there any sign of contamination?4) Are cells ready for feeding (see Section 13.6.2) or passage

(see Section 13.7.1)?5) Make a numerical estimate of cell density by calculating the

area of the 20× objective field and counting the number ofcells per field. This will be easiest if a digital camera andmonitor are used where the screen can be overlaid withcling film and each cell ticked with a fine felt-tipped marker.

6) Try to identify and count mitotic cells in these high-power fields.

Data

Qualitative1) Record your observations on morphology, shape, and

patterning for all cultures.2) Note any contaminations.3) Confirm healthy status or otherwise.

Quantitative1) Record cell density (cells/cm2) for each culture.2) Record mitotic index for each culture.

Analysis1) Account for differences in cell density.2) Account for differences in mitotic index.3) Compare appearance of cells from normal and transformed

cultures and high and low densities and try to explaindifferences in behavior.

Exercise 3 Aseptic Technique II: Preparing Mediumfor Use

Purpose of ProcedureTo maintain asepsis while handling sterile solutions.

Training ObjectivesAseptic handling: Training in dexterity and sterile manipula-tion.

Supervision: Continuous.

Background InformationObjectives of aseptic technique (see Section 6.1); elementsof aseptic environment (see Section 6.2); sterile handling (seeSection 6.3); working in laminar flow (see Section 6.4); visiblemicrobial contamination (see Section 19.3.1).

Demonstration of materials and operations: Demonstrate how toswab work surface and items brought into hood. Explain theprinciples of laminar flow and particulate air filtration. Showtrainee how to uncap and recap flasks and bottles and howto place the cap on the work surface. Demonstrate holding apipette, inserting it into a pipetting aid, and using it withouttouching anything that is not sterile and would contaminate

it, how to transfer solutions aseptically, sloping bottles andflasks during pipetting. Emphasize clearing up and swabbingthe hood and checking below the work surface.

ExerciseSummary of ProcedureAdd the necessary additions and supplements to 1× stockmedium.

Standard ProtocolPreparation of Medium from 1× Stock (see Protocol 11.7).Experimental variations to standard protocol1) Dispense 50 mL medium into each of 2 sterile bottles.2) Place one bottle at 4◦C.3) Incubate the other bottle for 1 week and check for signs of

contamination (see Section 19.3.1).4) Use these bottles in Exercise 4.

Exercise 4 Feeding a Monolayer Culture

Purpose of ProcedureTo replace exhausted medium in a monolayer culture withfresh medium.

ApplicationsUsed to replenish medium between subcultures in rapidlygrowing cultures, or to change from one type of mediumto another.

Training ObjectivesReinforces aseptic manipulation skills. Introduces one ofthe basic principles of cell maintenance, that of mediumreplenishment during propagation cycles. Makes traineeobserve culture and become aware of signs of mediumexhaustion, such as cell density and/or fall in pH, andalso looking for contamination. Awareness of risk ofcross-contamination. Comparison of preincubated withrefrigerated medium.

Supervision: Trainee will require advice in interpretingsigns of medium exhaustion and demonstration of mediumwithdrawal and replenishment.

Time: 30 min.

Background InformationReplacement of medium (see Section 13.6.2); monitoring forcontamination (see Section 19.4); cross-contamination (seeSection 19.5).

Demonstration material or operations: Exercise requires at leastthree semiconfluent flasks from a continuous cell line suchas HeLa or Vero, with details of number of cells seededand date seeded. Trainee should also be shown how tobring medium from refrigerator, and it should be stressedthat medium is specific to each cell line and not shared


among cell lines or operators. Also demonstrate swabbingand laying out hood, use of incubator, retrieving culturefrom incubator, and observing status of cells by eye andon microscope (freedom from contamination, need to feed,healthy status). Aspirator with pump for medium withdrawalor discard beaker will be required and the process of mediumwithdrawal and replacement demonstrated, with gassing with5% CO2 if necessary.

Safety: If human cells are being handled, a Class II biologicalsafety cabinet must be used and waste medium must bediscarded into disinfectant (see Section 7.8.5 and Table 7.7).

ExerciseSummary of ProcedureSpent medium is withdrawn and discarded and replaced withfresh medium.

Standard ProtocolFeeding a Monolayer Culture (see Protocol 13.1).

Ancillary Protocols: Preparation of Complete Medium (seeProtocols 11.7, 11.8, or 11.9 and Exercise 3); Preparation ofpH Standards (see Protocol 9.1); Handling Dishes or Plates(see Protocol 6.3).

Experimental VariationsThe flasks that are fed with the refrigerated and preincubatedmedium in this exercise should be used later for cell counting(see Exercise 12), and another identical flask should be keptwithout feeding, to be trypsinized at the same time.

Background: Complete media (see Section 9.5); replacementof medium (see Section 13.6.2).

DataCompare appearance and yield (cell counts in Exercise 12)from flasks that have been fed with refrigerated or preincubatedmedium with yield from the flask that has not been fed.

Routine maintenance should be recorded in a recordsheet (see Table 12.5) and experimental data tabulated inExercise 12.

Exercise 5 Washing and Sterilizing Glassware

Purpose of ProcedureTo clean and resterilize soiled glassware.

Training ObjectivesAppreciation of preparative practices and quality controlmeasures carried on outside aseptic area.

Supervision: Nominated senior member of washup staff shouldtake trainee through standard procedures.

Time: 20–30 min should be adequate for each session, butthe time spent will depend on the degree of participation by

the trainee in procedures as determined by his/her ultimaterole and the discretion of the supervisor.

Background InformationWashup area (see Section 4.5.2); washup (see Section 5.4.1);glassware washing machine (see Section 5.4.11; Fig. 5.21);sterilizer (see Section 5.4.4; Fig. 5.18,19); washing andsterilizing apparatus (see Section 11.3).

Demonstration material or operations: Trainee should observe allsteps in preparation and participate where possible; this mayrequire repeated short visits to see all procedures. Traineeshould see all equipment in operation, including stacking,quality control (QC), and safety procedures, although notoperating the equipment, unless future duties will includewashup and sterilization.

ExerciseSummary of ProcedureCollecting, rinsing, soaking, washing and sterilizing glasswareand pipettes.

Equipment and MaterialsAs in regular use in preparation area (see Protocols 11.1–11.3).

Standard Protocols1) Preparation and Sterilization of Glassware (see Proto-

col 11.1).2) Preparation and Sterilization of Pipettes (see Protocol 11.2).3) Preparation and Sterilization of Screw Caps (see

Protocol 11.3).

Ancillary Protocols: Sterilizing Filter Assemblies (see Proto-col 11.4).

DataTrainee should become familiar with noting and recordingQC data, such as numerical and graphical output from ovensand autoclaves.

Exercise 6 Preparation and Sterilization of Water

Purpose of ProcedureProvision of regular supply of pure, sterile water.

Training ObjectivesAppreciation of preparative practices carried on outsideaseptic area. Knowledge of need for purity of water andprocess of preparation.

Supervision: Intermittent.

Time: 30 min

Background InformationPreparation and sterilization of ultra pure water (UPW; seeSection 11.1.4; Figs. 5.17, 11.10).


Demonstration material or operations: Preparation supervisorshould discuss principles and operation of water purificationequipment and demonstrate procedures for collection,bottling, sterilization, and QC. Trainee participation atdiscretion of supervisor and instructor.

ExerciseSummary of ProcedurePurify water, bottle, and sterilize by autoclaving.

Standard ProtocolPreparation and Sterilization of Ultra Pure Water (seeProtocol 11.5).

Ancillary Protocol: Preparation of Glassware (see Protocol 11.1).

Data

AcquisitionResistivity (or conductivity) meter on water purifier andtotal organic carbon (TOC) meter. Automatic printout fromautoclave. Sterile tape on bottles. Sterility indicator incenter bottle.

RecordingEnter appropriate readings and observations in log book.

AnalysisReview log book at intervals of 1 week, 1 month, and 3 monthsto detect trends or variability in water quality or sterilizerperformance.

Exercise 7 Preparation and Sterilization ofDulbecco’s Phosphate-Buffered Saline (D-PBS)without Ca2+ and Mg2+ (D-PBSA)

Purpose of ProcedurePreparation of isotonic salt solution for use in an atmosphereof air.

ApplicationsDiluent for concentrates such as 2.5% trypsin, prerinsefor trypsinization, washing solution for cell harvesting orchanging reagents. As it contains no calcium, magnesium,sodium bicarbonate, or glucose, it is not suitable for prolongedincubations.

Training ObjectivesConstitution of simple salt solution. Osmolality. Bufferingand pH control. Sterilization of heat stable solutions byautoclaving.

Supervision: Continuous while preparing solution, thenintermittent during QC steps. Continuous at start andcompletion of sterilization and interpretation of QC data.

Time: 2 h.

Background InformationBalanced salt solutions (see Section 9.3; Table 9.2); buffering(see Section 9.2.3).

Demonstration material or operations: Use of osmometer orconductivity meter. Supervised use of autoclave or bench-top sterilizer.

Safety issues: Steam sterilizers have a high risk of burns andpossible risk of explosion (see Sections 7.5.2, 7.5.7). Simplebench-top autoclaves can burn dry and, consequently, havea fire risk, unless protected with an automatic, temperature-controlled cut-out.

ExerciseSummary of ProcedureDissolve premixed powder or tablet in UPW and sterilize byautoclaving.

Standard ProtocolPreparation of D-PBSA (see Protocol 11.6).

QC Data

Acquisition: Measure osmolality or conductivity and pH afterdissolving constituents.

Recording: Enter details into log book with date andbatch number.

Exercise 8 Preparation of pH Standards

Purpose of ProcedureTo prepare a series of flasks, similar to those in current use inthe laboratory, containing a simple medium or salt solutionwith phenol red, and adjusted to a pH range embracing therange normally found in culture.

ApplicationsAssessment of pH during preparation of medium and beforefeeding or subculturing.

Training ObjectivesFamiliarization with use of phenol red as a pH indicator.Sterilization with syringe filter.

Supervision: Continuous at start, but minimal thereafter untiloperation complete.

Time: 2 h.

Background InformationPhysicochemical properties, pH (see Section 9.2).

Demonstration material or operations: Use of pH meter. Principle,use, and range of syringe filters (see Fig. 11.12a,c).

Safety issues: None, as long as no needle is used on outlet.


ExerciseSummary of ProcedurePrepare a range of media at different pHs.

Standard ProtocolsPreparation of pH Standards (see Protocol 9.1 and Plate 22b),using option of 25-cm2 flasks.

Sterile Filtration with Syringe-Tip Filter (see Proto-col 11.11).

Exercise 9 Preparation of Stock Mediumfrom Powder and Sterilization by Filtration

Purpose of ProcedurePreparation of complex solutions and sterilization of heat-labile reagents and media.

Training ObjectivesTechnique of filtration and appreciation of range of options.Comparison of positive- and negative-pressure filtration.

Supervision: Instruction on preparation of medium. Constantsupervision during setup of filter, intermittent during filtrationprocess, and continuous during sampling for quality control.

Time: 2 h.

Background InformationPreparation of medium from powder (see Protocol11.9); sterile filtration (see Section 11.5.2; Protocol 11.12);alternative procedures (see Protocols 11.11, 11.13, 11.14).

Demonstration material or operations: Range of disposablefilters and reusable filter assemblies, preferably the itemsthemselves but if not, photographs may be used. Emphasizeconcept of filter size (surface area) and scale. Principlesand advantages/disadvantages of positive-/negative-pressurefiltration (see Section 11.5.2). Handling of filter, filtration,collection, and QC sampling should be demonstrated.

ExerciseSummary of ProcedureDissolve powder in UPW, filter-sterilize, bottle, and samplefor sterility.

Standard Protocol1) Prepare medium from powder (see Protocol 11.9)2) Sterilize 450 mL by vacuum filtration (see Protocol 11.12)3) Sterilize 550 mL by positive-pressure filtration (see

Protocol 11.13)

Ancillary ProtocolsPreparation of Customized Medium (see Protocol 11.10);Autoclavable Media (see Section 11.5.1); Reusable SterilizingFilters (see Section 11.3.6); Sterile Filtration with Syringe-Tip Filter (see Protocol 11.11); Sterile Filtration with Large

In-Line Filter (see Protocol 11.14); Serum (see Section 11.5.3;Protocol 11.15).

Experimental VariationsDivide dissolved medium into two lots and sterilize 550 mL bypositive-pressure filtration (see Protocol 11.13) and 450 mL bynegative-pressure filtration (see Protocol 11.12).

Background: CO2 and Bicarbonate (see Section 9.2.2);Buffering (see Section 9.2.3); Standard Sterilization Protocols(see Section 11.5).

Data1) Note pH before and immediately after filtering.2) Incubate universal containers or bottles at 37◦C for 1 week,

and check for contamination.

Analysis1) Explain the difference in pH between vacuum-filtered versus

positive-pressure-filtered medium.2) Does the pH recover on storage?3) When would you use one rather than the other?4) What filters would you use

a) For 5 mL of a crystalline solution?b) For 10 L of medium?c) For 1 L of serum?

Exercise 10 Preparation of Complete Mediumfrom 10× Stock

Purpose of ProcedureAddition of unstable components and supplements to stockmedium to produce a complete medium designed for aspecific task.

ApplicationsProduction of growth medium that will allow the cellsto proliferate, maintenance medium that simply maintainscell viability, or differentiation medium that allows cells todifferentiate in the presence of the appropriate inducers.

Training ObjectivesFurther experience in aseptic handling. Increased understand-ing of the constitution of medium and its supplementation.Stability of components. Control of pH with sodium bicar-bonate.

Supervision: A trainee who has responded well to Exercise 6should need minimum supervision but will require someclarification of the need to add components or supplementsbefore use.

Time: 30 min.

Background InformationMedia (see Sections 11.4.3, 11.4.4)


Demonstration material or operations: Set of pH standards(see Protocol 9.1). Range of bottles available for mediumpreparation.

Safety: No major safety implications unless a toxic (e.g.,cholera toxin or cytotoxic drug) or radioactive constituent isbeing added.

ExerciseSummary of ProcedureSterile components or supplements are added to presterilizedstock medium to make it ready for use.

Standard ProtocolPreparation of Complete Medium from 10× Concentrate(see Protocol 11.8). One or all of the options may be selectedfrom Protocol 11.8.

Ancillary Protocols: Customized Medium (see Protocol 11.10);Preparation of Stock Medium from Powder and Sterilizationby Filtration (see Protocol 11.9); Preparation of pH Standards(see Protocol 9.1).

Experimental Variations

Venting flasks1) Prepare medium according to Protocol 11.8A with-

out HEPES.2) Pipette 10 mL into each of four 25-cm2 flasks.3) Add 20 µL 1M HEPES to each of two flasks.4) Seal two flasks, one with HEPES and one without, and

slacken the caps on the other two.5) Incubate at 37◦C without CO2 overnight.

DataRecord pH and tabulate against incubation condition.

Analysis1) Check pH and account for differences.2) Which condition is correct for this low-bicarbonate

medium?3) What effect has HEPES on the stability of pH?4) When would venting be appropriate?

Bicarbonate concentration (see Section 9.2.2)1) Omit the bicarbonate from the standard procedure in

Protocol 11.8B and add varying amounts of sodiumbicarbonate as follows:a) Prepare and label 5 aliquots of 10 mL bicarbonate-free

medium in 25-cm2 flasks.b) Add 200 µL, 250 µL, 300 µL, 350 µL, 400 µL of 7.5%

NaHCO3 to separate flasks.2) Leave the cap slack (only just engaging on the thread), or

use a filter cap, on the flasks and place at 37◦C in a 5%CO2 incubator.

3) Leave overnight and check pH against pH standards (seeProtocol 9.1).

DataRecord pH and tabulate against volume of NaHCO3 added.

Analysis1) Explain what is happening to change the pH.2) Calculate the final concentration of bicarbonate in each

case and determine the correct amount of NaHCO3 to use.3) If none are correct, what would you do to attain the

correct pH?

Exercise 11 Preparation of Complete Mediumfrom Powder

See Protocol 11.9

Exercise 12 Counting Cells by Hemocytometerand Electronic Counter

There are several options in the organization of this exercise.It could be used as an exercise either in the use of thehemocytometer or in the use of an electronic cell counter,or it could be arranged as a joint exercise utilizing bothtechniques and comparing the outcomes. Alternatively, anyof these options could be combined with Exercise 11, tosave time. However, as the initial training in cell countingcan make the actual counting process quite slow, it isrecommended that cell counting is run as a stand-aloneexercise, utilizing cultures set up previously (e.g., fromExercise 9), and not as a preliminary to another exercise. Thecombined use of both counting methods will be incorporatedin the following description.

Purpose of ProcedureTo quantify the concentration of cells in a suspension.

ApplicationsStandardization of cell concentrations at routine subculture;analysis of quantitative growth experiments and cellproduction via growth curves and cell yields.

Training ObjectivesQuantitative skill. Counting cells and assessment of viability.Evaluation of relative merits of hemocytometer andelectronic counting.

Supervision: Required during preparation and examination ofsample and setting up both counting procedures. Countingsamples can proceed unsupervised, although the trainee mayrequire help in analyzing results.

Time: 45 min.


TABLE 2.2. Data Record from Exercise 12, Cell Counting

Cells per flaskat seeding

Hemocytometeror electronic

count at harvest

Dilution orsamplingfraction∗

Cell/mL oftrypsinate orsuspension

Cells harvestedper flask

Yield: Cellsharvested ÷cells seeded

∗Electronic counter dilution of 50× (e.g., 0.4 mL cell suspension in 20 mL counting fluid), with counter sample set at 0.5 mL, would give a factor of100. Hemocytometer chamber (Improved Neubauer) counts usually sample 1 mm2 × 0.1 mm deep, i.e., 0.1 mm3, so a factor of 1 × 104 will givecells/mL (see Section 21.1.1).

Background InformationCell counting, hemocytometer (see Section 21.1.1); elec-tronic counting (see Section 21.1.2).

Demonstration material or operations: Cell cultures used forcounting should be derived from Exercise 9. The use ofthe hemocytometer and electronic counter will requiredemonstration, with appropriate advice on completingcalculations at the end. The principles of operation of theelectronic counter should also be explained.

Safety: When human cells are used, handling should be ina Class II microbiological safety cabinet. All plastics andglassware, including the hemocytometer slide and coverslip,should be placed in disinfectant after use, and counting cupsand fluid from electronic counting should be disposed of intodisinfectant (see Section 7.8.5).

ExerciseSummary of ProcedureCells growing in suspension or detached from monolayerculture by trypsin are counted directly in an opticallycorrect counting chamber, or diluted in D-PBSA andcounted in an electronic counter. Cells may be stainedbeforehand with a viability stain before counting in ahemocytometer.

Standard ProtocolExercise should be performed first by electronic cell counting(see Protocol 21.2) with a diluted cell suspension and thenwith the concentrated cell suspension, using cell countingby hemocytometer (see Protocol 21.1), then repeated withthe same concentrated cell suspension, using estimation ofviability by dye exclusion (see Protocol 22.1).

Ancillary Protocols: Staining with Crystal Violet (see Protocol16.3); DNA Content (see Section 16.6); Microtitration Assays(see Section 22.3.5).

Experimental Variations1) Repeat counts 5–10 times with hemocytometer and

electronic cell counter and calculate the mean and standarddeviation (see Exercise 1).

2) Compare fed and unfed flasks from Exercise 9.

DataCell counts, with viability where appropriate, calculated perflask, by each counting method.

Details of routine maintenance should be recorded in arecord sheet (see Table 13.7) and experimental data in aseparate table (Table 2.2).

Analysis1) Calculate viable cell yield relative to cells seeded into flasks.2) Has changing the medium made any difference?

Exercise 13 Subculture of Cells Growingin Suspension

Purpose of ProcedureReduction in cell concentration in proportion to growth rateto allow cells to remain in exponential growth.

ApplicationsRoutine passage of unattached cells such as myelomaor ascites-derived cultures; expansion of culture forincreased cell production; setting up replicate cultures forexperimental purposes.

Training ObjectivesFamiliarization with suspension mode of growth; cellcounting and viability estimation.

Supervision: Initial supervision required to explain principles,but manipulations are simple and, given that the trainee hasalready performed at least one method of counting in Exercise10, should not require continuous supervision, other thanintermittent checks on aseptic technique.

Time: 30 min.

Background InformationPropagation in suspension, subculture of suspension culture(see Sections 13.7.4, 13.7.5); viability (see Section 22.3.1).

Demonstration material or operations: Trainee will require twosuspension cultures, one in late log phase and one in


TABLE 2.3. Record of Exercise 13, Subculture of Cells Growing in Suspension

Volume Cell count Viability

Sample

of cellsusp. incultureflask

fromhemocytometer

or electroniccounter

Dilutionor

samplingfraction1

Cells/mLin flask

(ratio ofunstainedcells tototal)2

Dilutionfactor forviability

stainViable

cells/mL Cells/flask

v c d c × d r f c×d×r×f c×d×r×f×v

e.g., cell counterwithout viabilitystain

20 15321 100 1532100 1 1 1532100 30642000

e.g., hemocytometerwith viability stain

20 76 10000 760000 0.85 2 1292000 25840000

1For electronic counting, 0.4 mL cell suspension in 20 mL counting fluid is a ×50 dilution, and ×2 as the counter counts a sample of 0.5 mL of thediluted suspension, giving a factor of ×100. For hemocytometer counting, if the center 1 mm2 is counted, the factor is 1 × 104; if all 9 fields of1 mm2 are counted (because the count was low) then the factor is 1 × 104 ÷ 9. In practice, it is better to count 5 fields of 1 mm2 on each side ofthe slide, whereupon the factor becomes 1 × 104 ÷ 10, or 1 × 103.2(Total cell count—stained cells) ÷ Total cell count

plateau, with details of seeding date and cell concentration,and should be shown how to add viability determinationinto hemocytometer counting (see Sections 21.1.1, 22.3.1,Protocol 22.1). If stirred culture is to be used rather thanstatic flasks, the preparation of the flasks and the use of thestirrer platform will need to be demonstrated.

Safety: Where human cells are used, handling should be in aClass II microbiological safety cabinet and all materials mustbe disposed of into disinfectant (see Exercise 12).

ExerciseSummary of ProcedureA sample of cell suspension is removed from the culture,counted electronically or by hemocytometer, diluted inmedium, and reseeded.

Standard ProtocolSubculture in Suspension (see Protocol 13.3); Viability (seeProtocol 22.1).

Ancillary Protocol: Scale-up in Suspension (see Protocol 26.1).

Experimental VariationsComparison of subculture from log-phase and plateau-phase cells.

Background: Cell concentration at subculture (seeSection 13.7.3).

DataDetermine cell concentration and viability in subculturesfrom log-phase and plateau-phase cells 72 h after subculture.Recording is best done in a table (Table 2.3) and transferredto a spreadsheet.

Analysis1) Calculate the cell yield as described in Table 2.4.2) Compare the yield from cells seeded from log and

plateau phase.

TABLE 2.4. Analysis of Exercise 13

Sample

Cells/flaskat

seeding

Cells/flaskat next

subculture


n c×d×v×r×f c×d×v×r×fn

Example 200,000 30642000 153.21

Log-phase cells

Plateau-phasecells


Exercise 14 Subculture of Continuous Cell LineGrowing in Monolayer

Purpose of ProcedurePropagating a culture by transferring the cells of a culture toa new culture vessel. This may involve dilution to reseed thesame size of culture vessel, or increasing the size of vessel ifexpansion is required.

Training Objectives

Assessment of culture: This exercise requires the trainee toexamine and assess the status of a culture. The traineeshould note the general appearance, condition, freedomfrom contamination, pH of the medium, and density ofthe cells.

Aseptic handling: Reinforces skills learned in Exercises 5, 7,and 8.

Subculture or passage: This exercise introduces the principleof transferring the culture from one flask to another withdilution appropriate to the expected growth rate. It showsthe trainee how to disaggregate cells by the technique oftrypsinization, and how to count cells and assess viability. Thetrainee is then required to determine the cell concentrationand select the correct concentration for reseeding, instillinga concept of quantitation in cell culture and enhancingnumeracy skills.

Supervision: Provided that the trainee has shown competencein aseptic technique, continuous direct supervisionshould not be necessary, but the instructor should beon hand for intermittent supervision and to answerquestions.

Background Information

Standard ProtocolsSubculture, Criteria for Subculture (see Section 13.7.1;Figs. 13.2, 13.3, 13.4); Growth Cycle and Split Ratios(see Section 13.7.2), Cell Concentration at Subculture(see Section 13.7.3; Fig. 13.4); Choice of Culture Vessel(see Section 8.2); CO2 and Bicarbonate (see Section 9.2.2,Table 9.1).

Experimental variations1) Cell concentration at subculture (see Section 13.7.3)2) Growth cycle (see Section 21.9.2)3) Effect of cell density (see Section 25.1.1).

Demonstration of materials and operations: The trainee shouldbe shown different types of culture vessel (see Table 8.1;Figs. 8.1–8.8) and photographs of cells, healthy (see Fig. 16.2,Plates 5, 6), unhealthy (see Fig. 13.1), contaminated (seeFig. 19. a–c), and at different densities (see Fig. 16.2; Plates5, 6). Instruction should be given in examining cells by

phase-contrast microscopy. A demonstration of trypsinization(see Protocol 13.2) will be required.

ExerciseSummary of ProcedureA cell monolayer is disaggregated in trypsin, diluted,and reseeded.

Equipment and MaterialsSee materials for standard subculture (see Protocol 13.2).

Standard ProtocolSubculture of Monolayer (see Protocol 13.2).

Experimental variationsApply in Protocol 13.2 at Step 11:

a) Seed six flasks at 2 × 104 cells/mL.b) Feed three flasks after 4 days.c) Determine cell counts after 7 days in two flasks that have

been fed and two that have not:i) Remove medium and discard.

ii) Wash cells gently with 2 mL D-PBSA, removecompletely, and discard.

iii) Add 1 mL trypsin to each flask.iv) Incubate for 10 min.v) Add 1 mL medium to trypsin and disperse cells by

pipetting vigorously to give a single cell suspension.vi) Count cells by hemocytometer or electronic

cell counter.vii) Calculate number of cells per flask, cells/mL culture

medium, and cells/cm2 at time of trypsinization.d) Fix and stain cells in other flasks (see Protocol 16.2).

Note: Pipettors should only be used for counting cells fromisolated samples and not for dispensing cells for subculture,unless plugged tips are used.

Ancillary Protocols: Using an Inverted microscope (see Protocol16.1); Cell Counting (see Protocols 21.1, 21.2); Preparation ofMedia (see Protocols 11.7, 10.8, 10.9); Staining with Giemsa(see Protocol 16.2).

Data1) Cell counts at start and in one set of flasks after 1 week.2) Examine and photograph stained flasks in Exercise 13. Best

resolution is obtained if examined before cell layer dries.

Analysis1) Calculate fold yield (number of cells recovered ÷ number

of cells seeded; Table 2.6) and explain the differences (seeSections 18.5.2, 21.9.3).

2) Is an intermediate feed required for these cells?3) Comment on differences in cell morphology of fed and

unfed cultures in Exercise 13.


TABLE 2.5. Record of Exercise 14

SampleVolume oftrypsinate

Cell count fromhemocytometer orelectronic counter

Dilution orsamplingfraction

Cells/mL intrypsinate Cells/flask

t c d c × d c×d×t

TABLE 2.6. Analysis of Exercise 14

Sample

Cells perflask atseeding

Cellsharvestedper flask1


n c×d×t (see lastcolumn ofrecord)

c×d×tn

Not fed 100,000

100,000

Fed 100,000

100,000

1Viability has not been taken into account in this instance astrypsinization, or at least the prewashes before trypsinization, tendto remove most of the nonviable cells when handling a continuouscell line. This is not necessarily the case with an early passage orprimary culture, when viability may need to be taken into account (seeRecording, Exercise 20).

Exercise 15 Staining a Monolayer Cell Culturewith Giemsa

Purpose of ProcedureStaining with a polychromatic stain like Giemsa reveals

Contents · 1916 Trypsinization and subculture of explants Rous & Jones, 1916 1920s/30s Subculture of ﬁbroblastic cell lines Carrel & Ebeling, 1923 1925–1926 Differentiation in

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