Quantitative Studies on Mammalian Cells in Vitro

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<ul><li><p>REVIEWS OF MODERN PHYSICS VOLUME 31, NUMBER 2 APRIL, 19S9</p><p>II l g~T. T. PUCK</p><p>Department of Biophysics, University of Colorado Medical Center, Denver 20, Colorado</p><p>~ 'HE major advances of the science of genetics havecome about through the use of two fundamental</p><p>types of operations. The first and oldest consists inmating of selected, multicellular plants or animals toproduce large numbers of offspring, among which thedistribution of various inherited characters is examined.This procedure has produced that body of knowledgeknown as classical genetics and which, beginningsystematically with the studies of Mendel, has deline-ated the processes which sustain and modulate bio-logical heredity in all living forms. This experimentalapproach, which essentially examines genetic mecha-nisms in the germ cells, has been extremely successfulin elucidating hereditary phenomena in organisms asdiverse as Drosophila and Zea mays, but is difhcult toapply to the slowly and less extensively reproducingorganisms like the mammals. Thus, while some notableadvances have been accomplished, it has not beengenerally possible to obtain suKciently large numbersof progeny from selected mammalian matings to providepopulations adequate for measurement of many im-portant genetic events.</p><p>The second major class of genetic operations involvesa more recent technique, which consists in study of theindependent micro- and ultramicro-organisms thefree-living cells and the viruses. Here the standard unitis a single cell or particle, and the fundamental operationinvolves examination of the distribution of genetictraits among the progeny of asexual reproduction. Theprincipal power of this method lies in the vast multipli-cation factor which it aBords. Instead of hundreds orthousands, millions or billions of progeny, all arisingfrom a single individual, can be rapidly produced andquickly scanned for a large variety of genetic characters,so that even rare events can be quantitatively examined.In the last twenty years, such genetic studies onmicroorganisms have produced a whole new field ofknowledge. Sexual methods of genetic analysis are notexcluded from these operations but can sometimes bearranged in a step preceding the clonal multiplicationof the single cells. In addition, however, new methodsfor nonsexual genetic analysis have been found whichpromise to be exceedingly productive. Among thefundamental results which have issued from use ofsingle-cell techniques applied to microorganisms duringthe last two decades are included: (a) the first system-atic demonstration of the direct relationship betweengenes and enzymes; (b) delineation of many specific,metabolic pathways involved in gene-controlled bio-</p><p>~ Contribution No. 79 from the Florence R. Sabin Laboratories.</p><p>syntheses; (c) demonstration of the direct exchangeof genetic materials between cells previously consideredto multiply only mitotically; (d) introduction of genesinto cells by means of temperate viruses or puredeoxyribonucleic acid; (e) the most accurate measure-ment of gene-mutation rates; (f) demonstration andquantitative analysis of the processes involved in en-zyme induction among genetically competent cells inresponse to specific environmental stimuli; (g) delinea-tion of the characteristics of linear inheritance in bac-teria; (h) systematic use of mitotic crossing-over incertain organisms to localize genes on their chromo-somes; and (i) the most detailed mapping at the levelof molecular dimensions of the linear sequence of geneticdeterminants, as accomplished in a bacteriophagechromosome.</p><p>An index of the fruitfulness of this latter approach tothe analysis of genetics and related metabolic processesis afI'orded by the fact that the great majority of thediscussion devoted to genetic processes in this bio-physical conference has been concerned with develop-ments arising directly from microbial genetics.</p><p>In our laboratory, a program was initiated, attempt-ing to develop a methodology that would make possiblestudies of mammalian cells by means of the techniquesof microbial genetics. If successful, such a methodologywould provide a new avenue for exploration of mam-malian genetic processes among the somatic cells ofmulticellular organisms, which would complementstudies on the genetics of the germ cells achieved byconventional mating studies. In addition, it wouldaGord all of the many kinds of analyses which an un-limited number of progeny provides. Such techniquesmight facilitate elucidation of the nature of any changesin the genome and phenome, respectively, which areresponsible for the characteristic behavior of the variousdifI'erentiated tissue cells; localization of diferent geneson the various chromosomes; provision of mutated cellscontaining specific biochemical blocks for the deline-ation of various enzymatic steps through quantitativestudy of specific biosyntheses, like that of hormones andantibodies in specialized cells; search for processes likesexual genetic exchange and transduction in somaticcells, and investigation of the genetic biochemistry ofnormal and malignant cells.</p><p>Participating in this program have been a series ofdevoted co-workers: Steven Cieciura, Harold Fisher,and Philip Marcus carried out studies which formed thebasis of their doctoral theses; and D. Morkovin, G.Sato, and N. C. Webb joined this program in a post-doctoral capacity. Chromosome studies have been</p><p>433</p></li><li><p>434 T. T. PUCK</p><p>(a)</p><p>(b)</p><p>FzG. 1. (a) Petri dish which was seeded with 100 single cells ofthe S3 strain of the HeLa culture, an aneuploid which originatedin a human cancer. (b) Petri dish seeded with single cells of aeuploid human strain. These cells tend more to spread and migrate,and so the colonies tend to run together unless a smaller numberof cells is plated or a larger plate is employed.</p><p>carried out initially in a collaborative arrangement withDr. Chu and Dr. Giles at Yale University, and morerecently with J. H. Tjio, who joined our laboratory lastyear. Dr. Arthur Robinson has conducted those aspectsof these studies which involved work on human patients.</p><p>The first step in this program, which, of course, hasdrawn a great deal on previously existing tissue-culturemethodology, was to develop means for plating singlemammalian cells, under conditions such that everysingle cell develops into a discrete, macroscopic colony. 'This is essential in order to quantitate growth of singlecells of a large population, and to make possible isola-tion of mutant clones. A simple, quantitative means forgrowth of single cells into colonies was achieved by a</p><p>procedure, identical in principle to, and only slightlymore complex in practice than, the plating methodwhich constitutes the basis of quantitative microbiology.Figure 1(a) illustrates a representative plating in which100 cells of the S3 HeLa clone, plated in a Petri dishin nutrient medium, have attached to the glass andproduced, within the limits of sampling uncertainty, adiscrete macroscopic colony from each cell inoculated.The counting of such colonies is completely objectiveand highly reliable, so that this methodology permits allof the types of experiments characteristic of quantita-tive bacteriology to be applied to mammalian cells.Cells taken from organs as diverse as skin, liver, spleen,bone marrow, testis, ovary, kidney, lung, and others,from man and other mammals, and from individuals ofa large variety of ages, all respond similarly. It can beconcluded that at least large numbers of cells exist invirtually every organ of the mammalian body whichretain the potentiality of growth as independent micro-organisms, if provided with an adequate physical andchemical environment.</p><p>By and large, two general types of cellular andcolonial morphologies result from such plating. Thatillustrated in Fig. 1(a) has been called "epithelial-like"because the cells form compact, tight colonies withrelatively smooth, well-defined edges. In our experienceso far, the cells that tend to grow stably in this fashionhave been polyploid, and. usually aneuploid. The othertype of colony, which has been called "fibroblast-like"but should perhaps be referred to simply as "stretched, "is illustrated in Fig. 1(b), which shows the tendency ofthe colonies to grow with rough edges, and of the cellsto line up as elongated parallel structures. This stretched,needle-like conformation is more characteristic of theeuploid cells grown by the methods developed in our lab-oratory. However, the molecular environment stronglyinfluences the morphology of cells grown in this manner. '</p><p>The course of developments in microbial geneticshas demonstrated the enormous utility of quantitativesingle-cell plating for the isolation of stocks with raregenetic markers that permit analytical experimentation.Unless each cell can grow in isolation to form a colony,it becomes impossible to apply the highly discriminatingscreening methods by which millions of cells are sub-jected to a stressful situation permitting growth of onlyoccasional rare mutants, for otherwise, the exceedinglyrare mutant which though potentially is capable ofreproduction is not part of a large reproducing popu-lation and may not be enabled to express its growthpotentiality. An effective single-cell plating techniquepermits ready recognition of even very rare geneticevents, and quantitation of their frequency.</p><p>Isolation of mutant clones from such plates is astraightforward process for which a variety of diferentmechanical methods have been developed. We haveestablished mutant clones which have arisen spontane-ously or have been induced as a result of x-irradiation.Among these are included forms with divergent nutri-</p></li><li><p>STUD I ES ON MAM MAL IAN CELLS IN VITRO 435</p><p>10090</p><p>o7060</p><p>~ 50CJ</p><p>403020100</p><p>0 ]0 15 20 25Percent human serum</p><p>(a)</p><p>-S3</p><p>Sl1</p><p>30 35</p><p>I I I I I I I I I I I I I I</p><p>60S/NDV</p><p>40</p><p>0</p><p>S3-940</p><p>20</p><p>p ~i::.::::;i:i:::-.@ma I I 1 I l I70 71 72 73 74 75 76 77 78 79 80 81 82 83 84</p><p>Chromosome number</p><p>(b)</p><p>Fro. 2. Demonstration of some representative mutant strainsisolated from the HeLa population. (a) Plating efficiency curvesin the presence of diferent amounts of human serum of 2 mutantsfound to occur spontaneously in the original HeLa population.The cellular and colonial morphologies of these two strains areidentical but their growth requirements are diferent. (b) Distribu-tion of chromosome number in 2 HeLa clones. The clone at thetop (S/NDV) has the same chromosome number as S3, but ischaracterized by its resistance to destruction by Newcastle diseasevirus.</p><p>tional requirements for colony formation, changedcolonial morphologies, resistance to destruction byspecific viruses like Newcastle disease virus, and di6er-ences in chromosomal constitutions (Fig. 2). Thesemutants have been grown for months or years incontinuous culture, during which they have producedastronomical numbers of progeny, and have exhibitedstability with respect to their identifying characteristicswhich is in every way comparable to that of thefamiliar mutants in bacteria and molds. "</p><p>Attention was next devoted to the development of adefined chemical medium which would promote growthin high efficiency of single mammalian cells, and so</p><p>make available means for isolating mutants with specificnutritional markers. Many laboratories have contrib-uted studies elucidating small molecular requirementsof mammalian cells in tissue cultures utilizing massivecell inocula. 4 ' In studying the growth requirements ofindividual, isolated cells, we found that single cells ofthe S3 clonal strain of the HeLa cell form colonies with100% plating efficiency, when a chemically definedsmall-molecular medium is supplemented with twomacromolecular serum fractions each migrating as asingle moving boundary in an electric field. The twoproteins of mammalian serum which, under the condi-tions of these experiments, complete the growth require-ments of single cells are serum albumin and an o.~-globulin with a molecular weight of approximately45 000, which appears to be a glycoprotein in compo-sition. Both substances are necessary for growth ofsingle cells under the specific conditions of our pro-cedures. The functions of these proteins are still understudy, but that of the a~-globulin has been found to befulfilled completely by the protein fraction namedfetuin, an a~-globulin which constitutes 45% of calffetal serum protein. ~</p><p>Albumin and fetuin have been purified by repeatedprecipitation to the point where preparations areapproximately 98% homogeneous electrophoreticallyand ultracentrifugally, although the ultimate purity ofsuch preparations is, of course, difficult to specify withcertainty, particularly since fetuin has been demon-strated to be heterogeneous immunologically. A typicalelectrophoretic pattern is presented in Figs. 3(a), and3(b) demonstrates the colonial development achievedfrom inoculation of single cells into a medium containingthe purified albumin and fetuin, amino acids, growthfactors, salts and glucose. The plating efficiency equalsthat in serum-containing medium. Experiments onmutants of S3 which exhibit different molecular nutri-tional requirements are now in progress.</p><p>It seems likely that fetuin and albumin may not berequired as true metabolites, but rather as conditioningfactors which permit establishment of the necessaryphysicochemical relationships between the cell andsurrounding medium. Thus, the fetuin fraction exercisesa specific action on the cell membrane, since trypsinizedcells added to a medium, complete except for thepresence of fetuin, remain as rounded spheres, unat-tached to the glass surface. With the addition of fetuinto such a medium, the cells rapidly attach to the glassand stretch out in a highly characteristic configurationLFigs. 4(a) and 4(b)]. As little as 1 mg/cc of fetuin canbe detected by means of this action. Fetuin has beenknown to be a powerful antitryptic agent, and at leastpart of the function of fetuin seems to be antiproteo-lytic in nature, because this substance prevents thetryptic loosening and rounding of glass-attached cells.The participation in the attachment reaction of aprotein not included in the fetuin fraction has bt;enclaimed by some workers. '</p></li><li><p>T. T. PUCK</p><p>\</p><p>5</p><p>(a) ENZ 18 Bi(b)</p><p>PxG. 3. (a) Klectrophoretic pattern of fetuin purified by repeated, fractional (NH4) 2SO4 precipitation from fetal calf serum. {b)Coloniesdeveloping on plate seeded with 100 S3 cells, in a medium containing known small molecular components, and purified fetuin and humanserum albumin.</p><p>In further development of tools for quantitative studyof the growth and genetics of the mammalian cell</p><p>in vitro, it became necessary to attempt to control thelability of karyotype which had been demonstrated toafI'ect cells cultivated by standard tissue-culture tech-</p><p>niques. Recent studies have shown that virtually everymammalian cell which has been established as a stabletissue-culture strain has a chromosomal constitutionradically diGerent from that of the parental speciesfrom which it originated. ' "Euploid cells were gener...</p></li></ul>