introduction · tissue culture nutrition andvirus propagation ... i. introduction plasma, serum,...

TISSUE CULTURE NUTRITION

JOSEPH F. MORGANLaboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada

CONTENTSI. Introduction................................................ 20

II. Recent Tissue Culture Methods .................................. 21A. Single Cell Isolations and Cell Strains............................................... 21B. Cell Suspension Methods............................................... 21C. Monolayer Tissue Cultures and Plaque Techniques...................................... 22D. Plating Methods and Cell Genetics .............................................. 22E. Nutritional Depletion Techniques............................................... 22F. Measurement of Packed Cell Volume ...................................... 22G. Chemical Measurement of Growth............................................... 23

III. Nutritional Requirements of Animal Tissue Cells .......................................... 23A. Amino Acids............................................... 23B. Carbohydrates............................................... 26C. Vitamins and Coenzymes............................................... 27D. Purines, Pyrimidines, and Nucleic Acid Components.................................... 28E. Lipids ............................................... 29F. Accessory Growth Factors............................................... 30G. Minerals............................................... 30H. Peptides, Peptones, Proteoses, and Proteins ............................................ 31I. Unidentified Factors................................................................... 33

IV. Tissue Culture Nutrition and Virus Propagation ........................................... 34V. Discussion and Conclusion.............................................. 35

VI. References................................................................................ 38

I. INTRODUCTION plasma, serum, ascitic fluid, and tissue extracts.Animal tissue culture, as an area of scientific The general methods evolved, which made use

endeavor, is generally considered to have begun of cover slips, specially designed flasks, or rotat-with the pioneer experiments of Harrison (76, 77) ing tubes, relied on the aseptic techniques ofabout 1906, although the feasibility of tissue cul- bacteriology to avoid microbial contamination.ture was suggested in some earlier publications Some of the success obtained with these methods(130, 187). In the fifty years that have elapsed was very probably due to the bactericidal activitysince Harrison's experiments, many investigators of the serum and other natural media employed.have employed tissue culture to obtain consider- Since these early methods have been describedable information on cell structure, morphology, in detail in several recent texts (11, 50, 130, 138,and development. However, the complexity of 163, 187, 191), they will not be discussed at anythe methodology developed by the earlier in- length in the present article.vestigators tended to restrict the application of It has long been recognized that the termtissue culture to selected areas of study. It is "tissue culture" is itself a misnomer. Althoughonly within recent years, with the advent of tissues, derived from many animal species, pro-antibiotics and the use of bacteriological prin vide the basic material for cultivation studies,ciples, that tissue culture has become recognized it is the constituent cells of the tissue that areas a basic research technique with potential propagated, rather than the tissues themselvesapplication to a wide variety of problems. as structural entities. This distinction has beenThe early tissue culture methods were designed heightened by the recent development of methods

primarily for studies on cell morphology and which permit the propagation of replicate cul-were based on the observation that many tissues tures from cell suspensions (44). For purposes ofcould be propagated almost indefinitely in media the present article, and to conform with thederived from the animal body, particularly enormous literature on tissue culture (123), the

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1958] TISSUE CULTURE NUTRITION 21

terms "cell cultivation" and "tissue culture" will more recent tissue culture techniques have beenbe considered as synonymous. designed specifically for cell nutrition studies.There seems to be a remarkable similarity A. Single cell isolations and cell strains. Lines

between the present status of tissue culture and of cells for cultivation purposes were originallythe status of bacteriology several generations derived through serial transfers, with the gradualago. At that time, complex natural materials, selection of specific cell types (130). Such cellsuch as blood or serum, were believed necessary lines were considered to be relatively homoge-for the growth of many bacterial pathogens and neous but not pure, since a mixed cell populationthe study of synthetic media for bacteria was in could not be excluded. One of the major advancesits infancy. From studies on bacterial nutrition in cell cultivation methods was the demonstra-have resulted the development of synthetic media tion by Sanford et al. (155) that strains of tissuefor many microorganisms and the opening up cells could be derived through single-cell isola-of the field of bacterial physiology. In the course tion, just like bacterial strains. Individual cellsof these studies, several new vitamins were dis- from cultures of mouse mesenchymal tissue werecovered and some insight was gained into the manipulated into fine capillary pipettes andcomplexities of intermediary metabolism. It propagated in a medium containing the fluidwould seem logical that future investigations removed from a large, metabolically active cul-with tissue culture might follow essentially the ture of the same tissue. In this way, the firstsame pattern of development. Specific knowledge clone strain of cells, strain L, no. 929, wasof the nutrition and metabolism of different cell obtained. It is interesting to note that thetypes seems to be required for future progress method which yielded a successful single-cellin many lines of cell investigation. The use of culture was based on the concept that presentbacteriological techniques and the background tissue culture media are inadequate to supportof information now available on microbial nutri- the growth of small inocula unless the mediation and metabolism may provide answers to have previously been altered by the metabolicsome of the problems in cell cultivation. activity of large, rapidly growing cultures. In

In writing a review article, certain arbitrary subsequent studies (100), a variety of otherdecisions must, of necessity, be made. The pres- single-cell strains were developed by this capil-ent article will deal only with the application of lary tube method. With further refinements ofbacteriological techniques to problems in tissue these techniques, it should ultimately be possiblecell nutrition and will not include the extensive to establish a whole spectrum of cell lines, com-literature on organ culture or plant tissue culture. parable to bacterial strains, for precise studies onThe application of tissue culture to virology tissue cell nutrition and metabolismWoffers subject matter sufficient for a separate ment of pure strains through single-cell isolation,reieadcnsqunly wl btuce.o.oi methods were required for the preparation ofbriefly in relation to cell nutrition problems. In le n ers ofquifor cultures.Ean anrevewigtemthdolgy f tsse cltue, he large numbers of uniform cultures. Evans and

reviewing the methodology of tissue culture, the associates (44) showed that cell suspensions coulddate of departure of modern from early methods be prepared from L strain cultures and used forhas been arbitrarily designated as 1946, a date the quantitative seeding of replicate culturecorresponding to the Tissue Culture Review vessels. The growth potential of the cells was notConference held at Hershey, Pennsylvania. As damaged by this procedure and actively growingfar as possible, this review has been restricted to cultures on either glass or perforated cellophanepapers published between 1946 and the end of substrates were obtained (37, 38, 43). Measure-1956. ment of proliferation in such replicate cultures

was carried out through counting of cell nuclei,*I.IUICENT TISSUE CULTURE METHODS following disruption of the cells by treatment

In discussing the application of bacteriological with citric acid (156). Adaptation of these pro-principles to problems in cell cultivation, it is cedures to synthetic media studies has beenimpossible to separate the methodology of tissue reported (133). The introduction of trypsin as aculture from the nutritional aspects of the prob- dispersing agent made it possible to prepare celllem. This is particularly true since many of the suspensions from decapitated whole embryos

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22 JOSEPH F. MORGAN [VOL. 22

(21), embryonic organ rudiments (122), monkey strains required the evolution of techniquestesticular tissue (57), actively growing cultures similar to the plating methods available forof human epidermoid carcinoma cells (162), and bacteria. This has recently been achieved by theepithelial-like cells from a variety of normal studies of Puck and his associates (104, 139-142).human tissues (16). This enzymatic dispersion It was found that growth of colonies from singletechnique has been most widely applied in the HeLa cells could be accomplished by a platingpreparation of monkey kidney cells for polio- procedure in which the test cells were placedmyelitis studies (195). over a layer of X-irradiated HeLa cells. These

It had long been thought that propagation of latter cells were unable themselves to multiplyanimal cells in vitro required a supporting frame- but exhibited active metabolism (139). It iswork, such as fibrin, cellophane, or glass. With interesting to note that the "feeder layer" pro-the demonstration that cell suspensions could be cedure of Puck is similar in principle to the useused to seed culture vessels, attempts were made of "conditioned medium" by Sanford et al. (155)to propagate such cells directly in a fluid medium. for the isolation of single-cell strains. In laterOwens et al. (126, 127) showed that mouse studies (104, 140), it was shown that the "feederlymphosarcoma cells could be propagated in layer" was unnecessary and that colony growthtubes of media under constant agitation by a from single cells could be obtained from normaltumbling procedure. Earle and associates (39-41) as well as malignant cultures. In an extension ofextended these studies to the growth of strain this work (142), mutants with different growthL and mouse liver epithelium cells, employing requirements were demonstrated to occur in theboth rapidly rotating tubes and large flasks agi- HeLa cell strain.tated by a commercial shaker. Similar prolifera- E. Nutritional depletion techniques. Cell nutri-tion of monkey kidney cells in rotating cultures tion studies have been complicated by the carryhas been reported (64). These methods make it over of nutritional substances from the organismpossible to prepare cultures of almost unlimited or by preliminary cultivation in highly enrichedsize from a variety of tissues and cell types. media containing uncharacterized materials.

C. Monolayer tissue cultures and plaque tech- Thus, in preliminary studies on a syntheticniques. Cell suspensions planted in stationary medium (110), freshly explanted chick embryonictubes or flasks develop into uniform and coherent tissues were cultivated in complex natural mediasheets. These monolayer tissue cultures have until the cultures became established and thebeen shown to be extremely susceptible to virus synthetic media could be added. Modificationsinfection, with the cell destruction accompanying of technique made it possible to start the culturesvirus multiplication resulting in plaque formation directly in synthetic media (120) but the carry(21). The studies of Dulbecco and Vogt (22-24) over of nutritional substances from the organismand of other workers (125, 136) have demon- could not be eliminated. Recent studies havestrated that plaque formation in tissue cultures shown (121) that freshly explanted tissues mayresults from the multiplication of single virus be depleted of nutritional reserves by initialparticles. Hence, it follows that the quantitative cultivation in a simple salt solution. Provided thetechniques already established for bacteriophage depletion period is not continued too long, thestudies may now be applied to the animal virus- cultures revive completely on the addition of thetissue culture system. synthetic media and survive for a normal period

D. Plating methods and cell genetics. With the without evident alteration in metabolic activity.long-term serial transfer of cell strains made This nutritional depletion technique has beenpossible by the newer cell suspension techniques, found to intensify greatly the cell demand forvariations within the cultures have become essential nutrients, such as coenzyme A, and itsapparent. Thus, Sanford et at. (159) found varia- use may make it possible to detect nutritionaltions in both transplantability and morphology requirements with increased precision and sensi-within a strain of mouse fibroblasts derived from tivity.a single cell, and the development of "altered" F. Measurement of packed cell volume. Manycell strains on repeated transfer of several types of the methods used for measuring populationof cultures has been reported (134). Quantita- increases in cell cultures involve destruction oftive evaluation of the population genetics of cell the cells. To obviate this difficulty, a simple

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hematocrit method has recently been proposed been widely applied in cell nutrition studies. Offor nutritional studies with the L strain (174). the methods recommended, determination of de-The cells are removed from the culture vessels by oxyribonucleic acid phosphorus (79) appears todispersion with trypsin, quantitatively trans- offer the most valid criterion of cell multiplica-ferred to a Van Allen hematocrit tube (171), and tion but the method is tedious and time-con-centrifuged. The packed cell volumes were found suming. Measurement of total protein (128) isto be in good agreement with total cell counts. simpler and more rapid but requires the assump-Similar measurement of packed cell volumes has tion that total cell protein remains constantbeen employed extensively to standardize vaccine throughout the growth cycle of the cultures.suspensions, and a somewhat similar method was Measurement of rate of incorporation of C14-used in earlier studies on tissue culture metab- labeled compounds (102) appears very promisingolism (58). It would appear that such techniques for future application because the method isafford a simple and rapid screening method for accurate and does not require destruction of thecell nutrition studies. cultures. Use of the color change of phenol red to

G. Chemical measurement of growth. Consider- indicate the presence of poliomyelitis virus or itsable effort has been expended in attempts to antibodies in monolayer tissue cultures has beenestablish a simple and reliable chemical method advocated (154) as a rapid chemical methodof determining growth in tissue cultures. Early based on the metabolic activity of the cultures.methods (130), based on dry weight, meas-urement of total metabolism by manometric III. NUTRITIONAL REQUIREMENTS OF ANIMALtechniques, lactic acid production, or glucose uti- TISSUE CELLSlization, proved to be variable and generally un- In the whole animal, the cells comprisingsuitable. As a consequence, attention has re- peripheral tissues receive their nutrients throughcently been focussed on the determination of the extracellular body fluids. Logically, therefore,specific tissue components, such as the nucleic acid ephosphorus. Willmer (190) introduced the meas- elymphs(76),uplasa (13), andetssueaextractsurmn of.cepaenpopou n h lymph (76), plasma (13), and tissue extracts,

valurem of nuclmepthotein phosphorusdb Danddthe particularly chick embryo extract (12, 13). Al-value of this method was confirmed byDavidsonis ,and Waymouth (18). This procedure was extended though these "natural" media permitted the

by Davidson et al. (19) to include fractionation long-term cultivation of a variety of cells andofthe.ucleoproiphosphorus.ino tissues, their complexity and variability made it

of theonucleoprotein pohrs into r ubnuic nearly impossible to identify individual growth-and deoxyribonucleic acids and a definite quantifaco reureet wtan ceaiy. Wittative relationship was found (20) between the the roduion ofth newertissue cuturate of protein synthesis and the rate of accumu- me .todsasiscusseinpeceding paragralatinorionuleiaci phsphrus Helymethods, as discussed in preceding paragraphs,lation of ribonucleic acid phosphorus. Healy, an thfomltnofsteicademy-

et al. (79), studying the multiplication of strain the me i ation o ncel rir-*els shwdtamesrmnofdxyio thetic media, specific information on cell require-

L cells, showed that measurement of deoxyribo- ments has been obtained for the first time. Thesenucleic acid phosphorus could be correlated with ments wilbe discussdeirelicellcouns.LandWinnck (02) oundthatspecific requirements will be discussed in relation

cell counts. Lu and Winnick (102) found that to geea.urtoa.ricpe n ilbchick embryonic cultures rapidly incorporated relatedetorelentrsties witcomlexanatrlbC14-labeled adenine from the culture medium med Foreaemore comreesive tementuofand that the rate of incorporation could be hmedea. For a more comprehensive treatment of

correlated with rate of growth. Oyama and Eagle referred torthreviewshbyWaymouth (172)rand(128) measured cell protein by the phenol reagent rgn(0)adto the tissue cutu bibliog-andfoudtathisdetrmiatin culdbe on-Morgan (109) and to the tissue culture bibliog-

and found that this determination could be con- raphy edited by Murray and Kopech (123).verted to dry weight, nitrogen, or cell counts by A. Amino acids. Possibly the first applicationappropriate conversion factors. of newer tissue culture techniques to the elucida-Although chemical determinations of growth tion of specific nutritional requirements was the

have the advantage of inherent precision, the demonstration by Eagle (25) that 12 amino acidsprocedures suffer from the disadvantage of gener- (arginine, cyst(e)ine, histidine, isoleucine, leu-ally requiring destruction of the tissue cultures. cine, lysine, methionine, phenylalanine, threo-For this reason, chemical procedures have not nine, tryptophan, tyrosine, and valine) were

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24 JOSEPH F. MORGAN [voL. 22

essential for growth of the L strain. The same 12 TABLE 1amino acids were found to be essential also for Comparative amino acid requirements ofthe human carcinoma HeLa cell (26). Omission various types of tissue cultures, ofof any one of these essential amino acids arrested Tetrahymena geleii, and of man*cell growth and this effect, in turn, could be TypeandReferencecompletely reversed by restoration of the missingamino acid. These studies established for tissuecultures the same basic principles on which Amino Acid ."_bacterial and animal nutrition responses are o 1ordinarily obtained. .W co

It should be borne mind that the medium _nAemployed by Eagle (25, 26) consisted of a limitednumber of chemically defined compounds, sup- Arginine + + + + + -S -Splemented with varying amounts of either whole Histidine + + + + + + -or dialyzed human or horse serum. The possibility Lynephan + + + + + + +cannot be excluded that breakdown of the serum Penylalanine + + + + + + +proteins influenced the amino acid requirements Tyrosine + + + + + - -Sof the cells under these conditions, since tissues Cyst(e)ine + + + + + - -Scultivated in plasma rapidly liquefy the coagu- Methionine + + + + + + +lum. This proteolytic activity can be prevented Serine _ _ _ - + -S -

by antitrypsin (53, 108). In earlier studies, in Threonine + + + + + + +which a dialyzed serum and embryo extract base Leucine + + + + + + +was used, Fischer et al. (52) found that the Isoleucine - + + + + + +absence of cystine, arginine, tryptophan, glu- Valine + + + + + -S +tamine, histidine, or proline decreased the radial Aspartic acid X .._ _ _ _outgrowth from chick heart explants. Alanine X ..___

Investigations on the amino acid requirements Proline X .._ _ _ _of chick embryonic heart fibroblasts cultivated Hydroxyproline X .- - - - -

in completely synthetic media (110, 115) by the Glycinenutritional depletion technique (121) have shown Glutamine - + + + +that omission of arginine, histidine, lysine, tyro- Asparagine +sine, phenylalanine, tryptophan, cystine, me- * Indicates essential, - indicates nones-thionine, threonine, leucine, or valine markedly sential, S indicates stimulating, X indicates in-reduced culture survival (118). Omission of hibitory.serine, isoleucine, glycine, or glutamine was t The media used to determine these require-without effect, but omission of glutamic acid, ments contained various proportions of serum.aspartic acid, alanine, proline, or hydroxyprolineimproved culture survival. Concurrent studies acid, aspartic acid, alanine, proline, and hydroxy-on the amino acid metabolism of these cultures proline, which were found to be somewhat in-by paper chromatography (135) showed patterns hibitory for survival of chick embryonic heartof amino acid uptake and release in the syntheticmedium that corresponded with the cell require- firbat 18,wr peiul on obmeniumtshfo survialntedessential amin acids nonessential for growth of the L strain and HeLawere foremovedfuromv thein-entialamn ,c. cell (25, 26). These differences may possibly bebiry ones accmute. Tese restssge attributed to differing requirements for cell

growth and survival or to the complicating effectthat active transamination may be producing of serum in the media used for the growthexcessive and possibly inhibitory end products experiments.of metabolism, a suggestion that is in agreement The amino acid requirements recently deter-with the finding by Westfall et al. (182) that minedfor chick embryonic heart fibroblasts (118),HeLa cells cultivated in serum and embryo the L strain (25, 32), HeLa cell (26, 32), Walkerextract cause an appreciable accumulation of 256 carcinosarcoma (106), and rabbit fibroblastsa-keto acids in the culture medium. Glutamic (67) are listed in table 1. For comparative pur-

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poses, the amino acid requirements of Tetra- (88, 89) have established the complexity of thehymena geleii (88) and of the adult human (148) amino acid interrelationships and have distin-are included. In general, there is a remarkable guished between the absolutely essential aminosimilarity in the pattern of essential and non- acids and those which are stimulating becauseessential amino acids for the various types of of a slow rate of synthesis by the organism. It istissue cultures: arginine, histidine, lysine, tryp- to be hoped that similar studies with tissue cul-tophan, phenylalanine, tyrosine, cystine, methio- tures will provide basic information on the in-nine, threonine, leucine, isoleucine, valine, and dividual metabolic pathways of the essentialglutamine are required for all culture types. amino acids.Exceptions to this common pattern are to be The importance of amide nitrogen for thenoted in the nonessentiality of isoleucine and growth and survival of tissue cultures has re-glutamine for chick fibroblasts, the necessity of cently been emphasized. Eagle and co-workersserine for rabbit fibroblasts, and the need of (32) found that both strain L and HeLa cellsasparagine by the Walker 256 cell. These differ- required L-glutamine for survival and growth.ences from the common pattern appear to be Glutamic acid, at high concentrations, could becharacteristic of the different cell types studied substituted for glutamine with the HeLa celland suggest that comparative biochemistry may but not with the L strain. With the Walker car-ultimately prove of value in characterizing differ- cino sarcoma 256, Neuman and McCoy (124)ent cells in tissue culture. found a dual requirement for glutamine andTwo major differences are apparent between asparagine. Neither -aspartic acid nor L-glu-

the requirements of tissue cultures and those of tamic acid, with or without ammonium chloridethe adult human. All cell cultures so far tested and adenosine triphosphate, was able to replacerequire both phenylalanine and tyrosine and also the corresponding amide. Massive quantities ofboth methionine and cystine. Extensive studies glutamine were incapable of replacing asparagine,on the sulfur amino acid metabolism of chick nor could asparagine replace glutamine. Withembryonic heart fibroblasts (114, 116) have two strains of mouse liver cells, Westfall andshown that these cultures have an absolute co-workers (183) found that, with glucose present,requirement for L-cystine, which cannot be com- both strains grew slowly in the presence of glu-pletely replaced by any other sulfur compound tamine and both died in its absence. In thetested, with the exception of L-cysteine. The absence of glutane, the cells of both liverrequirement for methionine, however, was found strains were unable to use glucose or amino acidsto be a supplementary one which could be demon- from the nutrient medium and could not producestrated only in the presence of L-cystine. Under lactic or keto acids. In contrast to these observa-these conditions, L- and D-methionine were tions, Morgan and Morton (118) found thatequally effective. This appears to be the first glutamine was not essential for the survival ofdemonstration of the biological activity of a either normal or nutritionally depleted chickD-amino acid in tissue cultures. In general, the embryonic heart fibroblasts. During cultivationi-forms appear to be active and the D-forms inac- of these tissues in completely synthetic media,tive but not toxic. It is of interest that in the glutamine increased in concentration in thenutrition of the chick (65) and of man (149), growth medium (135).D-methionine has also been found to be as effec- Most of the recent observations are in accordtive as the L- or DLformB. with the earlier findings of Fischer (54) on the

Nutritional studies with Tetrahymena have importance of glutamine in supplementary mediabeen suggested (68) as a pattern for the study of for tissue cultures. The relatively high blood leveltissue culture nutrition. The amino acid require- of glutamine in mammals and the involvement ofments of this ciliate, as listed in table 1, appear glutamine in amino acid transport across cellto occupy a position intermediate between the membranes, in transamination reactions, in trans-requirements of tissue cultures and those of man. peptidation, in stimulation of glycolysis, and inIn general, this organism appears to be less exact- providing nitrogen for purine synthesis (107) alling in its nutritional demands than the tissue support the concept that glutamine may be anculture systems so far tested. It should be noted essential factor for metabolism and growth ofthat extremely detailed studies with Tetrahymena many tissue cultures.

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TABLE 2 synthetic media for tissue cultures have containedCarbohydrate utilization by tissue glucose at the same 100 mg per cent level (25,

cultures* 26,45, 80, 110, 115). On the other hand, the early

Refer- synthetic media of White (184, 185) containedUtilized Reference Not Utilized ence 850 mg per cent glucose and, more recently (188),

a similar level of fructose has been substituted.A. Definitely Sucrose 2, 73 The defined nutrient medium of Waymouth (173)Glucose 2, 29, 73 Lactose 2, 73 contains glucose at a level of 500 mg per cent.

Manntose 2, 29, 73 Xylose 2 ' From these and earlier studies (172), it seemsFructose 2, 29, 188 Ribose 80B. Possibly Arabinose 2, 73 that the carbohydrate concentration is not a

Galactose 2, 29 Glucosamine 2 critical factor in cell nutrition studies and thatMaltose 2, 29 Starch 2 tissue cultures can tolerate much higher concen-

Glycogen 2 trations of glucose than the physiological level.I____-- Employing dialyzed serum and embryo extract

* These results are based primarily on studies as a base, Astrup et al. (2) found that fructose orwith chick heart fibroblasts cultivated in dialyzed mannose could be substituted for glucose. Slightmedia (2, 73), and on preliminary studies with L activity was found with galactose or maltose, butand HeLa cells (29). sucrose, lactose, xylose, arabinose, glycogen,

In considering recent studies on the amino starch, glucosamine, lactate, pyruvate, or f3-glyc-acid requirements of tissue cultures, it must be erophosphate were completely ineffective. In a

remembered that the method employed, in most recent re-investigation of this problem, employ-inssbeen the technique of omitting ing similar but more closely controlled conditionsinstances,has 72)Harris and Kutsk (73)found that D-glu-

single ammo acids individually from a complex (7), y ( )medium. The unsuitability of this procedure for cose, D-mannose, and D-fructose were utilized

studies has been emphasized by the cells but that L-glucose, D-galactose,bGlacstel tone(63) since complex interrelation-D-sucrose, D-lactose, D-ribose, D-xylose, andbyGladstoe(,secD-arabinose were not. The present information

ships between amino acids would not be detected. .tTheomplxitof uchinterelaionhipshason the carbohydrate requirements of tissue cul-The complexity of such interrelationships has trsi umrzdi al .Fo hs.- ST... ...... .tures is summarized in table 2. From these

been shown by the studies of Kidder and Dewey studies (table 2), it seems that the cells so far(88, 89) with Tetrahymena, and those of Morgan tandortn (14,116 wih cickheat.fbro tested have a restricted and highly selectiveand Morton (114, 116) with chick heart fibro-

reurmn o abhdaeblasts. The recent investigations on amino acidrequirements of tissue cultures must, therefore, Precise information on the pathways of carbo-

i hydrate utilization in tissue cultures is extremelybe regarded as largely preliminary stepsinm the limited. Wilson et al. (192) and Willmer (190)ultimate elucidation of precise information. have found that 60 to 70 per cent of the glucoseFurther detailed studies, preferably carried out utilized apeared as lactic acid but that the ratein the absence of added protein, are necessary to ..uncoveribsenterreton amiedprotein, ~noeacidsatoof utilization of glucose was a function of the glu-uncovripbeacose concentration and could not be correlatedand .to establish with certainty the absolute with rate of growth. Studies on the glucose util-

requirements of cell types. ization of mouse fibroblasts and mouse liverB. Carbohydrates. In contrast to the precisedatarecentydobtaed.Inontramino acd precir epithelial cells (178) showed that both cell typesdata recently obtained on amino acid require-reoe lcs rmth utr eimalittleqeremoved glucose from the culture medium atments of tissue cultures, relatively little nvestiga-' . anu~~~aroximately the same rate but that the 1lyco-tion has been made of the energy requirements gtyg y

of the cells. Early investigators, employing media gen contentsoThele rcesllsi25cte thatconsisting of serum and embryo extract, ordi- of the fibroblasts. These results idicate that thenarily incorporated glucose in the nutrient fluid distinctive metabolic activities of different cellat the normal blood level of approximately 100 mg types are maintained during cultivation in vitro.per cent. Much of this earlier work has been Studies by Pomerat and Willmer (137), employ-reviewed in detail by Waymouth (172) and, ing complex natural media, showed that respira-therefore, will not be discussed here. The major- tory poisons and conditions interfering withity of the recently developed synthetic and semi- phosphorylation did not affect growth. These

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results suggested that direct glucose breakdown, TABLE 3without preliminary phosphorylation, might be Vitamin relationships in tissue cultures*the major pathway in tissue cultures. On theother hand, recent experiments have shown thatmetabolic inhibitors, such as cyanide (17), azide, . . Chickiodoacetate, and dinitrophenol (10), inhibit Vitamn L Strain L embryonicand HeLa Strain fibroblastscellular growth as well as respiration. In addition, (27, 33) (4i6,8)0,Barban and Schulze (6) have shown that HeLa (184)(96 110)cells metabolize glucose terminally via the tri- Thiamin ++++ 0 0 0carboxylic acid cycle and that all the enzymes of Riboflavin ++++ 0 0 0this cycle are present in these cultures. Leslie Nicotinamide (Niacin) ++++ 0 0 0and Paul (98) found that insulin had a direct Pyridoxal (Pyrid- ++++ 0 0 0growth-promoting action on chick embryonic oxine)heart cultures by stimulating increased energy Pantothenic acid ++++ 0 0 0production through its control of pyruvate Choline ++++ 0 0 0metabolism. Inositol ++++ 0 0 0

It is evident that present knowledge of the Ascorbic acid ++ 0 0

energy requirements of cell cultures is extremely Vitamin A - 0 0 + +sketchy and that investigations employing com- Vitamin B12 ++pletely synthetic media should produce much p-Aminobenzoic acid - 0 0valuable and necessary information. Such in- Biotin - 0 0 0vestigations might determine the major path- Vitamin D - 0 0ways of carbohydrate utilization and establish Vitamin E - 0 0whether glycolytic mechanisms are of primary Vitamin K - 0 0importance or whether alternate shunt pathways * + +++ Indicates essential ++ indicatesexist. On the basis of the present knowledge, it stimulatory, 0 indicates vitamins added as adoes not seem likely that specific carbohydrate block to synthetic media as beneficial withoutrequirements of cells can be used as a basis for demonstration of individual requirement, -

identification of cell types. indicates nonessential.C. Vitamins and coenzymes. A considerable

literature exists on the effect of individual vita- been incorporated in most synthetic tissue cul-mins on growth and differentiation in various ture media (110, 184) but no direct effects fromtypes of tissue cultures (172). In general, how- this vitamin alone have been demonstrated.ever, the experiments were carried out in complex It should be borne in mind that the earlynatural media and unequivocal demonstration of synthetic media (110, 184) contained a completevitamin requirements was difficult. Recently, the supplement of vitamins. These were incorporateduse of improved quantitative methods and syn- as a group at physiological levels, without in-thetic or semisynthetic media have made it dividual experiments to establish actual require-possible to establish vitamin requirements with ments. The recent studies, under more precisesome degree of certainty. Eagle (27) has shown conditions (27, 33), have merely provided datathat specific vitamin deficiencies can be created which, in most cases, have justified the formulasin cultures of strain L and HeLa cells and that of the original media. These vitamin relationshipsthese deficiencies, in turn, can be corrected by in tissue culture are summarized in table 3.administration of the missing vitamin. Under Experiments to date have established precisethese conditions, seven vitamins were found requirements for certain of the water-solubleessential for survival and multiplication: choline, vitamins but similar studies with the fat-solublefolic acid, nicotinamide, pantothenic acid, pyri- group are still lacking. Fell and Mellanby (47)doxal, riboflavin, and thiamin. In an extension showed that hypervitaminosis A produced pro-of these studies (33), inositol was also found to be found changes in limb bud rudiments cultivatedessential. In addition to these compounds, vita- in vitro. Subsequently, these same investigatorsmin B12 has been shown (46, 188) to be beneficial (48) found that excess vitamin A in the culturefor growth of strain L cells. Ascorbic acid has medium of chick ectoderm prevented keratiniza-

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TABLE 4 the parent vitamin. Studies on nutritionallyInterrelationships between coenzymes and depleted chick heart fibroblasts cultivated in

vitamins in tissue cultures synthetic media (121) have shown a specific

Relative Activity requirement for coenzyme A in the presence ofCoenzyme Parent Vitamin of Coenzyme to excess pantothenate. It is also of interest that

Vitamin coenzyme A has been shown (7) to prevent theCocarboxylase Thiamin Equal (30, 89) partial mitotic inhibition caused by 6-mercapto-Diphosphopyri- Nicotinamide Equal (30, 89) purine in cultures of embryonic mouse skin anddine nucleotide Crocker mouse sarcoma 180. The interrelation-

Triphosphopyri- Nicotinamide Equal (89) ships between coenzymes and their parent vita-dine nucleotide Less (30) mins in tissue culture are shown in table 4.

Flavin mononu- Riboflavin Equal (30) In general, studies on vitamin and coenzymecleotide requirements of tissue cultures have not estab-

Flavin adenine Riboflavin Equal (89) lished whether significant qualitative or quantita-dinucleotide Less (30) tive differences exist between different cell types.Pyridoxal phos- Pyridoxal Less (30) Inclusion of the conjugated forms in the culture

Coenzyme A Pantothenic Equal (89) medium must depend, to a considerable extent,acid Less (30) on the degree of permeability of the cell mem-

Independent brane to these substances. This factor does not(121) appear as yet to have been adequately investi-

~~~~gated.

andeepithelium to differentiate D. Purines, pyrimidines, and nucleic acid com-tionandcausedthe ~~ponente. Studies on the purine and pyrimidineinto mucous membrane. Lasnitzki (96), extend- rqeentS of ti e cures hav yriededing~~~~~~~~~~~ ~ths.netgtoshoe htecs ia requirement of tissue cultures have yielded

equivocal results. Hopkins and Simon-Reuss (82)mun A in the culture medium of chick heart found that hypoxanthine, but not adenine, stimu-fibroblasts increased the mitotic rate, cell density, lated the growth of periosteal fibroblasts. Ehrens-and size of outgrowth. Although vitamin A was vIrd et al. (42) also found hypoxanthine, but notpresent in White's synthetic medium (184) and adenine or guanine, to be beneficial when incor-medium 199 of Morgan et al. (110), its concentra-tion in these media was less than 1 per cent of the m atsupplementy ediu Trowellactive level found necessary by Fell and Mellanby (6)cliaiglmhndsi yteimedia, found no beneficial effect from any of the(47, 48). purine or pyrimidine bases, including hypoxan-The relation between vitamins and the coen- thine and adenine.

zyme forms of the vitamins in tissue culture In the development of synthetic medium 199,nutrition is not clearly established. Healy et al. Morgan et al. (110) found the addition of adenine,(80) found that the addition of coenzyme A, di- g .nie xanthme hypoxanthe thymine andphosphopyridine nucleotide (DPN), and tri- uracil to be beneficial for the survival of chickphosphopyridine nucleotide (TPN) to a synthetic embryonic heart fibroblasts. Subsequent studiesmedium greatly accelerated the rate of multi- on the nutrition of strain L cells by Healy et al.plication of strain L cells, although responses to (80) indicated that the purine and pyrimidinethe individual three coenzymes could not be bases were inhibitory to growth. Replacement ofshown. Subsequently (81), three additional co- these bases by a mixture of the deoxyribosidesenzymes, flavin adenine dinucleotide (FAD), of adenine, guanine, and cytosine plus thymidineuridine triphosphate (UTP), and cocarboxylase .. )(TPP), were added to the medium and thiamin, A 5-methyndetidine, avcatedriboflavin, niacin, niacinamide, and pantothenate coplex synthetic mediumrrecentlyfoundomitted. In studies with the L strain and HeLa capable of supportig proliferation of strain Lcell, cultivated in the presence of serum, Eagle cells (45), contains deoxyadenosine, deoxyguan-(29, 30) found that a number of cofactors pos- osine, deoxycytidine, thmidine, and 5-methylsessed essentially the same activity as the corre- cytosine. In a comparison of various syntheticsponding vitamin but that in many cases the media, however, it was found that omission of theconjugates proved significantly less active than purine and pyrimidine bases decreased the sur-

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vival of chick heart fibroblasts (117). Whether TABLE 5these contradictory results, as summarized in Effect of purine and pyrimidine basestable 5, reflect varying purine and pyrimidine and conjugates on tissue cultures*requirements by different cell types or differ- TissueCultureandReferenceences in the testing methods still remains to bedetermined. Orotic acid, which is known (167) Compound Tested Chick heart fibroblasts Lto be a precursor of the mono-, di-, and triphos- (110 42 (101, (S8trin)phate pyrimidine nucleotides, the pyrimidine 117) 102)coenzymes, and the pyrimidine moieties in both Adenine + - + Xribo- and deoxyribonucleic acid, does not appear Guanine + - ' Xto have been studied in tissue culture media. Hypoxanthine + + X

Adenosine triphosphate (ATP) was included Xanthine + Xin the formula of Fischer's supplementary Thymine + Xmedium (52) and has subsequently been in- Uracil + Xcorporated, together with muscle adenylic acid, Adenosine-51-phosphate + Xin synthetic medium 199 (110) and modifications Adenosinetriphosphate + Xof this medium (115, 117). In synthetic media Uridine triphosphate +designed specifically for L strain cells (45, 80, 81), Deoxyadenosine +

Deoxyguanosine +these compounds have been omitted, as being Thymidine + +possibly inhibitory substances. Omission of these Deoxycytidine +compounds, as well as the purine and pyrimidine 5-Methyl Deoxycytidine +bases, was found to decrease the survival ofchick heart fibroblasts (117). Further studies on * + Indicates beneficial, = indicates doubtfulthe energy requirements and permeability of effect, - indicates no effect, X indicates in-tissue cultures are required before the role of ATP hibitory.and adenylic acid can be clarified,-and synthetic media will eventually provideTracer techniques have recently been employed answers to these questions.

in studies of the nucleic acid metabolism of tissue E. Lipi. Information on the possible lipidcultures. Thus, C14-labeled adenine was readily requirement of tissue cultures is scanty and diffi-utilized (101) for nucleic acid synthesis by chick cult to interpret. Jacquez and Barry (83) foundembryonic heart tissue cultivated in 25 per cent that free fatty acids were toxic to fibroblasts andembryo extract. The radioactivity was found in that serum albumin prevented this toxicity bythe adenine and guanine constituents of both the binding the fatty acids. Shaffer et al. (161) de-ribonucleic and deoxyribonucleic acids. Under veloped methods for the microdetermination ofthese conditions, C14-labeled guanine was poorly lipids in tissue cultures and demonstrated (66)utilized. Various stimulatory agents increased the that vitamin B12 and folic acid prevented therates of uptake of C'4-labeled adenine and thymi- accumulation of lipid in old cultures.dine, and the rate of growth could be correlated In the development of synthetic medium 199,with the rate of incorporation of these com- Morgan et al. (110) incorporated oleic acid, usingpounds (102). Chemical analysis of the nutrient Tween 80 as the source of fatty acid and also as a

medium during cultivation showed an extensive vehicle for dissolving cholesterol in a minimaluptake by the cells of nucleotides and nucleic amount of ethyl alcohol to avoid the toxicity of

acids,ak d the utilization ofCby-labeled nucleo this compound (131). Extensive studies (119)tides and tutlization of strabednucleo- showed that high concentrations of Tweens were

Atidesand nucleic acids was demonstratedt(103). toxic, unless used for only short periods of timeAt the present time, it is impossible to state (121) but that lower levels could be used without

definitely whether the purine and pyrimidmne deleterious effects on the cells. A clear-cut bene-bases, as such, are essential for cell nutrition, ficial action from the fatty acid components waswhether these materials are preferentially utilized not established but a slight general improvementas nucleotides, or whether the cells can synthesize in the culture medium was noted. Evans andtheir nucleic acids from smaller building blocks. co-workers (45) included the methyl esters ofIt is possible that the use of tracer techniques linoleic, linolenic, and arachidonic acids, as well

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as cholesterol and Tween 80, in the formula of a TABLE 6completely synthetic medium for strain L cells. Effect of inorganic ions on tissue cultures*Subsequently, these investigators (46) foundthat the unsaturated fatty acid group could beomitted from the medium without adverse effect. Inorganic Ion Chick heart Rat L S

fibroblasts articular LndStraiOn the basis of our present knowledge, it seems (74, 110, 111, femr and HeLathat a specific lipid requirement for tissue cul- 112, 184) (31)tures has not yet been demonstrated but, con- Na+ 0 0 ++++versely, the absence of a lipid requirement has K+ 0 ++++ ++++not been established with certainty. Ca++ 0 ++++ ++++

F. Accessory growth factors. Various accessory Mg++ 0 ++++ ++++growth factors have been incorporated into syn- Cl- 0 0 ++++thetic and semisynthetic media for tissue cul- H2PO4 0 0 ++++tures, without clear evidence for the essential HCOi- ++++ 0nature of these compounds. Thus, Fischer's sup- Ce-+ or Fe Xplementary medium (52) contained fumarate, BaC- Xmalate, oxalacetate, succinate, f3-glycerophos-phate, and fructose diphosphate. Considerable * ++++ Indicates essential, ++ indicatesemphasis was placed on the importance of fruc- stimulatory, - indicates non-essential, X in-tose diphosphate but confirmatory evidence for dicates inhibitory, 0 indicates ions normallythe value of this compound is lacking. Medium present in the culture medium but specific in-199 (110) and subsequent modifications of this dividual requirements have not been demon-medium (80, 81, 115, 117) contain sodium ace- strated.tate but the importance of this substance isuncertain. Sodium glucuronate has been incor- ural and synthetic, are prepared in an inorganicporated in the formula of a synthetic medium salt solution base designed to maintain the iso-designed for strain L cells (81) but the role of tonicity of the culture environment. It hasthis compound in cell nutrition has not been generally been assumed that the composition ofestablished. Similarly, glucosamine has been the balanced salt solution should approximateadded to another synthetic medium for strain L that of body fluids and, consequently, the mostcells (45) but an absolute requirement for this widely used solutions (36, 61, 70, 184) bear acompound alone has not been shown. It is known, close resemblance to the formula of Tyrode'showever, that cultures of connective tissue do solution (170). The complexity of these basal saltsynthesize hexosamine (8). In studies on mouse solutions has made it difficult to determine thesarcoma 37 grown in tissue culture, Rubin et al. precise inorganic requirements of tissue cultures.(153) found a deleterious effect from D-glucos- Thus, Harris (74), employing a dialyzed mediaamine but no toxicity from N-acetyl-D-glucos- base, found that bicarbonate was essential for theamine. Fjelde et al. (55) also reported the toxicity maintenance and outgrowth of chick heart fibro-of glucosamine for human tumor cells in tissue blasts. Shooter and Gey (164), using a complexculture. These results are of interest in view of natural medium together with chelating agents,the earlier report by Quastel and Cantero (143) found that calcium, magnesium, and potassiumthat injection of D-glucosamine inhibited sar- were essential. Recently, Eagle (31), using acoma 37 tumor formation in mice. defined medium supplemented with dialyzedAlthough evidence for the participation of serum protein, has shown that sodium, potas-

accessory growth factors in tissue culture nutri- sium, calcium, magnesium, chloride, and phos-tion is still inconclusive, the recent demonstra- phate ions are essential for growth and survivaltion by Barban and Schulze (6) that HeLa cells of a mouse fibroblast and the HeLa cell. No re-contain all the enzymes of the tricarboxylic acid quirement for bicarbonate was found. The effectcycle suggests that, under the proper conditions, of inorganic ions on tissue cultures is summarizedintermediates from that cycle might prove to be in table 6.beneficial ingredients of the culture media. Knowledge of the trace metal requirements of

G. Minerals. Tissue culture media, both nat- tissue cultures is still fragmentary, since the com-

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plexity of natural media and the doubtful purity tion and the exact role of the peptides was diffi-of salts used by early workers undoubtedly cult to determine. Thus, Fischer (51) studied thesupplied large quantities of undefined metals. effects of peptic and tryptic digests of lactoglobu-The first use of a trace metal in completely syn- lin and serum albumin, and of amino acid mix-thetic media was the incorporation of iron into tures made up in the same proportion as theWhite's nutrient solution (184). Fischer and unhydrolyzed proteins, on the growth of chickco-workers (52) included the chlorides of iron, heart tissues in dialyzed plasma. Growth wascopper, manganese, zinc, and cobalt in the for- significantly better in the presence of the pro-mula of their supplementary feeding mixture. tein digests and the beneficial effect was attrib-Morgan et al. (111) found that cobalt was very uted to the peptide content of these materials.toxic to cultures in natural media but was much Recently, Eagle (28), employing a syntheticless toxic to cultures in synthetic media. The pro- medium supplemented with dialyzed serum,tective effect of the synthetic media was shown found that amino acid deficiencies in strain L andto be due to complex formation between cobalt HeLa cells could be relieved by dipeptides con-and certain free amino acids in the medium, tainig the limiting amino acids. The effectiveparticularly histidine and cysteine (113). A concentrations of the dipeptides were of the samesimilar effect was shown with barium in natural order of magnitude as those required of the simpleand synthetic media (112). The trace metal con- amino acids and no stimulatory effect from thetent of natural and synthetic media was studied dipeptides was observed. Whether peptides ofby Healy et al. (78) and the high concentrations greater complexity are stimulatory to cell growthpresent in natural, as distinct from synthetic, under these conditions has not been determined.media was emphasized. The significance of these Considerable data exist on the use of gluta-findings in relation to cell growth remains to be thione in synthetic and semisynthetic media fordetermined. tissue cultures. This compound was included in

Further experimentation, particularly with the White's synthetic media (184, 185), and innewer and more precise cell culture methods and Fischer's supplementary feeding mixture (52).the use of chelating agents, is required before all In the latter case, it was observed that a cystinethe major and trace element requirements of the deficiency could be relieved by glutathione (51).cells can be established. The development of new However, in experiments with completely syn-and nontoxic buffer systems to replace bicar- thetic media, Morgan and Morton (114) foundbonate and phosphate is necessary before the role that glutathione could not overcome a cystineof these ions can be determined with certainty. deficiency. This tripeptide was added to syn-Initial experiments have shown (168) that tris- thetic medium 199 of Morgan et al. (110) and its(hydroxymethyl)aminomethane or glycylglycine concentration was increased in a subsequentcan be substituted for bicarbonate in synthetic modification by Healy et al. (80). In these studies,tissue culture media without major deleterious a similar beneficial effect on the growth of straineffects on the cells, and orthophosphite has been L cells was obtained by cysteine and it must berecommended (147) as a nontoxic buffer for tissue assumed that the stimulation observed was aculture studies. The development of suitable response to sulfhydryl groups in general. At thenonbicarbonate buffers for tissue culture media present time, no clear-cut requirement for gluta-might make it possible to employ cotton plugs thione itself has been established in tissue culturerather than rubber stoppers to close the culture nutrition.vessels. This would simplify the culture tech- Because of their wide use in bacteriologicalniques very considerably and would also avoid media, peptones were among the earliest com-the toxicity of most types of rubber stoppers for pounds employed in studies on tissue cell nutri-tissue cultures (132). tion. Thus, Baker and Carrel (3), Baker (4),H. Peptides, peptones, proteoses, and proteins. Baker and Ebeling (5), Wilson et al. (192), and

The early tissue culture literature (123, 172) con- Hanks (69) found that the addition of peptones,tains many references to the stimulatory action particularly Witte's peptone from fibrin, was ofof peptides on cell growth and survival. The value in promoting cell proliferation. Winnickmajority of these early experiments were carried and Winnick (193), carrying out nitrogen balanceout in complex natural media of variable composi- studies, found that partial proteolytic digests

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and complete acid hydrolyzates of C'4-labeled protein altered the physical nature of the cultureprotein were rapidly incorporated into tissue medium.cultures. Recently, Waymouth (175) has found Considerable uncertainty exists as to whetherthat Bacto-peptone, in the presence of a mixture the cells must first hydrolyze the protein in theof amino acids and defined growth factors plus culture medium to amino acids and peptidesserum albumin, appears to supply an essential before utilization or whether the protein can bethreshold factor for the growth of strain L cells. incorporated directly into the cells without priorThe chemical nature of the functional components hydrolysis. Sanford and co-workers (158) showedin the peptone has not yet been determined. that strain L cells failed to grow in a protein-con-

Proteoses of varying degrees of complexity taining medium unless amino acids were added.have also been used in many studies on tissue It thus appeared that the cells were unable tocell nutrition. Carrel and Baker (15) found that hydrolyze proteins in the culture medium. Simi-the proteoses of Witte's peptone possessed larly (49), horse serum residue proved incapablegrowth-promoting activity and Willmer and of supporting strain L cells unless supplementedKendal (189) isolated a heteroproteose from with amino acids and other growth factors.Witte's peptone that stimulated mitosis. More The problem of protein synthesis and aminorecently, Gerarde and Jones (60) observed that acid turnover in tissue cultures has recently beenBacto-proteose peptone would supplement the investigated through the use of proteins labeledactivity of dialyzed embryo extract in promoting with C'4-containing amino acids. Gerarde et al.collagen formation in chick embryo lung cul- (59) found that heart tissue had a low rate oftures. Ginsberg et al. (62) reported the value of amino acid turnover and that 25 per cent embryoBacto-tryptose phosphate broth in maintaining extract was necessary to stimulate protein syn-HeLa cell cultures. Up to the present time, no thesis and growth. Protein from the embryochemical fractionations leading to the identity extract in the medium was the principal source ofof the stimulating factor or factors in proteose protein for cultures of chick lung. Winnick andpeptone appear to have been successful. Winnick (193) observed that during cultivation

Sera of various animal species have been used of chick embryonic heart, both free and con-in the majority of the natural media for tissue jugated amino acids, as well as protein, werecultures (130), usually in conjunction with chick removed from the medium. Francis and Winnickembryo extract (14). With such a complex sys- (56) found that during the initial stages of growthtem, the exact role of the serum has been diffi- of chick embryonic heart cultures, tissue proteincult to determine although it has generally been was synthesized chiefly at the expense of aminobelieved that serum contributed to the main- acids in the nutrient fluid but that subsequentlytenance of survival rather than to active stimu- the main contribution was from the soluble pro-lation of growth. Thus, Parker (129) showed that tein of the medium. It was concluded that moststrains of chicken fibroblasts were able to pro- of the C14 of labeled proteins could be transferredliferate at a slow rate for long periods in a medium from nutrient medium to heart tissue proteinof chicken serum and Tyrode's solution. With without the release of free amino acids. Westfallthe development of improved quantitative cul- et al. (182) observed that considerably more thanture methods and chemically defined media, half of the total mass of HeLa cell cultures wasinterest in the role of serum has revived. Morgan derived from the protein supplied in the medium,et al. (115) showed that serum exerted only a rather than from the free amino acids present.minor effect on culture survival when added to Recent experiments with rat liver slices andsynthetic medium 199. Sanford and co-workers purified plasma proteins labeled with C14-(157) found that horse serum ultrafiltrate is less phenylalanine (146) have indicated that theseeffective than the serum residue for supporting proteins may penetrate the hepatic cell surfaceproliferation of strain L cells. Fractionation of without extensive' prior degradation.the serum (160) failed to segregate the activity At the present state of our knowledge of cellin any one component and it was concluded that nutrition, the question of the contribution fromeither a nonspecific growth-promoting factor was unhydrolyzed protein in the culture medium isresponsible for the effects observed or that the difficult to answer. Strain L cells have been

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propagated for extended periods of time in corm- cultures has also been observed in extracts ofpletely protein-free media (45) and, under these adult tissues but efforts to fractionate this mate-conditions, the cellular protein must have been rial and identify the active components have notsynthesized from amino acids and other small been successful (177).molecular compounds. On the other hand, when Considerable attention has been devoted tosoluble protein is present in the culture fluid, the the identification of the small molecular weightevidence suggests that such protein is incor- growth-promoting substances in chick embryoporated readily, without extensive hydrolysis. extract. Sanford and co-workers (157) found thatIt may be that cells cultivated in vitro possess a the ultrafiltrate of embryo extract was morenumber of alternative systems for protein syn- effective than the extract residue and was com-thesis and that the functioning of these systems parable in total activity to the whole extract.is regulated by the culture conditions. They concluded that the high molecular weight

I. Unidentified factors. The early tissue culture protein and nucleoprotein fraction could actuallyliterature contains almost innumerable reports on be eliminated from the culture fluid without dimi-the stimulation of tissue cultures by unidentified nution of potency. Westfall et al. (180) deter-components of biological materials (123, 172). mined the free and combined amino acids inThe discovery by Carrel in 1913 (14) that ex- embryo extract ultrafiltrate. Appreciable amountstracts of embryonic tissue possessed remarkable of the normal amino acids were found, in bothgrowth-stimulating properties for tissue cultures free and combined form, but no abnormal com-focussed attention upon this material and at- pounds were detected. The concentrations oftempts to identify its active components have pyruvic and a-ketoglutaric acids in the ultra-continued down to the present time. Fischer (50), filtrate were found (181) to be low in comparisonin an extensive series of investigations, employed with the normal values for blood. Rosenberga dialysis procedure to separate embryo extract et al. (150) employed alcohol fractionation andinto two major components, those of low molec- ion exchange methods to isolate from extractular weight and those of high. This latter frac- ultrafiltrate a fraction possessing the majortion was termed embryonin. The dialysis proce- growth-promoting activity of the original mate-dure of Fischer was later questioned by White rial. The identifiable components of this fractionand Lasfargues (186) and by Harris (72), and were found to be taurine, serine, glutamic acid,more controlled conditions were established. xanthine, uracil, glucose-6-phosphate, glucose,Wolken (194) subjected embryo extract to ultra- ferrous iron, and inorganic phosphate, but thesecentrifugation and observed a distribution of compounds in themselves did not account for thebiological activity in both the sedimented mate- biological activity of the original ultrafiltraterial and the supernatant fluid. Kutsky (94) (151, 152).isolated a nucleoprotein fraction from embryo Ultrafiltrate of ox serum has been recom-extract that contained most of the growth factors mended by Simms (165) and Simms and Sandersof high molecular weight originally present and (166) as a medium for promoting the early growthHarris and Kutsky showed (75) that this mate- of adult tissues, for studies on fat deposition, andrial exerted a synergistic action with the dialyzate for virus propagation in tissue cultures. Consider-of embryo extract. Subsequently, Kutsky and able interest has recently been shown in theco-workers (95) showed that nucleoprotein with chemical nature of the active ingredients in serumbiological activity could be isolated from a ultrafiltrate. Westfall and co-workers (179) ex-variety of embryonic tissues and that materials amined the composition of this material andcontaining cartilage represented the richest identified 23 amino acids or amino acid deriva-sources of activity. Although the growth-pro- tives, as well as at least 2 uncharacterized pep-moting property of the nucleoprotein fraction of tides. Eagle (33) has reported that the activeembryo extract appears definitely established, ingredient in serum ultrafiltrate is inositol.the possibility that this activity may be due to Preoccupation with the growth-promotingsmall molecular factors tightly bound to the substances in serum and embryo extract hasnucleoprotein molecules has not been completely tended to restrict somewhat the search for bio-excluded. Growth-promoting activity for tissue logical activity in other materials. Astrup et al.

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(1) demonstrated the occurrence of accessory between the nutrition of tissue cultures and virusgrowth substances in both animal and plant propagation within those cultures will be con-tissues. Morgan et al. (115) tested the activity of a sidered briefly.wide variety of natural materials incorporated at The use of chemically defined media for tissuegraded concentrations in synthetic medium 199 cultures has made it possible to study, under(110). When relative increase in cell survival controlled conditions, the effect of the extra-served as the criterion, the greatest activity was cellular growth medium on virus propagationfound in chick embryo extract, followed, in order within infected cells. Hare and Morgan (71)of decreasing activity, by whole liver extract, studied the growth of psittacosis virus in culturesbeef extract, horse serum, yeast extract, liver of minced whole chick embryo maintained in afraction L, and bovine amniotic fluid. On the balanced salt solution and found that sustained,basis of effective stimulating concentrations, long-term virus growth did not occur. The ad-greatest activity was found in whole liver ex- dition of either beef embryo extract or synthetictract, followed, in order of decreasing activity, medium 199 (110) greatly stimulated virus prop-by yeast extract, liver fraction L, beef extract, agation. Burr and co-workers (9), investigatingchick embryo extract, horse serum, and bovine the propagation of influenza and mumps virusesamniotic fluid. No activity was detected in corn in chorioallantoic tissues cultivated in syntheticsteep liquor, malt extract, distillers' dried solu- media, found that the extracellular medium hadbles, liver coenzyme concentrate, or ascitic fluid. little influence on virus propagation. Even afterMayyasi and Schuurmans (105) have shown the tissue cells were depleted of intracellularthat strain L cells can be propagated in 0.1 per nutrients by prolonged cultivation in a simplecent Bacto-yeast extract and 0.1 per cent Bacto- salt solution, good virus propagation was stillproteose peptone no. 3, supplemented with 20 per obtained and the yield of virus was not increasedcent horse serum. Kinsey and co-workers (90), by the addition of synthetic medium 199.with cultures of the ocular lens in synthetic Rappaport (144), investigating poliomyelitismedia, found that some essential substance was virus propagation in trypsinized monkey kidneydepleted in both the medium and the lens during cultures, found that good virus synthesis oc-the first 9 hr of cultivation. The depletion was curred in a synthetic medium containing onlynot corrected by various vitamins, coenzymes, cysteine, isoleucine, and D-ribose in a salt-or metabolites and the identity of the essential glucose medium buffered with tris(hydroxy-substance could not be established. These studies methyl)aminomethane. Eagle and Habel (34),make it evident that growth-promoting sub- also studying the propagation of this virus instances for tissue cultures occur in a wide spec- HeLa cell cultures, found that only minimaltrum of biological materials but the chemical amounts of virus were formed if the culturenature of these substances still remains to be medium did not contain glucose and glutamine,determined. even though a wide variety of other nutritional

factors were present. Levine et at. (99) have ob-IV. TISSUE CULTURE NUTRITION AND served that influenza viruses of the parent WS

VIRUS PROPAGATION and derived neurotropic NWS strains can be

The past ten years have witnessed remarkable propagated in chorioallantoic membrane cultures

developments in the application of tissue culture growing in a medium containing glucose. Themethods to virus propagation studies. Cell strains substitution of pyruvate for glucose as the sole

have been developed that are susceptible to in- carbon source permitted propagation of only the

fection with many of the major human and parent WS strain. Comparative biochemicalanimal viruses. Through the cell destruction, or studies (93) on normal and on poliomyelitiscytopathogenic effect, resulting from virus in- virus-infected tissue cultures have suggested thatfection, and the prevention of this effect by type- virus propagation is associated with changes inspecific immune serum, rapid diagnostic tests for the enzymatic activity of the infected cells. Themany of the common virus diseases have become complexity of the test system used makes itpossible. Discussion of the extensive literature on difficult to assess the significance of the bio-this subject (145, 176) is outside the scope of the chemical lesions reported.present article but certain interrelationships The results of these studies imply that the

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contribution of the extracellular medium to feasible. As a result, the large scale methodsintracellular virus propagation may be dis- employed in the antibiotics and fermentationtinctly different with different viruses and, industries may eventually be applied to mam-possibly, characteristic of each virus. At the malian cell cultures; and (f) Use of the modernpresent time, the importance of the composition methods of cell cultivation makes it possible toof the tissue culture medium used for virus employ serial subcultivation, as used in bac-propagation studies requires further clarification. teriology, to study the nutrition and metabolismThe extensive recent literature on the effect of of a variety of cell types.amino acid and vitamin analogues in virus-tissue The early synthetic media for cell cultures wereculture systems suggests that this method devised through the stepwise incorporation ofoffers a promising new approach to the chemo- blocks of nutrients without the establishment oftherapy of virus infections. definite requirements for the individual com-

ponents of the blocks. The objective of theseV. DISCUSSION AND CONCLUSION experiments was to devise media adequate to

The early tissue culture methods, which were support unlimited cell survival and multiplica-devised primarily for studies on cell morphology, tion and then, by a process of elimination, to

were not essentially bacteriological in principle arrive at the essential constituents. Althoughbut relied heavily upon the aseptic procedures of perfectly adequate media for all types of tissue

bacteriology and the bactericidal activity of the cultures have not yet been devised, the presentcomponents of the natural media employed, media make it possible to carry out many types

of nutritional and metabolic studies underWith the current emphasis on synthetic media . .and virus propagation studies, new methods have closely controlled conditions. As these media havebeen developed and techniques simplified, to become progressively more adequate and quanti-

the handling of large numbers of replicate tative tissue culture methods more accurate,pissermi ltures. In many instances, these new

specific nutritional deficiencies have beentissecltues.Inany nstnce, tesenew demonstrated and precise information has begunmethods have been devised through the applica- deaccumuate.athpresentrtime acontionofongestalised actriolgicl tch-to accumulate. At the present time, a consider-tion o long-established bacteriological tech- able body of information has been obtained onniques: (a) The development of pure cell strains

through single-cell isolations and the serialpropagation of such strains from cell suspensions (table 1), and a start has been made on theare primarily of bacteriological origin; (b) The vitamin and coenzyme requirements of cellpreparation of monolayer tissue cultures and the cultures (tables 3 and 4). Knowledge of the

formation of plaques in such cultures by animal carbohydrate requirements of the cells (table 2),viruses are analogous to the bacteriophage- of the role of the purine and pyrimidine basesbacterial cell system. These techniques make (table 5), and of the lipid, mineral (table 6), and

possible the application to animal virus studies of accessory growth factor requirements is stillall the quantitative methods evolved for bac- scanty and incomplete. Although protein, andteriophage work; (c) Plating and clone tech- protein degradation products, such as proteosesniques for animal cells are essentially similar to the and peptones, have proved stimulatory and, inconventional plating methods of bacteriology. some cases essential for cell cultivation, theAs a consequence of these methods, it is ossible exact function of these large molecular weight

individuals from a substances and their mode of penetration intoto select mduul rmaheterogeneouspopulation and study quantitatively the genetics the cell are still not clear. Knowledge of theof mammalian cells; (d) Nutritional depletion relative importance of glycolysis and respirationtechniques and simplified methods of measuring for cell growth in vitro is still incomplete andgrowth by packed cell volume or chemical pro- difficult to evaluate (86). All these areas requirecedures make it possible to carry out tissue cell intensive study with quantitative cell culturenutrition studies under the same rigid con- techniques.ditions as bacterial nutrition experiments; (e) The increasing realization that detailed knowl-The development of methods for the propagation edge of the nutritional requirements of cellof mammalian cells directly in fluid media has cultures is a prerequisite to the wider applicationmade the preparation of massive cell cultures of tissue culture in many fields of research has

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TABLE 7Summarized composition of various synthetic media for tissue cultures*

Medium and Composition Cell Type Growth orReference Copsto el~Survival

Rosenberg's S 27t 2 Amino acids; 1 purine, 1 pyrimidine; hexose di- Chick fibro- Growth(151) phosphate, B12, folic acid. blasts

Fischer's V 614t 10 Amino acids; glutathione, glutamine, hexose di- Chick fibro- Growth(52) phosphate. blasts

Eagle's Mediumt 12 Amino acids, glutamine; 9 B vitamins. L Strain and Growth(29, 33) HeLa

White's medium 15 Amino acids; 9 B vitamins, B12, 2 fat-soluble vi- Chick fibro- Survival(184, 185) tamins; low cysteine, ascorbic, and glutathione. blasts

Morgan's 199 and 19 Amino acids; 10 B vitamins, 6 fat-soluble com- Chick fibro- SurNivalM 150 (110, 115) pounds; acetate, glutamine; purines, pyrimidines, blasts

ATP, adenylic acid; low cysteine, ascorbic, andglutathione.

Healy's 703 (80) 19 Amino acids; 10 B vitamins, 6 fat-soluble com- L Strain Growthpounds; acetate, glutamine; 3 coenzymes; highcysteine, ascorbic, and glutathione.

Morgan's M 416 19 Amino acids; 10 B vitamins; 3 coenzymes; 6 fat- Chick fibro- Survival(117) soluble compounds; acetate, glutamine; purines, blasts

pyrimidines, ATP, adenylic acid; high cysteine,ascorbic, and glutathione.

Healy's 858 (81) 19 Amino acids; 6 B vitamins; 5 coenzymes; 6 fat- L Strain Growthsoluble compounds; acetate, glutamine; 5 deoxy-ribosides; glucuronate; high cysteine, ascorbic,and glutathione.

Evans' NCTC 107 22 Amino acids; 10 B vitamins, 6 coenzymes; 6 fat- L Strain Growth(45) soluble compounds, 3 fatty acids; acetate, gluta- and sur-

mine, asparagine, glucosamine; 5 deoxyribosides; vivalglucuronate, glucuronolactone; high cysteine, as-corbic, and glutathione.

* All media contained a generally similar salt solution with glucose present.t Supplemented with whole or dialyzed serum.

resulted in the development of many synthetic with varying amounts of whole or dialyzed serummedia, of varying complexity and adequacy. has been widely practiced. It is evident from theThe compositions of the synthetic media in most data of table 7 that media intended for supple-general use at the present time are summarized in mentation with serum are considerably lesstable 7. As this table illustrates, the general trend complex than those intended for use withoutin such media is to include as many as possible of supplementation. Thus, 2 media designed forthe nutritional factors already shown to be growth of strain L cultures, Eagle's mediumnecessary for man, animals, or bacteria. Current (29, 33) and Evans' NCTC 107 (45), contain 22emphasis has been placed to a considerable extent organic ingredients plus serum in the one caseon the inclusion of cofactors and conjugated forms and 63 organic ingredients in the other. Bothof nutrilites, on the assumption that cells in media were prepared in a similar basal saltvitro are unable to synthesize such compounds at mixture containing glucose. On the other hand,a sufficient rate. The observation that many media supplemented with serum have been usedcoenzymes are appreciably less active than the with considerable success (25-27) to demonstrateparent vitamins (30) raises some doubt as to the specific nutritional deficiencies and their cure.validity of this assumption and suggests that The actual contribution of the whole or dialyzedrelative permeability of the cells to these com- serum to the nutritional requirements of the cellspounds may be the limiting factor. is difficult to assess, since it is not known with

Supplementation of chemically defined media certainty whether the serum protein as such can

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penetrate the cell surface or must first be broken rapid multiplication is altogether advisable isdown to smaller fragments. The observation rendered somewhat questionable by the recent(117) that media designed for supplementation observation (134) that cell strains in continuouswith serum are completely inadequate to sup- propagation become completely altered in char-port cell survival in its absence suggests that the acter. The part played by adaptation of the cellscontribution of the serum protein is a major one. to a particular medium during rapid serial

In the development of synthetic media for propagation and the possible development of atissue cultures, two general criteria have been variety of nutritionally distinct sublines from oneemployed: long-term maintenance of survival or original strain remain to be determined.rapid multiplication. Failure to distinguish The enzymatic constitution of cell cultures hasbetween these two factors has been responsible not as yet been extensively investigated. Trans-for some conflicting results, since media designed amination activity has been demonstrated byfor the one purpose may not necessarily be Jacquez and co-workers (84) and by Westfallsuitable for the other (117). It seems essential et al. (182). Cholinesterases have been detected inthat all synthetic media recommended for use be chick embryonic cultures (87) and a variety ofclearly related both to cell type and to cultural enzymes related to nucleic acid metabolism haveconditions employed (table 7). been reported to occur in monkey kidney cultures

Although a number of cell strains have been (91, 92). Barban and Schulze (6) have observedcultivated through long-term serial passage in a that all the enzymes of the citric acid cycle occurcompletely synthetic medium (45), similar in HeLa cell cultures. These scattered observa-progress has not been achieved with fresh tissue tions emphasize the general lack of precise in-explants. The most adequate synthetic media for formation on the over-all metabolic activity offresh tissues (110, 117) support cell survival for tissue cultures. It is apparent that further studiesperiods of 40 to 60 days. Initially, such cultures of this nature are urgently needed.exhibit extensive areas of proliferation and these Tissue culture methods are finding increasingareas are subsequently maintained intact for application in the field of cancer research. Cellprolonged periods until degeneration and death cultures have been used to study the antagonisticensue (115, 117). The general picture is that of an effect of antimetabolites on normal and tumorinsufficient rate of synthesis of some factor or cells (7), to screen amino acid analogues forfactors necessary to maintain cell function and selective damage to mouse sarcoma cells (85), andintegrity. The nature of these factors has so far to investigate the cytotoxic action of carcinolyticdefied identification in terms of known nutritional agents (35). Underlying much of the syntheticsubstances and it is possible that some as yet media studies with tissue cultures has been theunrecognized type of growth factor may be hope of finding nutritional differences betweenresponsible. In spite of these drawbacks, how- normal and malignant cells that might proveever, it should be realized that maintenance and susceptible to treatment with specific analogues.survival of fresh tissues for 40 to 60 days in Recent investigations have shown (97) that thecompletely synthetic media provide conditions histological appearance of normal human cellsuitable for many types of study of cell function lines is indistinguishable from that of humanand metabolism. The precise control of cultural cancer cell strains. These studies raise theconditions made possible by the use of com- possibility that the recent techniques of rapidpletely synthetic media more than compensates cell cultivation may selectively propagate malig-for the nutritional inadequacy of these media. nant cell types, even from apparently normal

Considerable controversy has existed as to tissues, or that a transformation from normal towhether long-term survival or rapid multiplica- malignant cells occurs during the rapid culti-tion represents the more nearly normal physi- vation process. Until these factors are under-ological condition for cell cultures. Early cell stood and controlled, metabolizing but notnutrition studies were based primarily on survival actively propagating cultures from fresh tissueas the criterion but with the introduction of explants may possess some advantages over thequantitative cell suspension techniques the rapidly growing cell strains for these studies.emphasis has been shifted almost completely to Modern tissue culture provides a methodrapid growth. Whether this preoccupation with through which the morphology, biochemistry,

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metabolism, survival, and growth of individual ence in HeLa cells of the enzymes of the cit-mammalian cells, the progeny of these cells, or ric acid cycle. J. Biol. Chem., 222,665-670.mixed cell populations may be studied simul- 7. BIESELE, J. J. 1955 Antagonistic effects oftaneously under precisely controlled conditions. 6-mercaptopurine and coenzyme A onThe*increasinknowledge of specific nutritionl; mitochondria and mitosis in tissue culture.The increasing knowledge of specific nutritional J ipy.adBohm yo. ,19

requirements of tissue cultures, gainedthroughl126the application of quantitative bacteriological 8. BOLLET, A. J., BOAS, N. F., AND BUNIN, J. J.procedures, has already proved of considerable 1954 Synthesis of hexosamine by connec-value in virus research. Application of this basic tive tissue (in vitro). Science, 120, 348-349.knowledge of cell nutrition may make it possible 9. BURR, M. M., CAMPBELL, M. E., MORGAN,to control the metabolic activity of cell popu- J. F., AND NAGLER, F. P. 1954 Studies onlations and determine the factors underlying such the propagation of influenza and mumpsfunctions as hormone production and antibody viruses in tissue culture with chemicallyformation by sensitized cells. Tissue culture defined media. Canad. J. Microbiol., 1,techniques may also prove of further value 'M 158-169.techiqusmaalo prve f futhe vale m 10. CAILLEAU, R., MOSS, S., AND SIEGEL, B. V.studying the action of bacterial toxins at the '0'ALEUR. PMos'.N IGL . V.stlludyi theve actiono cterigaltoxinsath the- 1956 Effects of some metabolic inhibitorscellular level and i investigating the phe- on the growth and lipide composition ofnomena of hypersensitivity. Bacteriological Earle's strain L. cells. J. Natl. Cancerprinciples have contributed greatly to the de- Inst., 16, 1011-1019.velopment of quantitative cell cultivation 11. CAMERON,G. 1950 Tissueculturetechnique.techniques. In turn, the application of these Academic Press, Inc., New York.techniques may help to solve many problems in 12. CARREL, A. 1911 Rejuvenation of culturesbacteriological research. of tissues. J. Am. Med. Assoc., 57, 1611.

13. CARREL, A., AND BURROWS, M. T. 1911ACKNOWLEDGMENTS Cultivation of tissues in vitro and its tech-

nique. J. Exptl. Med., 13, 387-396.I wish to express my appreciation to my 14. CARREL, A. 1913 Artificial activation of

colleagues, Mrs. Helen Morton Coval and Dr. the growth in vitro of connective tissue. J.Evan T. Bynoe, for many helpful suggestions in Exptl. Med., 17, 14-19.the preparation of this manuscript. 15. CARREL, A., AND BAKER, L. E. 1926 The

chemical nature of substances required for

VI. REFERENCES cell multiplication. J. Exptl. Med., 44,503-521.

1. ASTRUP, T., FISCHER, A., AND VOLKERT, M. 16. CHANG, R. S. 1954 Continuous sub-culti-1945 Protein metabolism of tissue cells vation of epithelial-like cells from normalin vitro. 2. Accessory growth substances in human tissues. Proc. Soc. Exptl. Biol.animal and vegetable tissues. Acta Phys- Med., 87, 440-443.iol. Scand., 9, 134-161. 17. DANES, B., CHRISTIANSEN, G. S., AND LEIN-

2. ASTRUP, T., FISCHER, A., AND PHLEN- FELDER, P. J. 1954 Inter-relationshipsSCHLXGER, V. 1947 Protein metabolism between respiration and cell growth inof tissue cells in vitro. 5. The accessory tissue culture. J. Cellular Comp. Physiol.,growth substances in sugar-free malt ex- 43, 365-378.tracts. Acta Physiol. Scand., 13, 267-276. 18. DAVIDSON, J. N., AND WAYMOUTH, C. 1943

3. BAKER, L. E., AND CARREL, A. 1928 The Factors influencing the nucleoproteineffects of digests of pure proteins on cell content of fibroblasts growing in vitro.proliferation. J. Exptl. Med., 47, 353-370. Biochem. J., 37, 271-277.

4. BAKERL. E. 1936 Artificial media for the 19. DAVIDSON, J. N., LESLIE, I., AND WAYMOUTH,cultivation of fibroblasts, epithelial cells, C. 1949 The nucleoprotein content ofand monocytes. Science, 83, 605-66.

5. BAKER, L. E., AND EBELING, A. H. 1939 fibroblasts growing in vitro. 4. Changes inArtificial maintenance media for cell and the ribonucleic acid phosphorus (RNAP)organ cultivation. I. The cultivation of and deoxyribonucleic acid phosphorusfibroblasts in artificial and serumless (DNAP) content. Biochem. J., 44, 5-16.media. J. Exptl. Med., 69, 365-378. 20. DAVIDSON, J. N., AND LESLIE, I. 1950 A

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21. DULBECCO, R. 1952 Production of plaques 35. EAGLE, H., AND FOLEY, G. E. 1956 Thein monolayer tissue cultures by single cytotoxic action of carcinolytic agents inparticles of an animal virus. Proc. Natl. tissue culture. Am. J. Med., 21, 739-749.Acad. Sci., 38, 747-752. 36. EARLE, W. R. 1943 Production of malig-

22. DULBECCO, R., AND VOGT, M. 1953 Some nancy in vitro. IV. The mouse fibroblastproblems of animal virology as studied by cultures and changes seen in living cells.the plaque technique. Cold Spring Harbor J. Natl. Cancer Inst., 4, 165-212.Symp. Quant. Biol., 18, 273-279. 37. EARLE, W. R., EVANS, V. J., EDWARD, M. F.,

23. DULBECCO, R., AND VOGT, M. 1954 Plaque AND DUCHESNE, E. 1949 Influence offormation and isolation of pure lines with perforation design on growth of tissue cellspoliomyelitis viruses. J. Exptl. Med., 99, under perforated cellophane sheets in vitro.167-182. J. Natl. Cancer Inst., 10, 291-295.

24. DULBECCO, R., AND VOGT, M. 1954 One- 38. EARLE, W. R., EVANS, V. J., AND SCHILLING,step growth curve of western equine en- E. L. 1950 Extension of cellophane sub-cephalomyelitis virus on chicken embryo strate procedure to growth of in vitro cul-cells grown in vitro and analysis of virus tures of large areas. J. Natl. Cancer Inst.,yields from single cells. J. Exptl. Med., 10, 943-967.99, 183-199. 39. EARLE, W. R., SCHILLING, E. L., BRYANT,

25. EAGLE, H. 1955 The specific amino acid J. C., ANDEVANS, V. J. 1954 The growthrequirements of a mammalian cell (Strain of pure strain L cells in fluid-suspensionL) in tissue culture. J. Biol. Chem., 214, cultures. J. Natl. Cancer Inst., 14, 1159-839-8. 1171.

26. EAGLE, H. 1955 The specific amino acid 40. EARLE, W. R., BRYANT, J. C., AND SCHILLING,requirements of a human carcinoma cell E. L. 1954 Certain factors limiting the(Strain HeLa) in tissue culture. J. Exptl. size of the tissue culture and the develop-Med., 102, 37-48. ment of massive cultures. Ann. N. Y.

27. EAGLE, H. 1955 The minimum vitamin re- Acad. Sci., 58, 1000-1011.quirements of the L and HeLa cells in 41. EARLE, W. R., BRYANT, J. C., SCHILLING,tissue culture, the production of specific E. L., AND EVANS, V. J. 1956 Growth ofvitamin deficiencies, and their cure. J. cell suspensions in tissue culture. Ann.Exptl. Med., 102, 595-0. N. Y. Acad. Sci., 63, 666-682.

28. EAGLE, H. 1955 Utilization of dipeptides 42. EHRENSVARD, G., FISCHER, A., AND STJERN-by mammalian cells in tissue culture. HOLM, R. 1949 Protein metabolism ofProc. Soc. Exptl. Biol. Med., 89, 96-99. tissue cells in vitro. 7. Chemical nature of

29. EAGLE, H. 1956 Nutrition needs of mam- some obligate factors of tissue cell nutri-malian cells in tissue culture. Science, tion. Acta Physiol. Scand., 18, 218-230.122, 501-504. 43. EVANS, V. J., AND EARLE, W. R. 1947 The

30. EAGLE, H. 1956 Relative growth-promot- use of perforated cellophane for the growthing activity in tissue culture of co-factors of cells in tissue culture. J. Natl. Cancerand the parent vitamins. Proc. Soc. Exptl. Inst., 8, 103-119.Biol. Med., 91, 358-361. 44. EVANS, V. J., EARLE, W. R., SANFORD, K. K.,

31. EAGLE, H. 1956 The salt requirements of SHANNON, J. E., JR., AND WALTZ, H. K.mammalian cells in tissue culture. Arch. 1951 The preparation and handling ofBiochem. and Biophys., 61, 356-366. replicate tissue cultures for quantitative

32. EAGLE, H., OYAMA, V. I., LEVY, M., HORTON, studies. J. Natl. Cancer Inst., 11, 907-927.C. L., AND FLEISCHMAN, R. 1956 The 45. EVANS, V. J., BRYANT, J. C., FIORAMONTI,growth response of mammalian cells in M. C., MCQUILKIN, W. T., SANFORD, K. K.,tissue culture to L-glutamine and L-glu- AND EARLE, W. R. 1956 Studies oftamic acid. J. Biol. Chem., 218, 607-617. nutrient media for tissue cells in vitro. I. A

33. EAGLE, H., OYAMA, V. I., LEVY, M., AND protein-free chemically defined mediumFREEMAN, A. 1956 Myo-inositol as an for cultivation of strain L cells. Canceressential growth factor for normal and Research, 16, 77-86.malignant human cells in tissue culture. 46. EVANS, V. J., BRYANT, J. C., MCQUILKIN,Science, 123, 845-847. W. T., FIORAMONTI, M. C., SANFORD, K. K.,

34. EAGLE, H., AND HABEL, K. 1956 The WESTFALL, B. B., AND EARLE, W. R. 1956nutritional requirements for the propaga- Studies on nutrient media for tissue cellstion of poliomyelitis virus by the HeLa cell. in vitro. II. An improved protein-freeJ. Exptl. Med., 104, 271-287. chemically defined medium for long-term

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cultivation of Strain L-929 cells. Cancer report: cultivation of mesoblastic tumorsResearch, 16, 87-94. and normal tissue and notes on methods of

47. FELL, H. B., AND MELLANBY, E. 1952 cultivation. Am. J. Cancer, 27, 45-76.Effect of hypervitaminosis A on embryonic 62. GINSBERG, H. S., GOLD, E., AND JORDAN,limb-bones cultivated in vitro. J. Physiol., W. S., JR. 1955 Tryptose phosphate broth116, 320-349. as supplementary factor for maintenance

48. FELL, H. B., AND MELLANBY, E. 1953 of HeLa cell tissue cultures. Proc. Soc.Metaplasia produced in cultures of chick Exptl. Biol. Med., 89, 66-70.ectoderm by high vitamin A. J. Physiol., 63. GLADSTONE, G. P. 1939 Inter-relationships119, 470-489. between amino-acids in the nutrition of B.

49. FIORAMONTI, M. C., BRYANT, J. C., MCQUIL- anthraci8. Brit. J. Exptl. Path., 20, 189-KIN, W. T., EVANS, V. J., SANFORD, K. K., 200.AND EARLE, W. R. 1955 The effects of 64. GRAHAM, A. F., AND SIMINOVITCH, L. 1955horse serum residue and chemically defined Proliferation of monkey kidney cells insupplements on proliferation of strain L rotating cultures. Proc. Soc. Exptl. Biol.clone 929 cells from the mouse. Cancer Med., 89, 326-327.Research, 15, 763-766. 65. GRAu, C. R., AND ALMQUIST, H. J. 1943

50. FISCHER, A. 1946 Biology of tissue cells. The utilization of the sulfur amino acidsGlydendal, Copenhagen. by the chick. J. Nutrition, 26, 631-640.

51. FISCHER, A. 1948 Amino-acid metabolism 66. GRUNBAUM, B. W., SCHAFFER, F. L., ANDof tissue cells in vitro. Biochem. J., 43, KIRK, P. L. 1955 Lipides of chick heart491-497. fibroblasts. Effect of vitamin Bn2 and folio

52. FISCHER, A., ASTRUP, T., EHRENSVXRD, G., acid. Proc. Soc. Exptl. Biol. Med., 88,AND 0HLENSCHLGER, V. 1948 Growth 459-463.of animal tissue cells in artificial media. 67. HAFF, R. F., SWIM, H. E., AND PARKER, R. F.Proc. Soc. Exptl. Biol. Med., 67, 40-46. 1956 Amino acid requirements of rabbit

53. FISCHER, A. 1949 Application of soybean fibroblasts and their relation to vacciniainhibitor in tissue cultivation. Science, virus multiplication. Bacteriol. Proc.,109, 611-612. 1956, 76-77.

54. FISCHER, A. 1953 On the protein metabo- 68. HAMILTON, L. D., HUTNER, S. H., AND PRO-lism of tissue cells in vitro. J. Natl. Cancer VASOLI, L. 1952 The use of protozoa inInst., 13, 1399-1423. analysis. Analyst, 77, 618-628.

55. FJELDE, A., SORKIN, E., AND RHODES, J. M. 69. HANKS, J. H. 1948 The longevity of chick1956 The effect of glucosamine on human tissue cultures without renewal of medium.epidermoid carcinoma cells in tissue cul- J. Cellular Comp. Physiol., 31, 235-260.ture. Exptl. Cell Research, 10, 88-98. 70. HANKS, J. H., AND WALLACE, R. E. 1949

56. FRANCIS, M. D., AND WINNICK, T. 1953 Relation of oxygen and temperature in theStudies on the pathway of protein syn- preservation of tissues by refrigeration.thesis in tissue culture. J. Biol. Chem. Proc. Soc. Exptl. Biol. Med., 71,196-200.202, 273-289. 71. HARE, J. D., AND MORGAN, H. R. 1954

57. FRISCH, A. W., AND JENTOFT, V. 1953 Use Studies on the factors essential to the initi-of trypsin in preparing subcultures of ation and maintenance of multiplicationmonkey testicular tissue. Proc. Soc. of psittacosis virus (6BC strain) in differentExptl. Biol. Med., 82, 322-323. cells in tissue culture. J. Exptl. Med., 99,

58. GEMMILL, C. L., GEY, G. 0., AND AUSTRIAN, 461-479.R. 1940 The metabolism of tissue cul- 72. HARRIS, M. 1952 The use of dialyzed mediatures of Walker rat sarcoma 319. Bull. for studies in cell nutrition. J. CellularJohns Hopkins Hosp., 66, 167-184. Comp. Physiol., 40, 279-302.

59. GBRARDE, H. W., JONES, M., AND WINNICK, 73. HARRIS, M., AND KUTSKY, P. B. 1953 Util-T. 1952 Protein synthesis and amino ization of added sugars by chick heartacid turnover in tissue culture. J. Biol. fibroblasts in dialyzed media. J. CellularChem., 196, 51-68. ComlPsolnd2z449 l7.

60. GERARDE, H. W., AND JONES, M. 1953 The Comp. Physiol., 42, 449470.effect of cortisone on collagen synthesis in 74 HARRIS, M. 1954 The role of bicarbonatevitro. J. Biol. Chem., 201, 553-560. for outgrowth of chick heart fibroblasts in

61. Gur, G. O., AND GEY, M. K. 1936 Main- vtro. J. Exptl. Zool., 125,8598.tenance of human normal cells and tumor 75. HARRIS, M., AND KUTSKY, R. J. 1954 Syn-cells in continuous culture; preliminary ergism of nucleoprotein and dialysate

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19581 TISSUE CULTURE NUTRITION 41

growth factors in chick embryo extract. 89. KIDDER, G. W., AND DEWEY, V. C. 1951Exptl. Cell Research, 6, 327-336. The biochemistry of ciliates in pure culture.

76. HARRISON, R. G. 1906-07 Observations on In Biochemistry and physiology of Protozoa,the living developing nerve fiber. Proc. pp. 323-00. Edited by A. Lwoff. AcademicSoc. Exptl. Biol. Med., 4,140-143. Press, Inc., New York.

77. HARsION, R. G. 1910 The outgrowth of 90. KINSEY, V. E., WACHTL, C., CONSTANT, M.the nerve fiber as a mode of protoplasmic A., AND CAMACHO, E. 1955 Studies onmovement. J. Exptl. Zool., 9, 787-848. the crystalline lens. VI. Mitotic activity in

78. HEALY, G. M., MORGAN, J. F., AND PARKER, the epithelia of lenses cultured in variousR. C. 1952 Trace metal content of some media. Am. J. Ophthal., 40,216-223.natural and synthetic media. J. Biol. 91. KovAcs, E. 1956 Comparative biochemicalChem., 198, 305-312. studies on normal and on poliomyelitis

79. HEALY, G. M., FISHER, D. C., AND PARKER, infected tissue cultures. I. Observations onR. C. 1954 Nutrition of animal cells in synthetic nutrient mixtures incubatedtissue culture. VIII. Desoxyribonucleic with tissue cultures of normal kidney.acid phosphorus as a measure of cell multi- Can. J. Biochem. and Physiol., 34, 273-287.plication in replicate cultures. Can. J. 92. KovAcs, E. 1956 Comparative biochemicalBiochem. and Physiol., 32,319-326. studies on normal and on poliomyelitis

80. HEALY, G. M., FISHER, D. C., AND PARKER, infected tissue cultures. II. InvestigationsR. C. 1954 Nutrition of animal cells in of various enzyme systems in homogenatestissue culture. IX. Synthetic medium 703. of kidney tissue cultures of normal rhesusCan. J. Biochem. and Physiol., 32, 327-337. monkeys. Can. J. Biochem. and Physiol.,

81. HEALY, G. M., FISHER, D. C., AND PARKER, 34, 600-618.R. C. 1955 Nutrition of animal cells in 93. KovAcs, E. 1956 Comparative biochemicaltissue culture. X. Synthetic medium no. studies on normal and on poliomyelitis858. Proc. Soc. Exptl. Biol. Med., 89, 71- virus-infected tissue cultures. V. Profound77. alterations of acid and alkaline phosphatase

82. HOPKINS, F. G., AND SIMON-REUSS, I. 1944 activity in infected rhesus kidney cells. J.Effect of hypoxanthine on growth of avian Exptl. Med., 104, 589-613.tissue in vitro. Proc. Roy. Soc. (London), 94. KUTSKY, R. J. 1953 Stimulatory effect ofB132, 253-257. nucleoprotein fraction of chick embryo

83. JACQUEZ, J. A., ANDBARRY, E. 1951 Tissue extract on homologous heart fibroblasts.culture media. The essential non-dialyzable Proc. Soc. Exptl. Biol. Med., 83, 390-395.factors in hunan placental cord serum. J. 95. KUTSKY, R. J., TRAUTMAN, R., LIEBERMAN,Gen. Physiol., 34, 765-774. M., AND CAILLEAU, R. 1956 Nucleopro-

84. JACQUEZ, J. A., BARCLAY, R. K., AND SroCK, tein fractions from various embryonicC. C. 1952 Transamination in the me- tissues. A comparison of physicochemicaltabolism of P-2-thienyl-DL-alanine in nor- characteristics and biological activity inmal and neoplastic cells in vitro. J. Exptl. tissue culture. Exptl. Cell Research, 10,Med., 96 499-512. 48-54.

85. JACQUEZ, J. A., AND MoTrrTA, F. 1953 96. LASNITzxi, I. 1955 The effect of excessTissue culture screening of amino acid vitamin A on mitosis in chick heart fibro-analogs for selected damage of mouse blasts in vitro. Exptl. Cell Research, 8,sarcoma cells. Cancer Research, 13, 605- 121-125.609. 97. LEIGHTON, J., KLINE, I., BELKIN, M., LEGAL-

86. JONES, M., AND BONTING, S. L. 1956 Some LAIS, F., AND ORR, H. C. 1957 The simi-relations between growth and carbohy- larity in histological appearance of somedrate metabolism in tissue cultures. human "cancer" and "normal" cell strainsExptl. Cell Research, 10, 631-639. in sponge-matrix tissue culture. Cancer87. JONES, M., FEATHERSTONE, R. M., AND Rsac,1,3933B7ONTINGM, S. L. 1956 The effect of Research, 17, 359-363.

acetylcholine on the cholinesterases of 98. LESLIE, I., AND PAUL, J. 1954 The actionchick embryo intestine cultures in vitro. of insulin on the composition of cells andJ. Pharmacol. Exptl. Therap., 116, 114-118. medium during culture of chick heart

88. KIDDER, G. W., AND DEWEY, V. C. 1945 explants. J. Endocrinol., 11, 11S124.Studies on the biochemistry of Tetra- 99. LEVINE, A. S., BOND, P. H., AND ROUSE, H. C.hymena. III. Strain differences. Physiol. 1956 Modification of viral synthesis inZocl., 18, 136-157. tissue culture by substituting pyruvate

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for glucose in the medium. Proc. Soc. Interrelationships between cobalt andExptl. Biol. Med., 93, 233-235. amino acids in tissue culture. Arch.

100. LIKELY, G. D., SANFORD, K. K., AND EARLE, Biochem. and Biophys., 38, 267-274.W. R. 1952 Further studies on the pro- 114. MORGAN, J. F., AND MORTON, H. J. 1955liferation in vitro of single isolated tissue Studies on the sulfur metabolism of tissuescells. J. Natl. Cancer Inst., 13, 177-184. cultivated in vitro. I. A critical require-

101. Lu, K. H., AND WINNICK, T. 1954 Studies ment for L-cystine. J. Biol. Chem., 215,of nucleic acid metabolism in embryonic 539-545.tissue culture with the aid of C'4-labelled 115. MORGAN, J. F., CAMPBELL, M. E., ANDpurines. Exptl. Cell Research, 6, 345-352. MORTON, H. J. 1955 The nutrition of

102. Lu, K. H., AND WINNICK, T. 1954 The animal tissues cultivated in vitro. I. Acorrelation of growth with incorporation of survey of natural materials as supplementsradioactive metabolites into nucleic acids to synthetic medium 199. J. Natl. Cancerin embryonic tissue culture. Exptl. Cell Inst., 16, 557-567.Research, 7, 238-242. 116. MORGAN, J. F., AND MORTON, H. J. 1956

103. Lu, K. H., AND WINNICK, T. 1955 The Studies on the sulfur metabolism of tissuesroles of the nucleic acids and free nucleo- cultivated in vitro. II. Optical specificitiestides in chick embryonic extract on the and interrelationships between cystine andgrowth of heart fibroblast. Exptl. Cell methionine. J. Biol. Chem., 221, 529-535.Research, 9, 502-509. 117. MORGAN, J. F., MORTON, H. J., CAMPBELL,

104. MARCus, P. I., CIECIuIRA, S. J., AND PUCK, M. E., AND GUERIN, L. F. 1956 TheT. T. 1956 Clonal growth in vitro of nutrition of animal tissues cultivated inepithelial cells from normal human tissues. vitro. II. A comparison of various syntheticJ. Exptl. Med., 104, 615-631. media. J. Natl. Cancer Inst., 16, 1405-

105. MAYYASI, S. A., AND SCHUURMANS, D. M. 1415.1956 Cultivation of L strain (Earle) 118. MORGAN, J. F., AND MORTON, H. J. 1957mouse cells in bacteriological media com- The nutrition of animal tissues cultivatedbined with horse serum. Proc. Soc. Exptl. in vitro. IV. Amino acid requirements ofBiol. Med., 93, 207-210. chick embryonic heart fibroblasts. J.

106. McCoy, T. A., MAXWELL, M., AND NEUMAN, Biophys. and Biochem. Cytol., 3, 141-150.R. E. 1956 The amino acid requirements 119. MORTON, H. J., MORGAN, J. F., AND PARKER,of the Walker carcinosarcoma 256 in vitro. R. C. 1950 Nutrition of animal cells inCancer Research, 16, 979-984. tissue culture. II. Use of Tweens in syn-

107. MEISTER, A. 1956 Metabolism of gluta- thetic feeding mixtures. Proc. Soc. Exptl.mine. Physiol. Revs., 36, 103-127. Biol. Med., 74, 22-26.

108. MORGAN, J. F., AND PARKER, R. C. 1949 120. MORTON, H. J., MORGAN, J. F., AND PARKER,Use of antitryptic agents in tissue culture. R. C. 1951 Nutrition of animal cells inI. Crude soybean trypsin-inhibitor. Proc. tissue culture. V. Effect of initial treatmentSoc. Exptl. Biol. Med., 71, 665-668. of cultures on their survival in a synthetic

109. MORGAN, J. F. 1950 In R. C. Parker, medium. J. Cellular Comp. Physiol., 38,Methods of tissue culture, Ed. 2. Paul B. 389-400.Hoeber, Inc., New York. 121. MORTON, H. J., PASIEKA, A. E., AND MORGAN,

110. MORGAN, J. F., MORTON, H. J., AND PARKER, J. F. 1956 The nutrition of animalR. C. 1950 Nutrition of animal cells in tissues cultivated in vitro. III. Use of atissue culture. I. Initial studies on a syn- depletion technique for determining spe-thetic medium. Proc. Soc. Exptl. Biol. cific nutritional requirements. J. Bio-Med., 73, 1-8. phys. and Biochem. Cytol., 2, 589-596.

111. MORGAN, J. F., MORTON, H. J., AND PARKER, 122. MOSCONA, A. 1952 Cell suspensions fromR. C. 1951 Nutrition of animal cells in organ rudiments of chick embryos. Exptl.tissue culture. IV. Inhibition of cell activ- Cell Research, 3, 535-539.ity by cobalt and the protective action of 123. MURRAY, M. R., AND KOPECH, G. 1953 AL-histidine. Growth, 15, 11-22. bibliography of the research in tissue culture.

112. MORGAN, J. F., MORTON, H. J., HEALY, G. M., Academic Press, Inc., New York.AND PARKER, R. C. 1951 Nutrition of 124. NEUMAN, R. E., AND McCoy, T. A. 1956animal cells in tissue culture. VI. Low Dual requirement of Walker carcinosar-toxicity of barium. Proc. Soc. Exptl. coma 256 in vitro for asparagine and gluta-Biol. Med., 78, 880-882. mine. Science, 124, 124-125.

113. MORGAN, J. F., AND PARKER, R. C. 1952 125. NOYES, W. F. 1953 A simple technique for

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demonstrating plaque formation with Vol. 4. The Year Book Publishers, Inc.,virus of vaccinia. Proc. Soc. Exptl. Biol. Chicago.Med., 83, 426-429. 139. PUCK, T. T., AND MARCUS, P. I. 1955 A

126. OWENS, 0. VON H., GEY, M. K., AND GEY, rapid method for viable cell titration andG. 0. 1953 A new method for the culti- clone production with HeLa cells in tissuevation of mammalian cells suspended in culture: The use of X-irradiated cells toagitated fluid medium. Proc. Am. Assoc. supply conditioning factors. Proc. Natl.Cancer Research, 1, 41. Acad. Sci., 41, 432-437.

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132. PARKER, R. C., MORGAN, J. F., AND MORTON, synthetic medium. Application to polio-H. J. 1951 Toxicity of rubber stoppers virus synthesis. Proc. Soc. Exptl. Biol.for tissue cultures. Proc. Soc. Exptl. Med., 91, 464-470.Biol. Med., 76, 444-445. 145. RHODES, A. J., WOOD, W., AND DUNCAN, D.

133. PARKER, R. C., HEALY, G. M., AND FISHER, The present place of virus laboratory testsD. C. 1954 Nutrition of animal cells in in the diagnosis of poliomyelitis, withtissue culture. VII. Use of replicate cell special reference to tissue-culture tech-cultures in the evaluation of synthetic niques. World Health Organization, Mono-media. Can. J. Biochem. and Physiol., graph Series No. 26, 237-267.32 306-318. 146. ROBERTS, S., AND KELLEY, M. B. 1956

134. PARKER, R. C., CASTOR, L. N., AND MCCUL- Metabolism of plasma proteins in vitro. J.LOCH, E. A. 1956 Altered animal cell Biol. Chem., 222, 555-564.strains for quantitative culture work. 147. ROBERTSON, H. E., AND BOYER, P. D. 1956Anat. Record, 124, 18-19. Orthophosphite as a buffer for biological

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151. ROSENBERG, S., AND KIRK, P. L. 1953 saponified lipides of cultured tissue cells.Tissue culture studies. Identification of A microfractionation study of Earle'scomponents and synthetic replacements Strain L. Arch. Biochem. and Biophys.,for the active fraction of chick embryo 50,188-198.extract ultrafiltrate. J. Gen. Physiol., 162. SCHERER, W. F., SYVERTON, J. T., AND GEY,37, 239-248. G. 0. 1953 Studies on the propagation

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