thereby initiating a rapid change in the fumarate/succinate ratio, the free radical concentration increases to a maximum and then declines to zero Our results suggest, therefore, that the free radicals observed in heart particles arise from equilibrium processes catalyzed by succinic dehydrogenase and that their steady-state concentration is governed by the parameters which affect this equilibrium. The paper which follows presents quantitative data on the relation between these parameters and the free radical concentration, which confirm the foregoing conclusion and characterize the reaction mechanisms involved in free radical formation.

A minimal growth medium supplemented with dialyzed serum, which sufficed for the propagation of a wide variety of human cell strains in heavily inoculated monolayer and suspension cultures, did not permit the regular or optimal growth of small numbers of HeLa, HeLa S3, conjunctiva, or KB cells deriving from suspension cultures. At threshold concentrations of serum, the plating efficiency of single cells was greatly reduced as compared with their plating efficiency in a medium containing dialyzed serum instead of whole serum, and the clones which did develop grew at a slower rate. The nutritional deficiency could be overcome by adding the seven amino acids which are ordinarily not nutritionally essential. In most of the experiments serine alone sufficed.

The present article "is a progress report rather than a review and in large part summarizes studies from a single laboratory" on the minimal essential medium for cultivation of mammalian cells in either monolayer or suspension. Every cell culture examined, whether human or animal in origin, required at least 13 amino acids for survival and growth. All the cultured human cells examined were found to contain large amounts of glutathione, taurine, glutamine, ammonia, and glutamic acid. [The SCI® indicates that this paper was cited 2,255 times in the period 1961-1975.]

Growth of CeZZs-The cell lines used were strain HeLa, derived from uterine carcinoma (3), a variant HeLa strain which arose spontaneously (2), and two cultures derived from normal liver and conjunctiva (4). The cells were grown, adherent to glass, in a synthetic medium consisting of salts, vitamins, glucose, and the thirteen essential amino acids, supplemented with 5 per cent dialyzed human serum as previously described (5). Harvesting of Cells--In growth experiments, the cells were harvested during the logarithmic growth phase 6 or 24 hours after the last change of medium. In experiments which involve deficient media, the cells were harvested 24 hours after being overlaid with medium which lacked one or more of the essential amino acids. Preparation of Free Amino Acid Pools-The effects of temperature, mechanical disruption, and washing on the composition of the free amino acid pools were examined. When the cells were harvested by scraping them free from the glass in salt solution at room temperature before precipitation with cold trichloroacetic acid, the supernatant fluid contained up to 30 per cent of the protein nitrogen. This rapid and extensive autolysis was prevented by precipit,ating the protein without mechanically disturbing the cell layer. The 1 liter Blake bottles in which the cells were grown were drained for about 5 minutes, chilled rapidly in ice, and washed quickly with a balanced salt solution. Cold 8 per cent trichloroacetic acid was then added and the cell coagulum was scraped free of the glass, the vol

Monkey kidney cells tested in their first culture passage, 24 hours after their isolation from the animal host, required the same 13 amino acids for survival and growth as cell lines serially propagated in culture for years. Under the conditions of the present experiments, arginine, cystine, glutamine, histidine, and tyrosine proved necessary, over and above the 8 amino acids required for nitrogen balance in man. With the serially propagated lines, glutamic acid substituted for glutamine only at extremely high and non-physiological levels. In the monkey kidney cell cultures, however, glutamic acid and glutamine were interchangeable, mole for mole; and aspartic acid and asparagine were also effective as glutamine substitutes. Glycine was growth-stimulatory for monkey kidney cells in primary culture, and the cells grown in a glycine-deficient medium usually failed to survive subculture.

Of the many distinguished presidential adopinions and perhaps convictions on these dresses in the archives of our Society, some reprecomplex and multifaceted problems. I have, sent important contributions to the history of however, been too immersed in my own day-bymicrobiology; others have surveyed the conday preoccupations to be able to generalize with temporary scientific scene with humor and confidence, or to feel that my views are so obinsight; and a few have ventured to foretell the viously right that I can or should use this office shape of things to come. In any case, precedent and this occasion as a forum from which to almost requires that on this occasion your presipropagandize; and besides, this would not be the dent deal with the general rather than the right audience. Like the fictional juggler of Notre particular, and with the controversial rather than Dame I am therefore constrained to render my the factual. However, I must decline this oppresidential homage to the Society in terms of my portunity to philosophize. I choose to defer the work experience; but I take comfort in the fact thoughtful remembrance of things past; and it that many others before me have similarly chosen would be a venturesome man indeed who in to evade their presidential responsibility by today's expanding universe of science would dare offering their experimental data, with more or to extrapolate from the past into the future. less justifiable pride and with obvious affection. As to the present, there is to be sure much in the world about us that deserves and invites comment. A large segment of our population has For several years my associates2 and I have recently discovered that science is important. been studying the nutritional requirements and Unfortunately, that realization has been brought metabolic activity of human and animal cells in home by immediate and urgent practical needs. cultures. Surprisingly, every cell strain so far Even now, I am not convinced that the necessity examined, no matter what the species or organ for basic research unrelated to an immediate from which it was derived, and whether normal objective is understood or accepted by some of or malignant in origin, has proved to require those who most insistently bespeak its applicaessentially the same amino acids, vitamins, and tions. The importance of this pursuit of knowlsalts, and at approximately the same conedge for knowledge's own sake is not a rationalicentrations. This metabolic uniformity, and the zation by unworldly scientists attempting to difficulties so far encountered in the production escape reality. It is a truism that the esoteric and or identification of biochemical mutants, conapparently useless information of today untrast sharply with the extraordinary diversity and predictably becomes the essential keypiece in variability of microorganisms. As shown in table the practical application of tomorrow, or next 1, a total of only 28 defined growth factors (13 year, or 20 years hence. But even if this were as amino acids, 8 vitamins, 6 ions, and glucose) self-evident to all as it is to most scientists, the supplemented with serum protein, have so far problems remain of how to achieve a proper proved adequate for the cultivation of all the balance between applied research and the mainhuman cell lines examined; and a total of 32 stream of scientific progress, and of how to profactors provide for the sustained propagation of vide the proper educational, economic, and every mammalian cell strain so far isolated. political climate for the most fruitful developOn the omission of a single one of these essential

Three tissue culture cell lines deriving from normal human tissue (liver, conjunctiva, and intestine) and two lines deriving from human cancer [KB (nasopharynx), and J-111 (monocytic leukemia)] have been examined with respect to their amino acid requirements in comparison with those of a HeLa cell and a mouse fibroblast. With the possible exception of tryptophan, all seven cell lines required the same amino acids (arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, valine); and in the absence of any one of these 12, cytopathogenic changes developed which culminated in the death of the cell. The provision of non-essential amino acids, purines, pyrimidines, and NH4+ had a glutamine-sparing effect, but did not eliminate the need for this amino acid. Extremely high and nonphysiological concentrations of glutamic acid (20 mM) did, however, substitute for glutamine.

myo-Inositol (also called meso-inositol) is generally considered a member of the B vitamin complex and is required for growth by a number of yeasts (1) and fungi (2). An exogenous inositol requirement has, however, not been demonstrated in any bacterial species, and, although deficiency symptoms have been produced in animals, evidence for its vitamin function in man remains inconclusive. Inositol deficiency has been found to produce alopecia in rats (3, 4) and mice (5) and decreased growth in mice, rats, cotton rats, and hamsters (3, 6, 7). Several investigators have, however, been unable to confirm this growth-promoting effect (8, 9) and the role of inositol in the production or cure of experimental alopecia has also been questioned (9, 10). An interrelationship between inositol and a number of other vitamins in the production of deficiency symptoms (6, 11) is a complicating factor. A lipotropic effect in rats, independent of that of choline, has been reported by several investigators (12). Although a similar lipotropic effect has been found in man (13), there has been no demonstration of a deficiency syndrome resulting from lack of inositol. In the light of these results, it is of interest that myo-inositol has proved essential for the survival and growth of every cultured human cell examined, whether normal or malignant, as well as of a mouse sarcoma. With every one of eighteen cultured human strains, growth ceased in inositoldeficient media, there was cytopathogenic evidence of cell injury, and the cells eventually died. The quantitative aspects of that requirement and the degree to which myo-inositol could be replaced by its isomers and by related compounds are described here.

Only minimal amounts of poliomyelitis virus were formed by HeLa cells placed in a medium free from glucose and glutamine, even if the medium contained an otherwise full complement of essential and non-essential amino acids, purines, pyrimidines, NB4 +, and serum protein. Conversely, within one log of the optimal yield of virus was formed by HeLa cells in a medium containing only glucose, glutamine, and salts, even if the cells had been starved in this medium for 12 hours prior to their inoculation. The presence of glucose alone caused an average 170-fold increase in viral output beyond the amounts formed by the glucose- and glutamine-depleted cells. The addition of glutamine alone caused an average 2000-fold increase; and the addition of both increased the viral formation 40,000-fold. Qualitatively similar results were obtained with unstarved cells, not previously depleted of glucose and glutamine. It follows that only a small proportion of HeLa cells are capable of forming virus unless either glucose or glutamine, or both, are present in the medium. The elaboration of virus was significantly delayed in media containing glucose but no glutamine. The absence of glucose and glutamine did not prevent the fixation of poliomyelitis virus by the cell. When these compounds were added to previously depleted cells even 6 hours after inoculation, and after the excess free virus had been removed by washing and by the addition of specific antiserum, normal amounts of virus were formed despite the degenerative changes caused by the previous glucose and glutamine deprivation. Possible functions of glucose and glutamine in the elaboration of virus are discussed in the text. Such factors other than glucose, glutamine, or salts (e.g. amino acids, purines, pyrimidines, vitamins, protein, or NH4 +) as may be needed by HeLa cells for the propagation of poliomyelitis virus, need not be present in the medium and cannot be easily washed out of the cell. Even 12 hours' total deprivation of the cells in salt solution prior to inoculation only slightly decreased their virus-synthesizing capacity in a similarly deficient medium, provided only that adequate amounts of glucose and glutamine were retained.

Twelve n-amino acids (arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine) have been found essential for the growth of two mammalian cell lines in tissue culture, a mouse fibroblast' (strain L) (1) and a human uterine carcinoma cell (strain HeLa) (2).The minimal vitamin requirement of these cell lines has also been defined (3).As will be shown here, under the conditions of the present experiments, glutamine proved similarly essential for the survival and growth of both cell lines.In a medium containing the twelve amino acids previously shown to be essential, the seven demonstrably essential vitamins, glucose, electrolytes, and serum protein, both the mouse fibroblast and the human carcinoma cell degenerated and died unless the medium was supplemented with glutamine.The quantitative aspects of this glutamine requirement, and the limited degree to which it could be satisfied by glutamic acid, are described. Methods and MaterialsThe methods of maintaining stock cultures of the two cell lines, of setting up replicate cultures with the various experimental media, and of cell enumeration to evaluate the growth response have been described in previous papers dealing with the amino acid and vitamin requirements of the mouse fibroblast (1, 3) and the HeLa carcinoma cell (2, 3).A helpful modification in the cell-counting, procedure has been the use of disodium Versenate (disodium salt of ethylenediaminetetraacetic acid) to disperse the cells, instead of mechanically scraping them from the surface of the culture flask.The flask was drained, and 3 ml. of fresh medium at pH