SIGNIFICANCE OF ASCORBIC ACID (VITAMIN C) FOR THE GROWTH IN VITRO OF CROCKER MOUSE SARCOMA 180

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1 SIGNIFICANCE OF ASCORBIC ACID (VITAMIN C) FOR THE GROWTH IN VITRO OF CROCKER MOUSE SARCOMA 180 JOHANNES P. M. VOGEI,AAR, D.M.S., AND ELEANOR ERLICHMAN, B.S. (From t h Instit~itr ~ of Canrrr Rcseurrh, Colz4~izhia University, and St. L~rke's Hospital, New York City l) It is a conspicuous fact that rather limited attention has been given to the significance of vitamins for the growth in elitro of both human and animal tissues. Although Burrows (9), as early as 1925, performed studies to determine the biological significance of vitamins for the growth of tissues in vitro, it is only recently that a beginning has been made with the study of explants in media to which one or more vitamins in chemically pure form has been added. There can be hardly any doubt about the significance of such studies for increasing our knowledge of these, in more than one respect, elusive substances. In an article (24) dealing with the composition of a feeding solution for human fibroblasts, published several years ago, the statement was made that this probably could be improved by the addition of vitamins and certain hormones. At that time it was known to the writers, from previous unpublished experiments, that the growth of chicken fibroblast cultures could be increased substantially by the addition to the culture medium of fluids rich in one or more of the vitamins. Milk, fruit juices, and a yeast extract were used. A very dense growth was obtained, especially with the yeast extract, making it appear probable that the addition of vitamins stimulated the growth of chicken fibroblasts. The complexity of these media, however, made it impossible to be certain on this point. Similar experiments were later performed with cultures of human fibroblasts, but no improvement in growth was observed. There is undoubtedly a great difference in behavior between embryonic chick tissues and adult human tissues, and great care should therefore be taken in drawing generalizations from results obtained with explants. The remarkable progress in our knowledge of the chemical structure of the vitamins and the successful synthesis of several of them have made possible an exact study of the significance of these biologically important substances for growth and differentiation of cells and tissues outside the organism. In the past few years, Lillian E. Baker has published the results of a series of experiments apparently undertaken to improve the feeding solution mentioned above, by the addition of various vitamins. She obtained an excellent growth of cultures of chicken monocytes in an artificial medium containing, besides Witte peptone, cysteine, glucose, hemin, thyroxin, and insulin, all of which are components of the feeding solution for human fibroblasts, vitamins A, B,, B1, and D, but not vitamin C (3). In a medium recommended for the cultivation of both epithelial cells and With the assistance of a grant from the Anna Fuller Fund. 283

2 2 84 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN fibroblasts, the main additions were vitamins A, C, and D, and glutathione (2). These artificial media needed the addition of some blood serum in order to assure a maximal activity. Complete fornlulas are given (S), also, of two media for the cultivation of fibroblasts, epithelial cells, and monocytes. Both contain, besides the chemicals mentioned above, homologous blood serum, glutathione, and the vitamins A, C, and D when the medium is to be used for chicken fibroblasts and epithelial cells; and the vitamins A, B,, B2, C, and D when chicken monocytes are to be cultivated. All three types of cells grow well in these media. As justly remarked by I3aker, these are at present the best artificial media known for the cultivation of the animal cells and tissues used in her experiments. Whether this holds true for human tissues seems rather doubtful, since culture media very similar in composition to those of Raker, and containing the same vitamins, did not prove to be better than our regular culture medium for human fibroblasts. It should also be emphasized that human fibroblasts have now been grown over a period of a year with the feeding solution as originally described ( 12 ). It is not yet possible to ascertain the specific action of the various vitamins on cells growing in a culture medium. In the papers referred to, Raker makes no mention of this, but gives only the composition of the artificnl media and the results secured. She proved, however, that vitamin A has a stimulating effect on the growth of chicken fibroblasts ( l), confirming similar observations by Bisceglie on liver and spleen cultures of new-born guinea-pigs (6). Gordonoff and Ludwig (14) observed a stimulating action of vitamin A on fibroblast cultures of the chick embryo and on mouse cancer explants. A corresponding action was observed by Baker for vitamin C on cultures of chicken monocytes (4). The best results were obtained with a concentration of mg. ascorbic acid per 100 C.C. of medium. The influence of the vitamin on the cells became obvious only after about two weeks, probably because by that time the explants had used up their reserves of vitamins. The cells in the media containing vitamin C showed less granulation than those of the control cultures. The proliferation was more rapid; the colonies were thicker and occupied a larger area. Since the culture media contained glutathione in addition to the ascorbic acid, control experiments were performed with each of these substances. It was found that the marked improvement of the culture nlediuni must be ascribed to the ascorbic acid. Glutathione had been added to the medium in order to prevent the oxidation and consequent destruction of the vitamin. Without the presence of this tripeptide, the ascorbic acid oxidizes completely within twenty-four hours. A solution containing both ascorbic acid and glutathione, however, could be kept in the refrigerator for practically three weeks without showing any loss of re- ducing power. It is a well known fact that ascorbic acid has the tendency to oxidize into dehydroascorbic acid, a substance which also has pronounced antiscorbutic properties. This oxidation is a reversible reaction. The dehydroascorbic acid may change irreversibly into 2, 3-diketo-1-gulonic acid, which is inactive in respect to scurvy. v. Jeney and Toro (15) state that ascorbic acid plays a cardinal part in the formation of fibers in fibroblast cultures of the chick embryo. Their cul-

3 ture media contained vitamin C in a concentration varying from to 0.18 per cent. In regard to the function of the ascorbic acid, the writers remark that one may assume that in the absence of this substance the connective tissues cannot form intercellular substance. This supposition is in full agreement with the fact that skin wounds of guinea-pigs suffering from scurvy do not show signs of healing even after three weeks. There is a formation of new tissue, but neither collagenous fibers nor capillary buds are formed (v. Jeney). In scorbutic animals there is insufficient formation of cement substance between the endothelial cells (Aschoff, Koch, Hojer). v. Jeney and Toro do not state whether the fibroblasts grew better under the influence of the vitamin. Gordonoff and Ludwig (14) reported recently that vitamin C may have an inhibiting action on the growth of chick embryo fibroblasts. Since vitamin C forms part of the artificial culture medium recommended by Baker for growing chicken fibroblasts, it seems that further experiments will have to be performed to determine the significance of the vitamin for the growth of these cells. It follows, however, from the preceding observations, that there can be little doubt as to the importance of vitamin C for the growth of various cells and tissues. As far as the other vitamins are concerned, Bornstein (7) and Erdmann (11) studied the relationship between vitamin B and tissue respiration in vitro. There is a close alliance between vitamin B,, tissue respiration, and carbohydrate metabolism (Peters and Sinclair, 19). Vitamin B, is a component of the oxidation-pigment-enzyme system of tissues discovered by Warburg. Gordonoff and Ludwig (14) observed a total inhibition of the growth of chicken fibroblasts in plasma devoid of the vitamin B complex. Vitamin D was studied by Croxatto (lo), Nordmann and Horger (la), Rix (2 I), and v. Brand and Nauck (8 j. This vitamin stimulates the growth of both animal fibroblasts and epithelial cultures and may cause calcification processes in the explants. Gordonoff and Ludwig (14) did not observe any special influence of vitamin I) on the growth of their cultures. Juhasz-Schaffer (16), in a study of vitamin E, observed a stimulating action of this vitamin on the growth of cultures made from various organs of chick embryos. Rossi (22, 23) made similar observations. The present article deals with the significance of ascorbic acid for the growth of primary cultures of Crocker mouse sarcoma 180. Little is known about the chemical requirements of this interesting tumor. The explants were obtained from nine to twelve-day-old tumors and were cultivated in Carrel flasks. The culture medium consisted of a mixture of equal parts of beef plasma, calcium Ringer, feeding solution, and a modified magnesium Ringer. The ascorbic acid was added in the form of Cebione solution (Merck), which is a partly neutralized solution of ascorbic acid of a ph of about 6. Each cubic centimeter of this solution contains 50 mg. ascorbic acid. This solution was diluted 1000 times with a modified magnesium Ringer of the same ph as the Cebione solution. The dilution evidently contained 5 mg. ascorbic acid per 100 C.C. Three different culture media were used, the composition of which follows : (a) 25 per cent plasma, 25 per cent calcium Ringer solution, 25 per cent feeding solution, 25 per cent modified magnesium Ringer.

4 286 JOHANNES P. M. VOGELArZR AND ELEANOR ERLICIIMAN (0) 25 per cent plasma, 25 per cent calciuil~ Ringer solution, 25 per cent feeding solution, 20 per cent modified magnesium Ringer, 5 per cent diluted Cebione solution. (c) 25 per cent plasma, 25 per cent calcium Ringer solution, 25 per cent feeding solution, 15 per cent modified magnesium Ringer, 10 per cent diluted Cebione solution. From the above forlnulae it is evident that the media contained the same amount of plasma and feeding solution. The ph and the osmotic pressure of all three were practically the same. The media b and I: contained, respectively, 0.25 mg. and 0.50 mg. ascorbic acid per 100 C.C. Since medium c proved to be slightly better than medium 6, in most experiments only media a and c were used. More than 200 cultures were observed. The sarconlatous tissue grows well in both the control medium and in a medium containing ascorbic acid. The beef plasma used in these experiments may have contained some ascorbic acid. The an~ount probably was small in comparison to that of the ascorbic acid purposely added. No data could be found in the literature concerning the vitamin C content of beef plasma. Since, as will be pointed out later, the addition of ascorbic acid proved to be beneficial to the growth, the question is of minor importance. The tumor tissue shows a good activity in all three media. Within a few hours after the preparation of the explant, ameboid cells may be seen emigrating from the tissue. These rapidly increase in number both by actual division and by the continued emigration of similar cells from the central part of the explant. Within a few days a dense zone of these cells is formed (Fig. 1 In the meantime, a cellular network develops, comparable to the network of fibroblasts growing out from normal tissue (Fig. 2). It is evidently composed of pathological fibroblasts. At the periphery ameboid cells may be seen splitting off from it. Besides these pathological cells, apparently normal fibroblasts and cells of the macrophage and nlonocyte type may be seen. They form, however, by far the minor part of the new-growth. Freshly made explants of tumor tissue sometimes seem to be composed almost exclu-

5 sively of ameboid cells, whereas in other cases the network of pathological fibroblasts dominates. The above remarks hold both for cultures grown in the control medium and for those in a medium containing ascorbic acid. There is, however, a difference in character between the two series of cultures in that those grown in the medium containing ascorbic acid are slightly larger and have a denser structure than those in the control medium. This difference is most pronounced after about four or five days growth in vitro. These observations are similar to those made by Baker on cultures of monocytes, to which reference has already been made. Vitamin C evidently stimulates both the emigration of cells and the frequency of cell division. That the increase in size of the cultures of sarcoma 180 was due not only to a more rapid emigration, but mainly to an actual increase in cell number, fol- lows from the fact that the central part of the explants had not become less dense in structure after the new growth had developed. The increase in size of the control cultures, on the contrary, was not infrequently due more to the emigration of cells than to cell multiplication. This was conspicuous from the fact that the zone of emigrating cells sometimes practically lost its connection with the central and original part of the explant, due obviously to failure of the latter to provide new cells. In this case the plasmatic area between the zone of ameboid cells and the original explant contained only a small number of loosely arranged cells which rapidly degenerated in the liquefying zone of plasma. This area of liquefaction should not be interpreted as due to a tendency of the tumor tissue to liquefy the culture medium, since it occurs only when the growth is not satisfactory in every respect. When plasma liquefies under the influence of actively growing tissue the first signs always occur at the periphery of the explant and not within. Mouse sarcoma 180

6 288 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN should be regarded as a tumor which does not liquefy the plasma. This statement is, of course, made only in regard to beef plasma. Besides the stimulation of emigration and of cell division, still another property of the vitamin in regard to the explant was noticed, namely a retarding effect on the degeneration of the cells. At a time when most of the ameboid cells in the control medium were degenerating most of those in the medium containing the vitamin still appeared in a good condition. They were moving around and dividing. This observation is in complete agreement with the observation made by Baker on monocytes. All in all, it may be said that in explants vitamin C fulfills at least three important functions: it stinlulates cell movement and cell division, and has a retarding effect on cell degeneration. It also seems to be necessary for the formation of intercellular fibers. The observations for mouse sarcoma 180 were made on primary cultures of the tumor. It is quite possible that the differences between cultures grown in the two media will become more pronounced when the cultures are grown over an appreciable length of time. It is interesting to note that the influence of the ascorbic acid became apparent within a few days, whereas in the case of the monocytes described by Baker it was evident only after two weeks. This could be explained by assuming that the cells at the beginning of the experiment still had a certain amount of the vitamin at their disposal and only after exhausting this became dependent upon the culture medium. In a few series of cultures the difference in behavior between those grown in medium a and those in medium c was less pronounced than in other series, although it was always in favor of the medium with ascorbic acid. Whether in these cases the tumor tissue contained any appreciable amount of the vitamin, thus enabling the cells of the control series to be nearly as active as those in the medium containing vitamin C, is impossible to say. Not much is known about the vitamin C content of tumors and their requirements, which seen1 to be different for various neoplasms. Kellie and Zilva (17) proved that allnost if not all the indophenol-reducing capacity of the Jensen rat sarconla is due to 1-ascorbic acid. Fodor and Kunos (13) reported that subcutaneous injection of vitamin C in tumor mice resulted in a great increase in the size of Ehrlich carcinoma, whereas Pollia (20) apparently did not observe a similar effect on the growth in z~ivo of a malignant dibenzanthracene tumor of the rat. To what extent the growth of sarcoma 180 in animals can be influenced by injection of vitamin C is not known. From the experiments mentioned in this article a stimulating action may be expected. 1. BAKER, L. E. : Proc. Soc. Expcr. Uiol. & Med. 33 : 124, BAKER, L. E.: Compt. rend. Soc. dc biol. 120: 932, BAKER, L. E.: Compt. rend. Soc. de bid. 120: 1160, BAKER, L. E.: Compt. rend. Soc. de biol. 121: 427, BAKER, L. E : Science 83: 605, BISCEGLIE, V.: Arch. f. Entwicklungsmech. d. Organ. 108: 708, B~RNSTEIN, K.: Biochcm. Ztschr. 200: 176, 1928.

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