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THE INTERCHANGEABILITY of the ELEMENTS CHLORINE and IODINE in CULTURE MEDIA for HUMAN THYROID FIBROBLASTS Among Those Elements W

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THE INTERCHANGEABILITY of the ELEMENTS CHLORINE and IODINE in CULTURE MEDIA for HUMAN THYROID FIBROBLASTS Among Those Elements W THE INTERCHANGEABILITY OF THE ELEMENTS CHLORINE AND IODINE IN CULTURE MEDIA FOR HUMAN THYROID FIBROBLASTS JOHANNES P. M. VOGELAAR, D.M.S., AND ELEANOR ERLICHMAN, B.S. (From the Institute of Cancer Research, Columbia University, and St. Luke's Hospital, New York City') Among those elements which occur only .in minor amounts in the human body, none has such an interesting history or has been the subject of so much research as iodine. Although there can be no doubt concerning its physiologi- cal significance, actually very little is known about the requirement of various cells and tissues for this element in both its organic and inorganic forms. In view of the fact that the human body not only contains chlorine and iodine, but also fluorine and bromine, the question naturally arises whether all are vital elements and to what extent they can replace one another. The experi- ments reported in this article were undertaken to determine the interchange- ability of chlorine and iodine in culture media for human thyroid fibroblasts. The effect of iodine and iodides on tissue cultures has been studied by various investigators. Pitini ( 12) observed a stimulating effect of potassium iodide on the growth of spleen and sciatic nerve of young guinea-pigs. Glass capillaries filled with a 10 per cent solution of the iodide were placed in close proximity to the cultures. In the interpretation of the results obtained with such a method it must be borne in mind that divers uncontrollable factors may affect the tissue growth, such as local changes in the osmotic pressure and in the pH of the culture medium. Lambert (8), in an investigation of the comparative resistance of bacteria and human tissue cells to certain common antiseptics, determined the effect of iodine on pieces of tissue taken from human spleen and lymph nodes. After having been immersed for some time in iodine solutions of various concentra- tions, the pieces were washed with a salt solution and then placed in a culture medium. A good growth occurred after exposure to a 1:2000 solution of iodine for one hour, a strength sufficient to sterilize the tissue completely in most instances. Lambert and Meyer (9) made similar experiments with pieces of rabbit spleen which were exposed for only twenty minutes to the antiseptic. The tissue cells survived an exposure to iodine in the concentration 1:2500, but were killed by iodine in the concentration 1: 1000. In interpreting their re- sults the writers remark that the surviving tissue cells may have come from the center of the small fragments, where they were partially protected. This seems plausible and makes it difficult to formulate a definite statement as to which concentration can be endured by the cells. Salle and Lazarus (13) also studied the resistance of bacteria and em- bryonic tissue to germicidal substances. They found that iodine in a concen- tration 1:1800 or higher completely inhibits the growth of embryonic chick 1 With the assistance of a grant from the Anna Fuller Fund, 242 INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 243 tissue. The iodine was applied in the form of a Lug01 solution, which was mixed with chick embryonic fluid and placed over tissue fragments ,embedded in plasma. Cervello and Levi (3) studied the effect of iodine on tissues from various organs of chicks and chick embryos. This was done either by adding an iodine-potassium iodide solution to the culture medium or by using plasma taken from a chicken previously injected with the salt solution. The writers found that both connective-tissue cells and epithelium grow well in a medium containing a considerable amount of iodine (0.125% I,, 1.25% KI). The presence of free iodine in the medium was demonstrated by the addition of starch. It should be kept in mind that an iodine-iodide solution has iodine partly in the form of a tri-iodide, a fact which necessarily complicates the in- terpretation of the results. Olivo (1 1) used the heart and skin of chick embryos for his experiments with iodine. Small pieces of tissue were kept for several hours in isotonic solutions of sodium chloride, potassium iodide, and several mixtures of these before being placed in the culture medium. No appreciable difference be- tween these cultures and the controls was observed except that the emigration of cells began sooner in cultures previously treated with potassium iodide. In other experiments cultures were grown in media containing different amounts of the iodide. Cultures grew well in the presence of 0.25 per cent potassium iodide, whereas in a medium with 0.45 per cent potassium iodide the outgrowth of the explants was decidedly less than that of the controls. Buchsbaum and Bloom (2) observed an approximately normal growth of embryonic chick fibroblasts in a culture medium containing iodine in a con- centration 1:4000 or,less. Strickler and Fowler (14) determined the effect of iodine vapor on the growth of chick fibroblasts and malignant cells of the rat. The cultures were exposed to vapors arising from iodine crystals or from an iodine-potassium iodide solution. The iodine vapor did not stimulate the growth of the fibro- blasts and was frequently harmful. Tumor cultures treated with iodine al- ways grew less than the controls and showed a more extensive degeneration. How much iodine reached the growing cells, and in what form could not be ascertained. Zepponi and Crainz (17) used a novel method for studying the effect of iodine on chick fibroblasts, placing some gold previously exposed to iodine vapor'in the vicinity of the explants. The gold thus treated exerted a strongly stimulating effect, probably caused by a direct action of the iodine on the cells, since untreated gold did not influence the cells in any way. Hirashima (6) secured a vigorous growth of heart fibroblasts from the chick embryo in a medium containing sodium iodide tantamount to 25 X M (0.037 per cent). With an increasing concentration of the iodide, the growth was correspondingly less. When a value of 1/10 M (1.5 per cent) was reached, a majority of the cultures did not show any growth. Baker and Ebeling (1) maintained cultures from a pure strain of chick embryo fibroblasts in vital condition over a period of 56 days in a medium con- taining 130y per cent potassium iodide. This is equal to 783 X lo-* M po- tassium iodide. 244 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN Crainz (4) studied the effect of iodine on heart fibroblasts and liver epi- thelium of the chick embryo. The iodine was added to the culture medium in the form of Lugol’s solution. The addition of a small amount of iodine to the culture medium was advantageous, whereas with greater quantities an inhibi- tory effect on the growth activities was noticed. The latter, however, was lessened when the cultures had been treated previously with a lower concentra- tion of the halogen. Iodine was applied in concentrations ranging from 1:4000 to 1 :20,000. With the latter value a strongly stimulating action was observed, whereas with the former the cultures showed slight activity. Crainz exposed some cultures to iodine vapor arising from Lugol’s solution and, like Strickler and Fowler, noted a harmful effect on the cells. The epithelial cells showed a lesser sensitivity to iodine than the fibroblasts. Lambert (7, 8) and German (5) exposed cultures to the action of potas- sium mercuric iodide using, respectively, human fibroblasts and those of the chick embryo. The composition of this compound, so different from the chemicals used by the aforementioned authors, makes a comparison of the results with those of others impossible. EXPERIMENTAL Fibroblasts from fresh human thyroid tissue were grown for fourteen days in a mixture of equal parts of citrated beef plasma (16), calcium Ringer (15), feeding solution (15), and magnesium Ringer (15). After liquefying the plasma with trypsin (16) the pieces of tissue were equally divided over four culture media containing different amounts of iodine. In the preparation of three of the media, modifications of calcium Ringer, magnesium Ringer, and feeding solution were used. These were made by replacing part or all of the chlorides of the original solutions by equivalent amounts of the corresponding iodides. The composition of the various salt solutions follows : Ca. Ringer solution A Ca. Ringer solution R Ca. Ringer solution C 73.8 C.C. 1 M NaCl 73.8 C.C. 1 M NaI 73.8 C.C. 1 M NaI 121.6 C.C. M/100 KCl 121.6 C.C. M/100 KC1 121.6 C.C. M/100 KI 162.2 C.C. M/lO CaClz 162.2 C.C. M/10 CaCl2 162.2 C.C. M/10 CaIz 142.4 C.C. dist. water 142.4 C.C. dist. water 142.4 C.C. dist. water 500.0 C.C. Ca. R. A 500.0 C.C. Ca. R. B 500.0 C.C. Ca. R. C Salt solution A Salt solution R 70.8 C.C. 1 M NaCl 70.8 C.C. 1 M NaI 22.0 C.C. M/lO KCl 22.0 C.C. M/10 KI 12.5 C.C. M/10 CaCl2 12.5 C.C. M/10 CaI2 51.4 C.C. M/100 MgClz 51.4 C.C. M/100 MgI2 11.7 C.C. M/100 KaSOa 11.7 C.C. M/100 KZSO, 50.0 C.C. M/100 NaZHP04 50.0 C.C. M/lOO Na2HP04 281.6 C.C. dist. water 281.6 C.C. dist. water 500.0 C.C.
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