Metaplasia produced in cultures of chick ectoderm by high vitamin A / by Honor B. Fell and E. Mellanby.

  • Fell, Honor B.
Date:
[1953?]
Catalogue details

Licence: In copyright

Credit: Metaplasia produced in cultures of chick ectoderm by high vitamin A / by Honor B. Fell and E. Mellanby. Source: Wellcome Collection.

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    avoleum (kindly supplied by British Drug Houses Ltd.) containing 1,500,000 i.u./g. This was dissolved in ethanol to give a solution containing 20,000 i.u./ml. The ethanol solution was then added to normal plasma to raise the vitamin A content to the desired amount, which varied in different experiments between 1000 and 3000 i.u./lOO ml. In adding the extra vitamin A, the amount present in the plasma itself must be taken into account, and this for normal fowls varied between 200 and 350 i.u./lOO ml. When vitamin A acetate was added, the crystalline substance was dissolved in ethanol to give concentrations similar to those obtained with vitamin A alcohol. The strength of the solution, both of the vitamin A alcohol and the vitamin A acetate, was such that the plasma finally contained about 0-2% ethanol; the same quantity of ethanol was added to the normal control plasma. Because of the instability of the vitamin A in alcoholic solution under some conditions, it was necessary, after adding the vitamin A alcohol or acetate to the plasma in the calculated amount, to determine its vitamin A content again. This was especially important with vitamin A acetate in ethanol, since a stock solution, even when kept in the dark at 0° C, soon loses its potency. Vitamin A alcohol is fairly stable in plasma, but the acetate slowly diminishes so that, for instance, AH plasma containing 2040 i.u. vitamin A per 100 ml. may, at the end of 7 days, contain only 1459 i.u. The estimation of the vitamin in plasma was made colorimetrically (Fell & Mellanby, 1952). It may be pointed out that the vitamin A, whether added as alcohol or acetate, is not in the identical physical state normally present in plasma. In normal plasma, the vitamin A is in a water-soluble form probably in combination with a protein as a lipoprotein, and can only be removed from the plasma by petrol ether extraction as a fat-soluble substance after the plasma proteins have been denatured by 50 % ethanol. On the other hand, vitamin A alcohol and acetate when added in alcoholic solution to plasma can be extracted directly by shaking the plasma with petrol ether. Thus in the enriched plasma used in the present experiments, the greater part of the natural vitamin A was water-soluble (250-350 i.u./lOO ml.), whereas the remainder was fat soluble (2250-2150 i.u./lOO ml., on the assumption that the total plasma content was 2500 i.u./lOO ml.). In our previous work on bone development in tissue culture (Fell & Mellanby, 1952), it was found that vitamin A in artificial hypervitaminotic plasma had the same kind of action as a similar concentration of vitamin A in natural hypervitaminotic plasma, but that the former was much more effective and influenced the bone changes more rapidly. In other words, this action of vitamin A was greater in the fat-soluble form than in its natural water-soluble combination. Tissue culture - The culture medium consisted of three parts of fowl plasma: one part of a concentrated extract of a 13- to 14-day chick embryo; the Tyrode solution with which the extract was made contained 1 % glucose, giving a concentration of 0-25 % in the final medium. The best results were obtained with the vitamin A acetate; although the vitamin A alcohol produced the same changes in the ectoderm as the acetate, it seemed to be slightly toxic and caused rather more degeneration from the 9th day onwards. Watch-glass cultures (Fell & Robison, 1929; Fell & Mellanby, 1952) and large hanging-drop preparations were used. In the former, nine or twelve drops of plasma were mixed with three or four drops of embryo extract. The tissue fragments were first placed in embryo extract made with Tyrode containing the usual concentration (0-1 %) of glucose and diluted to half the strength of the extract in the culture medium; they were then explanted, with the ectoderm uppermost, on the surface of the clot in the watch-glass and the excess extract was sucked off with a fine pipette. The tissue was removed from the old clot and transplanted to fresh medium four times a week; it was found desirable to cut off the sheets of epithelium which spread over the clot since during transplantation they sometimes became folded over the under surface of the explant, keratinized and obstructed nutrition. The hanging-drop preparations were made with 1£ in. square cover-slips and 3 x 1^ in. hollow ground slides. The explants were grown in a mixture of 0-06 ml. of plasma and 0-02 ml. of embryo extract per cover-slip and were subcultivated 3 times a week.