Lillian E. Baker

Effect of vitamin A on proliferation of fibroblasts.

The purpose of the experiments reported here was to ascertain whether or not vitamin A is one of the substances needed by fibroblasts for their life and proliferation in vitro. Two previous investigators have already attempted to answer this question. Unfortunately, however, they arrived at opposite conclusions. Thus, Burrows 1 compared the change in concentration of vitamin A in chick embryos, as they increased in age, with the change occurring simultaneously in the growth-promoting power of the juice of the embryos. He concluded that vitamin A inhibited growth. Bisceglie, 2 on the other hand, added a trace of the “A factor” to hanging drop cultures of liver and spleen and found it to be growth stimulating. No information was given as to the source of the “A factor”, or the extent to which it had been purified. In the present investigation, crystalline carotene was first tried as a source of vitamin for the cells. No increase in growth was obtained.∗ Vitamin A was, therefore, prepared from halibut liver oil. Two different concentrates were made. In preparing the first, the oil was saponified, and the vitamin extracted with petroleum ether. The ether was evaporated, and the residue taken up in methyl alcohol. Sterols were then removed by cooling the methyl alcohol solution to 0°C. After this, the vitamin was redissolved in hexane, and more sterols were removed by cooling to —10°C. An Oily residue was obtained on evaporation of the hexane. The second preparation was obtained on evaporation of the hexane. The second preparation was purified to a further extent. In this case, a mixture of CO2 ice and alcohol was used to freeze out the sterols from the methyl alcohol solution. The residue from the methyl alcohol was then dissolved in pentane and the freezing with CO2 ice repeated.

Chemical nature of some substances required for the growth of fibroblasts and epithelial cells.

Pure strains of fibroblasts or of epithelial cells increase in mass in an unlimited manner, when they are cultivated in plasma and embryonic tissue juice. For 14 years, colonies of a strain of fibroblasts have doubled in size every 48 hours in such a medium. Pavement and thyroid epithelium also have been found to manufacture unlimited amounts of protoplasm from the constituents of embryonic juice. Neither epithelial cells nor fibroblasts multiply in serum proteins, egg albumin, crystallized egg albumin, amino acids from embryonic juice, or artificial mxtures of amino acids for a longer time than in Tyrode solution. So far, embryonic juice is the only material which has been found to maintain epithelial cells and fibroblasts in a conditoti of true cultivation. Investigation of the chemical nature of the nutritive materials in the embryo juice has led to the conclusion that the nitrogenous substance utilized by the tissues is the protein itself. The amino acids and other ultra-filtrable and dialyzable constituents slightly stimulated the migration and multiplication of the cells, but failed to produce an increase in the mass of the tissues. Since the protein of the embryo juice is utilized by the cells, it seems evident that it must be hydrolyzed and some intermediate product absorbed. Complete digestion by trypsin and pepsin produced only toxic substances. However, some of the higher cleavage products formed by the partial hydrolysis of protein have been found to produce the same effect as the protein of the embryo juice, causing continuous multiplication of cells and increase in the mass of tissue for long periods of time. In fact, some preparations of these protein hydrolytic products have given a larger increase in the mass of the tissue than has ever been obtained by embryo juice.

Maintenance of Fibroblasts in Artificial and Serumless Media

The importance of developing artificial media, which can be used in place of serum for maintaining the life of organs and tissues outside the body, hardly needs to be emphasized. Many of the studies for which the organ-culture technic 1 was invented, as well as others that can be carried out by the simpler methods of tissue-culture, depend for their success on the creation of suitable artificial media. These media are needed to reduce the cost of experimentation, to make possible extensive work with human organs and those of small animals from which serum in large quantity cannot be obtained, and for all studies in which the production of serum and other protein substances is to be investigated. For cultivating organs and for all work with tissues in which function rather than growth is the subject of study, it is important that these media maintain the cells without causing proliferation. All the artificial media previously reported have been designed to promote growth. 2 3 The media to be described in this paper were designed for maintenance. One of them is serumless. In the others, a very small amount of serum has been incorporated. The results obtained when these media were used to maintain a pure strain of fibroblasts in tissue-culture are described below. Experiments in which they were used for cultivating organs will be reported in another communication. 4 The compositions of the media are as follows: MEDIUM I. Whole-blood digest to give either 30 or 60 mg% nitrogen Serum, 2 or 3% Tyrodes solution To bring the vitamin A into solution, it was necessary to dissolve it at high concentration in serum and then use a small amount of this serum in the medium 5 The concentration required proved to be only 0.07%