THE CULTIVATION OF MONOCYTES IN FLUID MEDIUM

Monocytes from blood and from spleen have been cultivated in fluid medium in Carrel flasks for over 2 months. Diluted serum supplied all the essential nutritive substances. Cultivation in fluid was made possible by adjusting the initial pH of the fluid to 7.4, and not allowing it to fall below 7.0 or 6.8. The cells remained in good condition when the pH was adjusted with either lactic acid, hydrochloric acid, or carbon dioxide. Adjustment with carbon dioxide was found to be more convenient and also more practical, since it does not destroy the buffer action of the medium. After 2 months of cultivation, the monocytes were in excellent condition and still proliferated actively. They gave every indication that indefinite multiplication could be maintained under the conditions of these experiments. It is hoped that this method of cultivation, with some modifications, will prove useful in studying the metabolism of these cells

ARTIFICIAL MAINTENANCE MEDIA FOR CELL AND ORGAN CULTIVATION : II. THE CULTIVATION OF ORGANS IN ARTIFICIAL MEDIA

Experiments have been described in which organs cultivated in the Lindbergh apparatus have been kept alive in artificial media for periods ranging from 3 days to 3 weeks, with life continuing in portions of some glands for from 35 to 62 days. The media used varied in composition from very simple and inexpensive ones in which some serum was used to a rather complex medium containing only that negligible amount of serum that is necessary as a solvent for vitamin A. The experiments performed demonstrate that artificial media can be used for organ cultivation, thus making it possible to study the behavior of human organs; to control the composition of the media at will; and to reduce the cost of experimentation

NITROGEN METABOLISM OF NORMAL AND SARCOMATOUS FIBROBLASTS IN PURE CULTURES

1. Both normal and sarcomatous fibroblasts of the rat utilize many different fragments of the protein molecule for their growth in vitro. Alpha and beta proteoses have approximately equal growth-promoting power. 2. A mixture of peptones, peptides, and amino acids, containing a negligible quantity of proteose, produces a temporary proliferation of normal fibroblasts, and an unlimited multiplication of sarcomatous fibroblasts, provided these substances are derived from liver which contains products of unknown nature that complete the nutritive effect of the protein degradation products. 3. Amino acids contribute to the nutrition of the cells, but are unable without the addition of peptides or polypeptides to support their life. 4. The proteolytic products are more toxic to normal than to sarcomatous fibroblasts. The hypothesis is suggested that the greater acidity produced by the large glycolysis of the sarcomatous cells may account for this difference through altering the speed of action of protein synthetizing enzymes

EFFECT OF LIVER AND PITUITARY DIGESTS ON THE PROLIFERATION OF SARCOMATOUS FIBROBLASTS OF THE RAT

1. A media containing all the essential constituents for the cultivation in vitro of sarcomatous fibroblasts of the rat has been prepared by digesting calf liver and also the anterior lobe of the pituitary body with pepsin. 2. The nutritive action of the pituitary digests is not altered by thorough extraction with ether. 3. After a pure strain of sarcomatous fibroblasts had been cultivated for 3 months in a liver digest, its proliferative activity was as great as at the beginning of the experiment. The same was true of the colonies cultivated for 1 month in a digest of pituitary gland. The increase in the volume of the colonies which takes place in the digests is about as great as that produced by chick embryo juice. 4. Normal chicken fibroblasts also proliferate in both digests, but they undergo fatty degeneration after a more or less prolonged period of cultivation

LIPOIDS AS THE GROWTH-INHIBITING FACTOR IN SERUM

The growth-inhibiting action of serum has been shown to be due largely to the lipoids. Serum from which the lipoids have been removed is much less inhibiting to the growth of fibroblasts in vitro than is the original serum, and only slightly more inhibiting than Tyrode solution. The lipoids extracted from the serum are toxic and more inhibiting to the growth of fibroblasts than the original serum. Lipoids extracted from chicken brain, chicken liver, egg, and embryonic tissue have likewise an inhibiting action

THE EFFECT OF DIGESTS OF PURE PROTEINS ON CELL PROLIFERATION

1. The pepsin hydrolytic products of the pure proteins, crystalline egg albumin, crystalline edestin, and purified fibrin, are utilized by fibroblasts for their proliferation. It appears, therefore, that the growth of fibroblasts in fibrin digests and in the proteose of Witte's peptone is due to the split products of the protein itself and not to accompanying cellular constituents or other impurities. Preliminary denaturation of the protein, as carried out in these experiments, does not alter the nutritive properties of the digest. 2. The digests of the pure proteins employed are deficient in certain substances and do not meet the entire nutritive requirement of the cells for an unlimited period of time. Some supplementary nutritive substances are present in fresh embryonic heart tissue. This circumstance explains the growth of fibroblasts from fresh embryonic heart in digests which do not promote the growth of a pure strain of fibroblasts. 3. Glycocoll and nucleic acid have been found to supplement the nutritive action of pure protein digests for sarcomatous fibroblasts, and to increase greatly the length of life of the tissues. 4. Vegetable proteins, as well as animal proteins, yield proteolytic products which promote the multiplication of fibroblasts

ACTION ON FIBROBLASTS OF THE PROTEIN FRACTION OF EMBRYONIC TISSUE EXTRACT

The above experiments indicate that the growth-stimulating substance found in embryonic tissue extract, which has been responsible for the continuous growth of fibroblasts in vitro for 14 years, is either protein in nature or closely associated with the protein of the extract and adsorbed by it. If any specific hormone responsible for cell division is present, it is united to the protein or carried along with it in its first precipitation. It seems probable that the tissues utilize this protein for the nitrogen which they build into protoplasm. Whether it is first hydrolyzed before adsorption by the tissues has not been ascertained as yet. It has been shown in other experiments reported in the following paper that the amino acids of the tissue juice do not suffice for the growth of fibroblasts and that hydrolyzed tissue juice is toxic in the same way that a too concentrated mixture of amino acids is toxic. The results of the foregoing experiments may be summarized as follows: 1. Fractionation of embryo tissue juice has shown that it is the protein fraction that contains the activating substance. 2. Tissues continue to grow for a long time in the protein of the extract precipitated by CO2 and at a rate approximately equal to that in the original extract diluted to the same nitrogen concentration. 3. The non-protein nitrogen gives slight stimulation to growth. 4. Purification of the protein by repeated precipitation destroys its growth-promoting properties, but whether this is due to a denaturing of the protein,—which occurs very readily,—or to loss of some substance possibly an enzyme attached to it, has not been ascertained. 5. Preparations of purified proteins from embryonic tissue and egg white have shown no marked nutritive or stimulating action. A number of other pure substances have been tried without effect

EFFECT OF THE AMINO ACIDS AND DIALYZABLE CONSTITUENTS OF EMBRYONIC TISSUE JUICE ON THE GROWTH OF FIBROBLASTS

The ultrafilterable constituents of embryonic tissue extract are unable to support cell life in vitro. They stimulate cell migration and possibly multiplication, without increasing the mass of the tissue. Embryonic tissue extract freed from amino acids by dialysis still retains a considerable part of its growth-promoting properties. The area of growth of tissues in embryonic tissue extract free from amino acids is appreciably less than that with the whole extract, probably owing to the denaturation of part of the protein, or perhaps to the inactivation or loss of an enzyme. The addition of either the ultrafilterable components or an artificial mixture of amino acids to this dialyzed extract increases the area of cell migration but does not restore all the activity lost on dialysis. The observed differences in growth of tissue, due to the addition or removal of dialyzable and ultrafilterable constituents of the extract, prove that the amino acids produce a more active cell migration and possibly multiplication, but no building up of new protoplasm

THE CHEMICAL NATURE OF SUBSTANCES REQUIRED FOR CELL MULTIPLICATION

1. Fibroblasts and epithelial cells in pure culture obtain the nitrogen, which they build into protoplasm, from proteoses and possibly other primary derivatives of proteins. These proteoses have been prepared from embryo tissues, egg white, commercial fibrin, rabbit brain, Witte's peptone, etc. 2. The presence in embryo juice of a hormone that stimulates cell division is improbable. 3. Proteoses separated from peptic digests of fibrin by sodium sulfate determine a more abundant and prolonged multiplication of the fibroblasts than is produced by embryo juice. Peptones and the smaller split products appear to furnish some nutrient material, but do not cause the rapid proliferation characteristic of proteoses, and are sometimes toxic for tissue cells. 4. Possibly the effect of embryo juice on fibroblasts and epithelium is due to the splitting of the protein of the juice into proteoses by the cell enzymes, or by other enzymes activated by the presence of living cells

ARTIFICIAL MAINTENANCE MEDIA FOR CELL AND ORGAN CULTIVATION : I. THE CULTIVATION OF FIBROBLASTS IN ARTIFICIAL AND SERUMLESS MEDIA

Several media designed for maintaining the life of cells and organs outside the body have been described. Cultures from a pure strain of fibroblasts have been maintained in these media in vital condition and with little or no proliferation for periods varying from 43 to 56 days. One of these media is very simple, inexpensive, and easy to prepare; and one is serumless