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The present report is concerned with changes in the pyroninophilic structures of the liver cell, as an early effect of carbon tetrachloride poisoning.In the hepatic cells of various mammals and lower animals a peculiar type of granulation has repeatedly been observed and described.1 These structures are characteristic in that they stain an intensive red with methyl green-pyronin (Pappenheimer's method). They vary in size and shape and may have the form of spheres, plump rods or lumps. They are often accumulated in the immediate neighborhood of the nucleus, but occasionally are spread over the entire cytoplasm. There is a clear relationship between the pyroninophilic granule content of liver cells and the nutritional state of the animal. In livers of fasting animals or in animals fed on carbohydrate or fat only the pyroninophilic structures disappear; in animals kept on a diet rich in proteins and products of protein cleavage they are especially numerous. The pyroninophilic granules can be digested by prot...

Various authors (Borrel,1 Roskin,2 Lipschutz,3 Fischer,4 Zweibaum5) have indicated that the structure of Rous sarcoma cells shows certain peculiarities, which differentiate them from normal mesenchyme cells.These indications are very fragmentary and there is little agreement between the various descriptions. The morphological features of the Rous sarcoma cell are still not sufficiently known.The structural peculiarities of the Rous sarcoma cells appear with great clarity and they are easily detectable in pure culture of sarcoma cells. Our studies of cell strains, cultured without addition of normal tissue revealed that the sarcoma cells in vitro constantly show profound morphological alterations of their cytoplasma and nuclei, often associated with marked, indeed extraordinary hypertrophy of the entire cells.In this note we report on the changes taking place in the nuclei of the sarcoma cells. The studies were made on cultures (preparations in toto and serial sections) fixed in Carnoy and stained with Gie...

Material and Technic The strain of malignant mesenchymal cells described in this paper originates from a rat tumour induced by 1:2-benzpyrene. This tumour is a sarcoma which developed in a rat after four injections of the carcinogenic substance over a period of two months. The tumour thus produced was inoculated from rat to rat. Up to the time of writing it has reached its fourteenth passage and is still nearly 100 per cent virulent on inoculation. For the tumour culture small fragments of a benzpyrene tumour were used which had been transferred from animal to animal four times previously. The material was taken eighteen days after inoculation, when the tumour was the size of a plum, was clearly delineated from the surrounding tissue, and of fairly hard consistency. The cut surface was perfectly homogeneous and gave no evidence of necrosis. Microscopic examination showed a typical fibrosarcoma, composed of long spindle-shaped cells, arranged in narrow bundles taking a rather irregular course in parts (Fig. 1). The nuclei of the spindle cells are oval in form and rich in chromatin. There are extreme variations in their shape and size. Numerous smaller histiocytes are scattered among the spindle cells, their nuclei being smaller and denser than those of fibroblasts, with a basophilic cytoplasm. Many cells are in a stage of mitotic division and 2–5 mitoses are to be found in many visual fields. The tissues were cultivated in a hanging drop or in flasks according to the classical methods of Carrel. After numerous preliminary experiments, in which chicken plasma, rat plasma, and a mixture of both in various proportions were used, chicken plasma was found to be the most suitable medium for the culture of our strain of rat tumour cells. As a growth-promoting factor chicken embryonic juice (third centrifugate exclusively) was employed. The method of Fischer and Parker proved especially favourable for culture of this strain of tumour cells. According to these authors, cultures are prepared in Carrel flasks on Tyrode plasma and cultivated without addition of embryonic juice. Every third day cultures are fed on heparinized plasma (1:1000) after previous washing. The plasma is completely removed after being one hour in a flask. Coverslip cultures must not be transferred more often than every third or fourth day. In most instances they have not reached a sufficient size at this time to permit division. If a rapid increase in the number of cultures is desired, the following method proves successful: On transferring a culture, it is divided into two portions, both of them being cultivated together in the same plasma drop. After two or three days the two daughter cultures fuse into one colony which is able to attain a fair size in the course of further development. Such a colony may be further subdivided without difficulty.

We have previously reported some nuclear alterations in Rous sarcoma cells grown in pure culture.1 The following is a brief account of the cytoplasmic changes which are generally developed in Rous sarcoma cells cultivated in vitro.The most outstanding changes take place in the central area of the cell. The cytoplasm of the central cell region at first takes on a cloudy appearance. Indefinite at first, this zone gradually becomes better defined and finally assumes a very sharp outline (Fig. 1). In general, the segregated central area is 3-6 times as large as the nucleus, is spherical in shape and always appears to be denser than the surrounding cytoplasm, but shows no pronounced structural peculiarities. In the first stage of its development the central area is always somewhat eosinophilic; in the more advanced stage of the segregation process its tinctorial characteristics become rather indefinite; with Giemsa it stains at times slightly red and at times slightly blue, and mostly in mixed tones. The nucle...

Studies on the effect of X-rays upon cells growing in vitro have shown that with sufficiently high X-ray doses the outgrowth of the irradiated cell culture can be checked immediately and completely. For colonies of chicken fibroblasts, 250,000 r units are the minimum dose which produces this effect (“the immediate lethal dose”).1It is also possible to produce a lethal effect on cells in vitro by exposure to considerably smaller X-ray quantities. X-ray doses, which are themselves too low to cause immediate cessation of growth of the irradiated culture, are capable of exerting a lethal action after a latent period (Fischer and Baastrup,2 San-tesson,3 Spear,4 Cox5). Cultures treated with such X-ray doses continue to grow for some time; but after a certain number of passages, cell growth ceases.The mechanisms of action of the immediate and the delayed lethal doses, respectively, are, in all probability, fundamentally different. The immediate lethal doses stop the outgrowth in the irradiated culture by alterin...

SummaryChick embryo heart fibroblasts cultured in vitro, when immersed in colchicine solutions 10-4 or 10-5 M, show a tendency to assume a spherical shape. Their surfaces display a considerable activity as manifested by rapid protrusion and withdrawal of pseudopodia. Subsequently the zone of out-growth of the culture splits up into detached elements, comprising one or more cells. This whole process is not lethal to the cells and is, to a great extent, reversible.

SummaryThe effect of direct irradiation with X-rays on mitoses in cultures of rabbit's bone-marrow was investigated. The X-rays in all doses applied (250, 500, 1000, 2500, 5000 r units) produced at first a profound decrease in the number of mitoses. In cultures irradiated with doses of 1000 r units and less the decrease in mitoses was followed by complete recovery of the mitotic activity. The recovery of mitoses in cultures irradiated with 2500 r units and more was only incomplete and the irradiation resulted in aplasia of the bone marrow.

SummaryThe appearance of an osteoid sarcoma in a series of chickens injected intravenously with the causative agent of Rous sarcoma derived from sarcoma cell cultures, is reported.

IN investigations on the physiology of neoplastic growth, the conceptions are often encountered that the malignant cell has an unusually high growth capacity as compared with the normal cell, and that neoplastic tissue is especially rich in growth-promoting factors.

THE following is a brief account of the cultivation of the causative agent of fowl leukosis (haemacyto-blastosis strain T1, Engelbreth-Holm) in the presence of normal cells proliferating in vitro.

A FRAGMENT of tissue removed from the body and explanted in vitro does not commence growing immediately. A certain period of time must elapse before proliferation sets in. In a paper some years ago which has been overlooked in the literature on tissue culture, Cohn and Murray1 reported that the period between the moment that the tissue fragment is explanted in vitro and the first appearance of new cells varies with the age of the embryo. As the embryo develops, this latent period becomes progressively longer. The latent period of adult tissue explanted in vitro is considerable as compared to that of embryonic tissue. We have demonstrated2 that it is approximately three times longer than that of tissue taken from an embryo in a late developmental stage. Furthermore, we have shown that during postnatal growth the latent period increases gradually3. It reaches its maximum when the animal ceases growing and remains constant thereafter, not even changing during senescence3,4.

Embryo extract is a powerful growth-stimulant for cell colonies in vitro. It is generally accepted that its influence is manifested in, and limited to, the activation of cell-division. Our observations on the effect of X-rays on cell cultures make it necessary to revise this view. The investigations reported here demonstrate that cell cultures deprived of mitotic capacity by previous irradiation with X-rays will also grow at a considerably increased rate after the addition of embryo extract.The experiments were performed on standardized cultures of chicken fibroblasts derived from the hearts of 7-days-old embryo The cell colonies were cultivated in hanging drops according to the method of Carrel. After the irradiation, they were planted into Carrel flasks. Growth curves of the culture were constructed from planimetric measurements of outline drawings of the surface areas made every 24 hours. Irradiation was carried out using a demountable X-ray tube which worked at a tension of 35 KV on currents 20 MA. Th...

SummaryThe occurrence of mitosis in the liver cells of rats receiving daily intraperitoneal injections of thiourea is reported. Both mitosis of normal configuration and pathological mitosis were encountered. Thiourea induced mitotic activity of the liver seemed to be at its height during the period of 48 to 72 hours after the first injection.

SummaryRats with experimental skin wounds received intraperitoneal injections of saline extract of adult chicken heart. The course of wound healing in animals treated in this way was compared with the healing process in wounds of equal size in untreated rats. The experiments showed that treatment with cell growth-promoting extracts of adult hearts caused a significant reduction (21.8%) in the mean time required for healing, as compared with the controls.

AN extensive growth of cell colonies in vitro is only obtained if tissue extracts are added to the culture medium. It has been generally accepted (Levi1, Verne2, Willmer3) that extracts of growing tissues (embryonic and neoplastic tissues) and extracts of organs with active cell formation (for example, bone-marrow) chiefly possess these growth-activating properties. Extract of embryonic tissue is considered to be the best growth activator (Willmer3).

IN a previous note from this Laboratory1, it was reported that extracts of heterologous adult tissues possess the ability to stimulate in vitro the growth of various cells including human epithelium, and the use of adult tissue extracts in wound treatment was suggested. We report below the results of experiments carried out to purify the growth-promoting principles present in adult tissue.

NUMEROUS experiments on gamma irradiation of cells cultivated in vitro have demonstrated their high radio-resistance to doses designed to produce a lethal effect. Sub-lethal doses have the effect of producing changes in the mitotic activity of the culture, while the lethal dose has so far only been observed as a delayed effect.

Effect of Extracts of Heterologous Adult Tissue on Cell Growth in vitro and their Use in Wound Healing

THE procedure adopted for the cultivation of the Rous sarcoma in vitro in accordance with the usual method described by A. Fischer1 is as follows: A fragment of tumour is incubated in a medium consisting of chicken plasma and dilute embryonic extract. After a time the growing culture begins to digest the surrounding medium and also itself. Such a culture will die out if it is not provided with fresh tissue by means of subculture. The sarcoma cells infiltrate the added tissue fragment within which they remain for a time embedded. If normal tissue is regularly added, the sarcoma cells can be cultured over an unlimited period and will retain their malignancy.

THE following is a report on the inactivation of the causative agent of fowl leucosis (haemacyto-blastosis, Strain T Engelbreth-Holm) by means of X-rays.