George O. Gey

Human Hormone Production in vitro

A new in vitro hormone-synthesizing cell system, line BeWO, derived from human gestational choriocarcinoma, has been permanently established; a high degree of synthesis of functional hormone has been maintained for 18 months in tissue culture. Predominantly glycolytic metabolism, dependent on the level of glucose in the medium, has been observed. Many facets of placental and tumor-cell metabolism, growth, and differentiation are found in this unique multipotential cell.

Long-term growth of chicken fibroblasts on a collagen substrate☆

Abstract In 3 separate experiments, cells derived from chick embryo muscle explants have been grown in either Waymouths medium or Lings (AN.54) medium with 20% human placental cord serum. Continuous transfer and new outgrowth from a succession of 2 × 2 mm fragments has continued for periods of 24 to 44 months. Continuous growth was achieved only on a collagen substrate, and no continuous growth was obtained when cells were transferred to glass. When incubator temperature was raised to 43 °C over a period of 1 month, new cell types developed and had the capacity to both survive and grow directly on glass for several months.

Humoral factors involved in the induction of liver regeneration in the rat.

Summary On the basis of a comparison of the action of blood sera from partially hepatectomized and control rats on the growth response of tissue cells in vitro an hypothesis was formulated with regard to the induction of the regenerative process in the liver which follows partial hepatectomy. According to this hypothesis certain constituents of normal blood serum exert a growth inhibitory action at their normal concentrations. Partial hepatectomy reduces the amount of functioning hepatic tissue thereby resulting in a decrease of the serum concentration of these constituents. This in turn results in the initiation of regenerative growth in the liver. Further evidence supporting this hypothesis was obtained by the induction of cell division in the intact liver of adult rats by decreasing the concentration of serum constituents in vivo through plasmapheresis.

ACTIVITIES AND RESPONSES OF LIVING CELLS AND THEIR COMPONENTS AS RECORDED BY CINEPHASE MICROSCOPY AND ELECTRON MICROSCOPY

Some of the very remarkable properties of the protoplasm of living cells must be related to the basic structure of the fibrous gels of which it is composed. The free and purposeful movement of the organized structures and of different cell components, apparently differing in function, can now be visualized and studied quite well with advanced methods of physical optics. By the proper use of phase optics and time-lapse photomicrography,’ a remarkable comparison of the living cell’s composition and structure can be made with similar but fixed structures examined in the same or an homologous cell, a t much higher powers, under the electron microscope.23 9 A series of static images and short motion picture scenes were used to illustrate some of the similarities and differences in the structural organization and the behavior of various components of the tumorous cell strain of the rat (T-333) derived in vitro from a normal fibroblast strain 1 4 ~ . ~ Cinephase studies were also made of the 23-year-old strain of human chondromyxosarcoma, strain D-1 Re; of the 15-year-old human fibrosarcoma, strain A.Fi.; and of the 3-year-old strain of human epidermoid carcinoma of the cervix, strain HeLa, when grown in thin tissue-culture slides. These motion pictures show clearly the structural transformations of the very active superficial plasmagel layer of these cells. Coarse fibrous processes and fine microfibrils, especially numerous in the normal strain 14p cells, reveal their long filamentous character as shown in PLATE I, FIGURES 1 and 2, and reveal their narrow dimensions (some about 0.2 microns in diameter). These microfibrils show their remarkable ability to grow rapidly in crystalline fashion, then stiffen and often break off, or even “deliquesce” into the receding less polymerized or solated base from which they originally appeared. In PLATE I, FIGURE 1, the upper edge of this thinly spread out normal rat fibroblast shows three distinct and two fainter microfibrils, all of which began as tiny protrusions from the cell edge or from the surface and within a few minutes became greatly elongated. Some of them may become several times longer than those shown here.6 Such an image can be compared directly with the microfibrils seen in thin sections with electron microscopy as shown in PLATE 1, FIGURE 2, where one long and two shorter ones may be seen projecting from the plasmagel membrane. In some cases the microfibril may be seen to be composed of two components or represented as two states of gelated protoplasm, a stiff fiber component and a softer beaded component, It may often be seen to recede toward the cell membrane like a tiny droplet of water falling down a single spider-web fiber or the entire fibril may “deliquesce” and fuse with plasmagel

RESPONSES OF A VARIETY OF NORMAL AND MALIGNANT CELLS TO CONTINUOUS CULTIVATION, AND SOME PRACTICAL APPLICATIONS OF THESE RESPONSES TO PROBLEMS IN THE BIOLOGY OF DISEASE

For about half a century now, published reports show that investigators have progressively widened their interests in, and have more seriously examined the behavior of, explanted living tissues and their constituent cells, and that these investigators have tried in a number of ways to influence and control cellular activity. These biopsy studies of primary explants and continuous cell cultures have described work on tissues, especially from avian and mammalian hosts, in various stages of development up to senescence and death, and from experimentally altered and diseased hosts. Especially during the latter half of this period, there has developed an expanding interest in improving the techniques of tissue culture and a more serious application and planned projection of these methods to certain problems in the biology and control of disease. We have tried to contribute to these areas during this period.‘, 2 , 3 4 . The technical achievements to date, and their potential applications, demonstrate the feasibility of comparing the derived normal or controlled state with a diseased state of the tissues or cells examined freshly during several days or more following biopsy, or as a more or less stabilized cell population during continuous cultivation. The tissue and cell responses in vilro, however, should be expected to vary somewhat from the natural state following varying periods of maintenance in vitro. In fact, whatever may have been the original state of the tissue prior to explantation, and however dependent, of course, it may have been upon the conditions imposed, one should be prepared to expect a variety of responses following explantation, including considerable shifts in cell composition. The responses of certain cells to continuous cultivation might be: a continuation of a previous rapid growth, as in a tumor; a continuation of an original dormant state (an apparently difficult state to maintain in vilro) ; a relative reversal of a previous state of increased physiological activity; a new transformation or adaptation;6. or, perhaps, whether wanted or unwanted, varying degrees of cell degeneration and death. In this latter category, we must hold ourselves responsible for the degenerative changes that have set in and attempt to account for them in a true spirit of scientific endeavor. Socalled ‘Lnonspecific” degeneration, for example, often plagues those wishing to keep cells in a good s t d e of health during continuous cultivation. Are the

GROWTH OF CELLS IN AGITATED FLUID MEDIUM

One of the difficulties arising from the use of tissue cultures in physiological studies has been the accurate evaluation of growth under experimental conditions. The methods which have been developed, in most cases, have been confined to a particular type of measurement which gives an indirect measure of the actual number of cells present a t any one time. When compact cell colonies are formed, the determination of cell number may be difficult or impossible, and the evaluation of the conditions of the environment to which each individual cell of the colony is exposed often cannot be determined. The cells on the edge of the colony are exposed directly to the nutrient fluid, while those in the center are exposed partially or exclusively to living or handicapped cells. In the method to be reported here, cells which do not form colonies and are maintained in suspension have been used. The cell suspension is kept agitating so that the medium is mixed continually and all of the cells are exposed to uniform environmental conditions. Samples of the suspension are removed for cell counts. Since Harrison’O 9 l1 developed the first method for growing animal tissues in vitro, it was thought by many investigators that a supporting framework consisting of a substance such as fibrin was necessary for optimum growth of tissues in * Since then, it has been found that, in the case of many cell types, good growth occurs on a glass surface in a liquid l2 It has been found now that cells can be grown without being permanently attached to a surface. This method was first reported a t the meetings of the American Association for Cancer Research held in April 1953.

Chemically characterized concentrated corodies for continuous cell culture (the 7C's culture media)

A series of concentrated chemically characterized media has been developed for continuous culture of mammalian cells and designated as 7Cs media. They contain amino acids at levels from 27 to 162 mM/l comparable to the amino acid content of 5 to 30 per cent of serum or 0.3 to 1.5 per cent of lactalbumine hydrolysate, but about 5 to 30 times the amino acid concentrations contained in most of the chemically defined media currently in use. The 7Cs media contain fat-soluble vitamins, the polyunsaturated fatty acids, and trace elements known to be nutritional essentials for the mammalian organism. The proportionality pattern of the amino acids in these media are, in general, similar to those found in animal tissues and food proteins of high biological values and, therefore, avoids gross imbalance. The levels of the vitamins were based on their known biological potency determined for whole organisms and taking into consideration the concentrations found in nonstorage human or animal tissues. The trace elements content are in proportion similar to those existing in plasma or body fluids. The buffering capacity of the 7Cs media has been improved compared to most other CDM due to two factors: (1) substitution of the bicarbonate buffer with β-glycerophosphoric acid disodium corrects the tendency of pH to rise during incubation because of the loss of CO2 and since the calcium salt of glycerophosphate is soluble in water it does not form precipitates with calcium like other inorganic phosphate buffers; (2) the high levels of amino acids in these media increase the buffering capacity due to the zwitterionic character of these substances [19]. Continuous culture studies with WRC-256 and MBIII lymphoblast cells demonstrate that the high amino acid levels of 7Cs media AN-54 and AN-81 are able to support satisfactory growth of these cell lines without supplements of serum or any other tissue extracts. These two cell lines have been maintained in these serum-free media for five and two years, respectively.

Utilization of Serum Lipids by Cultured Mammalian Cells.

Summary 1) A variety of mammalian cells growing in tissue culture took up appreciable amounts of lipid from the serum used in the medium. For the MB III strain of mouse lymphoblasts, triglycerides were most rapidly utilized followed by phospholipids and thea cholesterol. This preferential utilization of triglycerides was also shown by 4 other cell strains. 2) The MB III cell utilized saturated and unsaturated lipids equally readily, and there was little net breakdown of lipid to satisfy cell energy requirements. There was however considerable conversion of triglycerides to phospholipid following absorption. 3) Cholesterol uptake appears to be a general phenomenon, and appreciable amounts ranging from 0.6% to 2.8% of the dry weight were found in all strains of cultured cells examined. Cholesterol content of cells was related to the type of serum used in the growth medium. 4) The composition of the lipid taken up by the cells differed markedly from that in the medium. This is discussed from the point of view of: (1). A specific process of uptake or (2). A non-specific uptake by pinocytosis, followed by incorporation and a non-specific excretion. Evidence is presented in favor of this second mechanism.

In vitro identification of the trophoblastic stem cell of the human villous placenta

Abstract An in vitro search for th e epithelial stem cell of the placenta has been made. The cytotrophoblast has been selected for tissue culture from germinal beds of cell column cytotrophoblast of the first and early second trimester placenta. These beds have been shown by Wislocki and Padykula 9 to represent the generative source of new and expanding placental villi in vivo. Th e morphologic charact eristics of these cell columns confirm their cytotrophoblast composition in hematoxylin and eosin sections. Under the dissecting microscope comparable areas were stlected and ex planted in tissue cultures. Primary growth of cytotrophoblast with characteristic micro villi migrated from these cell columns and have been followed by daily microscopic observation. HCG was detected in the medium in which these cells were grown for a limited period of time, roughly comparable to the period during which mitosis occurred in cytotrophoblast. It is concluded that these cells represent the functioning cytotrophoblast of the normal placenta.

A fibrillar structure in rat fibroblasts as seen by electron microscopy.

During the course of comparative tissue culture studies of a strain of normal rat fibroblasts and its malignant cell derivative, 1 we obtained preparations of both of these cell types which under the electron microscope† showed an unmistakable fibrillar structure in the thinly spread cytoplasm. The fibrils, of an estimated thickness of 10 to 100 mμ, may converge and diverge in fan-like formations of great regularity (Fig. 1) but are more often seen gathered together in long bands of varying widths (Fig. 2). The composite bands are seen clearly in phase microscope movies of the cells, 3 but it is only by the increased resolution available with the electron microscope that the nature of the bands becomes clear. They are apparently composed of long thin converging fibrils which in general radiate from the dense central area. That the fibrils are not produced by a wrinkling of the formvar membrane on which the cell is stretched is indicated by their uniformity, by their sharp change in direction in certain areas of the periphery, and by their fine converging and diverging structure. Besides this, no wrinkles were seen in the membrane outside of the cellular area where clear visualization is possible. Of interest are the relations of these bands to the general structure. The mitochondria, often long and thin, seem frequently to be associated with individual fat droplets. Some of the elongated mitochondria are found in areas which show many microsomes, with the whole area bounded by parallel fibrous bands. This appears to be a static representation of the streams of material which can be seen in the movies of these cells. In other areas, the great masses of mitochondria may be seen crossing those fibrous bands.