MIXED CULTURES OF PURE STRAINS OF FIBROBLASTS AND EPITHELIAL CELLS

1. Strains of epithelium and fibroblasts cultivated side by side in the same medium keep their individual characteristics. When sectioned and stained by the Van Gieson method, the cultures show the epithelium stained greenish yellow and the fibroblasts and their fibrillæ pink. 2. There are no transition forms between the epithelial cells and fibroblasts. 3. The epithelial cells belonging to an older strain are still able to form primitive structures of winding tubules, with typical glandular epithelium. 4. Under the conditions of the experiments, no dedifferentiation takes place.     

THE DEVELOPMENT OF PURE CULTURES OF FIBROBLASTS FROM SINGLE MONONUCLEAR CELLS

1. Most isolated guinea pig mononuclear exudative cells in tissue culture become typical migrating macrophages, but a small proportion take on fibroblastic characteristics, and produce pure colonies of fibroblasts. These fibroblasts maintain their morphological characteristics through repeated subcultures. 2. It is suggested that the subsequent development of individual mononuclear cells in tissue culture is conditioned at the time of explantation. 3. Apposition with other cells is not necessary for the initiation of mitotic cellular division. 4. There is a definite optimal relationship between the bulk of the medium, the number of explanted cells and the extent of proliferation. The presence of other cells in the vicinity enhances cellular division. 5. Mitosis in the isolated explanted cell is preceded by a latent period. The rate of division varies in different colonies of fibroblasts. 6. Admixed erythrocytes in the mononuclear suspension definitely inhibit proliferation of fibroblasts in tissue culture. The inhibiting factor in disintegrating erythrocytes is apparently present in the stroma.     

A SIMPLE METHOD FOR THE ISOLATION OF PURE CULTURES FROM SINGLE BACTERIAL CELLS

A method is described for the isolation of pure cultures from single bacterial cells, which is simple in operation and which requires no complicated apparatus. In addition to its simplicity, the method possesses the further advantage that several selected colonies can be isolated from the same preparation.     

PURE CULTURES OF LARGE MONONUCLEAR LEUCOCYTES

1. Pure strains of mononuclear leucocytes were isolated from the blood of adult chickens and keptin active condition for nearly 3 months. 2. The cultures were composed of large mononuclear leucocytes which migrated and proliferated in vitro at a slower rate than fibroblasts. The cells had no tendency to form a tissue, as do fibroblasts and epithelial cells. They were much less resistant than fibroblasts. 3. Differentiation of the large mononuclears into cells assuming the appearance of fibroblasts took place under certain conditions. 4. The activity of the large mononuclears was increased by embryonic tissue juice and inhibited by homologous serum.     

IMMUNOLOGICAL STUDIES ON PURE CULTURES OF VARIOUS SPIROCHETES

Experiments were carried out for the study of culture spirochetes in their relation to various immunity reactions in vitro. Several strains of Treponema pallidum and one each of Treponema calligyrum, Spirochata refringens, Treponema microdentium, and Treponema mucosum were used. Tests were made of immune substances responsible for agglutination, complement fixation, spirocheticidosis, and opsonization. In cases of agglutination and complement fixation, cross titrations were made. 1. In the sera derived from rabbits immunized with various spirochetes agglutinins were demonstrated in varying quantities for the homologous antigens. The amounts of agglutinins developed were considerably higher in the pallidum immune sera than in the other groups. There was no parallelism between the amounts of antigens injected and the amounts of agglutinins developed. 2. Cross titrations among different pallidum strains revealed that the agglutiantion is not necessarily strongest when homologous antigens and immune sera are brought together. 3. On the other hand, the reactions between the immune sera and antigens belonging to different species were sufficiently specific to justify the grouping. 4. Certain degrees of group reactions were observed between the pallidum immune sera and the calligyrum, and occasionally very faintly also between the pallidum and the refringes antigens and vice versa. There was a much more pronounced group reaction between the calligyrum and refringes. The immune serum and antigen of the microdentium showed a slight affinity for the mucosum but none for the pallidum, calligyrum, or refringes, while the mucosum immune serum caused a slight agglutination with many members of the other groups. Hence, it appears that the pallidum is more or less related to the calligyrum, while the affinity between the calligyrum and refringes, and possibly also between the calligyrum and mucosum in a much smaller degree, seems close. The microdentium showed the least relation to any other spirochetes. 5. Titration of agglutinins in the sera obtained 3 months after the cessation of immunization revealed that the agglutinin contents were already greatly reduced, having fallen roughly to 0.01 of the original strenght. The rates of disappearance were irregular in different animals and bore no direct relation to the initial titers. Titration made of the immune sera which had been preserved aseptically in a refrigerator (6°C.) during the same period (3 months) indicated that the original strength of these sera was reduced to about one-tenth. The agglutinins for spirochetes disappear from the rabbit''s body much more rapidly than they are reduced in the separated sera by deterioration on standing at 6°C. 6. Titration of the immune sera for complement fixation power showed with a few exceptions, in which there was only slight complement binding, that the titers were high enough to indicate the presence of this principle. The anti-pallidum sera possessed higher average titers than the other immune sera tested with correspondingly homologous antigens. The least active were the anti-refrigens sera. 7. Cross titration of anti-pallidum immune sera for complement fixation showed that a given serum with a high titer for its own strain of antigen was also strong with most of the other strains of the pallidum. Instances occurred also in which the titers with heterologous pallidum antigens fell far below those of the homologous. Group reactions between the different spirochetes) such as the pallidum and the calligyrum, the calligyrum and the refringens, and the microdentium and the mucosum, were also indicated. The mucosum and the pallidum showed a slight degree of group reaction. No anti-pallidum serum fixed complement with the microdentium. 8. The immune sera were tested for their spirochetiddal properties in vitro against the correspondingly specific and heterologous varieties with and without the addition of complement. Many of the anti-pallidum sera killed their own strains. Normal rabbit serum exhibited only a slight degree of inhibition. Without complement, the immune sera caused a considerable reduction in the number or density of colonies, but not a complete suppression of growth. Complement alone had no injurious effect upon the pallidum strains. The antisera for the calligyrum, refringens, and mucosum showed feeble spirocheticidal action, while the antisera for microdentium was stronger. A syphilitic rabbit serum tested against a strain of culture pallidum gave a feeble inhibitory effect. 9. Under the influence of immune sera and complement, the spirochetes undergo within a few hours complete disintegration or granular degeneration. Without complement, they are more powerfully agglutinated, but no disintegration occurs, even after 20 hours, and complement alone has no effect. 10. In the presence of homologous immune serum and complement, the culture pallidum may be ingested by the leukocytes, but phagocytosis is slight, possibly on account of the filamentous nature of the organisms. The spirochetes in such a mixture disintegrate within a few hours, disintegration being especially rapid when the immune leukocytes are used. In the absence of immune serum, phagocytosis is not noticeable, while without complement but in the presence of immune serum and leukocytes, some phagocytosis, without subsequent lysis, occurs. A virulent strain of pallidum, obtained from syphilitic orchitis in a rabbit, exposed to agglutination, lysis, and phagocytosis by an immune serum prepared by means of culture pallidum strains, showed only slight agglutination and phagocytosis but rapid immobilization without disintegration in the presence of complement.     

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.     

USE OF THE SINGLE CELL METHOD IN OBTAINING PURE CULTURES OF ANAEROBES

The pipette method has proved a feasible method of obtaining one-cell pure cultures of anaerobes. Both bacilli and spores may be used as seeding material, but spores give a much higher percentage of positives. Boiling alone affords a sufficient degree of anaerobiosis to the medium for initiating one-cell growths, and semisolid agar is the most convenient form of medium. Exposure to air during isolation apparently has no effect on the viability of spores of anaerobes, but young bacilli of some species suffer from a comparatively short exposure to free oxygen.     

A PURE STRAIN OF CARTILAGE CELLS IN VITRO

1. A strain of cartilage cells, obtained from the pars cartilago scleræ of the eye of chick embryos, has been cultivated for more than 3 months in vitro. 2. The initial growth of the cartilage was possible only on the free surface of the coagulum. 3. The hyaline substance disappeared during cultivation in vitro. The succeeding stages of a transformation from small, lymphocyte-like cells into large, spindle-shaped cells were observed. The cartilage cells were spindle-shaped and grew in close contact, forming thin membranes. In surface-grown cartilage cells, the nucleus, usually containing one large nucleolus, stained less deeply than the cytoplasm. 4. The rate of growth of cartilage was slower than that of fibroblasts and epithelium. After cultivation on the surface of the coagulum, the cartilage cells could multiply even when embedded in the coagulum. But their growth was less extensive and uniform.     

GIANT CENTROSPHERES IN DEGENERATING MESENCHYME CELLS OF TISSUE CULTURES

1. Degenerating mesenchyme cells in tissue cultures cultivated in various modifications of Locke''s solution often have giant centrospheres. 2. These giant centrospheres develop gradually around the centriole and in their most complete condition consist of centriole, medullary zone, and cortical zone. 3. Preceding the appearance of the centrosphere the centriole is surrounded by degeneration granules or granules and vacuoles, and as the centrosphere develops these are forced first to the periphery of the medullary zone and latter, when it develops, to the periphery of the cortical zone. 4. The mitochondria become orientated about the centriole and centrosphere, at first more or less radially, later concentrically. They change from threads to rods and granules. 5. The giant centrospheres and cancer cell inclusions (Plimmer''s bodies, etc.) are identical.     

A PURE STRAIN OF THYROID CELLS AND ITS CHARACTERISTICS

1. A pure strain of thyroid epithelium was isolated and maintained in active condition for 7 months. At the end of the experiment, the rate of cell multiplication was as great as at its beginning. 2. The thyroid cells grew at the surface of the coagulum as pavement epithelium, and within the coagulum as a glandular structure. 3. The cells did not dedifferentiate, and the lumen of the acini in cultures from a strain over 4 months old contained colloid secretion similar morphologically to that from a freshly extirpated thyroid gland.     

PROLIFERATION OF B AND T CELLS IN MIXED LYMPHOCYTE CULTURES

Electrophoretically fractionated CBA/Ca spleen T cells alone respond to allogeneic cells in one-way MLC and to PHA. They do not respond to E. coli LPS. B cells alone do not respond to allogeneic cells nor to PHA, but do respond to LPS. When karyotypically distinguishable syngeneic mixtures of T and B lymphocytes are stimulated with allogeneic cells, at the most 5% of mitoses on 5–9th culture day are of B cell origin. This indicates that B cells are not substantially recruited to proliferate in the MLC.     

GENETIC STUDY OF HUMAN CELLS IN VITRO : CARBOHYDRATE VARIANTS FROM CULTURES OF HELA AND CONJUNCTIVAL CELLS

The isolation of carbohydrate variants from cultures of HeLa and conjunctival cells was described. Factors inherent in the cell culture system, such as parent populations and dialyzed serums, have been shown to influence the outcome of variant isolations. Established stable variants incorporated significantly more pentoses or lactate into various cell fractions than the parent cultures. Besides their abilities to propagate continuously in the selecting environments, the variants multiplied slower, were more susceptible to sub-zero preservation and the cytotoxic effect of D-2-deoxyglucose, showed lower cloning efficiencies and were less susceptible to the deleterious effect of glucose oxidase. The ribose variants also differed from the parent cultures in morphological appearance such as formation of multinucleated cells and ring-shaped colonies. They converted more ribose into other component sugars of mucopolysaccharides than the parent cultures. Preliminary analyses of the mucopolysaccharides extracted from the ribose variants and parent cultures showed large difference in their carbohydrate (Molisch-positive materials) and DNA ratios. Evidence suggests that a sequence of interrelated events from genetic selection to primitive morphogenesis has been established.     

GIANT CELLS IN CULTURES FROM HUMAN LYMPH NODES

Giant cells resembling those found in tuberculous nodes appeared in cultures of various normal and pathological human lymph nodes cultivated in plasma. They migrated out from the explants from normal and tuberculous nodes, from nodes from acute and chronic lymphadenitis and Hodgkin''s disease, and from a metastatic sarcoma. They were most abundant in cultures from tuberculous nodes. The giant cells are similar in structure to the large wandering cells and probably arise from them. We are uncertain, however, as to how the giant cells develop. There is no evidence of fusion of the large mononuclear wandering cells; on the other hand, there is some evidence that they arise by amitosis of the nuclei without division of the cytoplasm, which increases in bulk. They contain a large central area of more or less granular character which takes up neutral red with great avidity. This central area probably consists of dead material, the waste products of metabolism and of digested foodstuffs such as lymphocytes that the cells are unable to get rid of in the abnormal environment, and perhaps also of accumulated non-living foreign substances that have penetrated into the cells and become segregated. The central area is surrounded by a conspicuous zone of fat globules in which the nuclei are embedded. The nuclei vary in number from 2 or 3 to 50 or 60. Usually, however, there are not more than 10 or 20, and in the cells that are flattened out on the cover-slip they often have a horseshoe-like arrangement about the equator of the central area. Mitochondria are abundant and usually in the form of wavy or curly threads. They seem to be most numerous in the ectoplasm immediately about the fat zone. They also extend in among the fat globules and out into the ectoplasm. A distinct, clear, homogeneous ectoplasm envelops the cell.     

THE EFFECT OF DILUTION OF PLASMA MEDIUM ON THE GROWTH AND FAT ACCUMULATION OF CELLS IN TISSUE CULTURES

1. Dilution of plasma with isotonic solutions causes a more extensive migration in cultures of cells of the actively migratory type, such as those of spleen and bone marrow. Dilution with a limited quantity of distilled water produces the same effect. Less actively motile cells are influenced little or not at all by dilution. The effect on cells of the first type is probably due to the reduction in the quantity of fibrin in the clot producing lessened resistance to cell locomotion. 2. Dilution of plasma with either isotonic solutions or distilled water is without effect on cell multiplication, as is shown by records of the number of mitoses in living culture preparations. 3. Dilution of plasma with suitable quantities of Ringer''s solution causes a marked diminution in the quantity of fat accumulated by the cells. This reduction is to be attributed to the decrease in the quantity of fat in the medium. The accumulation of fat by cells in cultures is therefore not to be regarded as the result of a cell degeneration, but as an accumulation, the source of the fat being the medium in which the cells are growing.     

CLONAL CHARACTER OF F1 HYBRID LYMPHOCYTE SUBSET RECOGNITION OF PARENTAL CELLS IN ONE-WAY MIXED LYMPHOCYTE CULTURES

Proliferation of F₁ hybrid lymphocytes in mixed lymphocyte cultures is stimulated by mitomycin-blocked parental cells. The demonstration of this phenomenon using F₁ hybrids derived from congenic lines of mice establishes that the stimulation is controlled by genes in or closely linked to the major histocompatibility locus chromosome region. In agreement with the finding that tumor-bearing mice have an increased capacity for primary alloantigen recognition, it was observed that the F₁ hybrid response to parent was also augmented by tumor bearing. Chromosomal analysis of dividing cells in one-way mixed cultures confirms that F₁ cells, and not the blocked parental cells, enter mitosis. Stimulation of F₁ cells by a soluble mediator liberated by the parental cells was not observed and mitomycin blocking of parental cells seems to be a completely effective blocking agent ensuring that parental cells can not enter DNA synthesis. The specificity and clonal nature of F₁ recognition of parent was demonstrated using a 5-bromodeoxyuridine-suicide procedure. Distinct clones of lymphocytes in F₁ spleen cell populations seem to recognize one or the other parent, but not both, in such experiments. These observations and others in tumor systems suggest that most or all heterozygous organisms may possess potentially self-reactive clones of lymphocytes.     

WANDERING CELLS,ENDOTHELIAL CELLS,AND FIBROBLASTS IN CULTURES FROM HUMAN LYMPH NODES

Wandering cells migrate from cultures of human lymph nodes after 2 or 3 hours incubation; they are actively ameboid and phagocytic. Endothelial cells appear in the plasma clot after 24 to 48 hours incubation. They are relatively inactive and less phagocytic. Fibroblasts are seen after 48 hours incubation. They are inactive and not phagocytic. The probable origin of wandering cells from endothelial cells is discussed.     

GRANULES IN THE CELLS OF CHICK EMBRYOS PRODUCED BY EGG ALBUMIN IN THE MEDIUM OF TISSUE CULTURES

It is difficult to understand what factors may be concerned in the formation of the al. granules. The phenomenon may be concerned with changes in the cell membrane due to an abnormal environment; that is, material which would otherwise be excluded may be permitted to enter the cell, or, on the other hand, certain substances may be prevented from passing out of the cells. Previous investigators have shown that mesenchyme cells sometimes engulf certain foreign bodies, and it is possible that the solution of white of egg is ingested in the same manner. When a solution of peptone was placed on the cells instead of egg white, the phenomenon did not occur (Fig. 13); the cell remained normal and degenerated in the usual manner (Fig. 14). This would seem to indicate that the al. granules are not formed from peptone. Regardless of the factors involved, it is evident that egg albumin in the medium of tissue cultures of chick embryos causes the formation of numerous large granules in the cytoplasm of the connective tissue cells. This phenomenon is associated with unfavorable conditions for the life of the cells and results in the rapid death of the cultures.     

THE FORM AND FUNCTION OF SYNOVIAL CELLS IN TISSUE CULTURES : II. THE PRODUCTION OF MUCIN

1. Synovial cultures are differentiated in tissue cultures from other tissues of mesenchymal origin by their type of growth and cell function. 2. In these respects they are more closely allied to chondroblasts and osteoblasts than to fibroblasts. 3. Synovial cells in tissue cultures develop marked globular cytoplasmic granulations that stain easily with neutral red and sometimes with toluidine blue; they show marked polymorphism with all transitions from round to spindle, polygonal and star shapes and eventually form an epithelial-like membrane, composed of cells with numerous syncytial bridges. 4. In cultures of typically growing synovial cells a mucin-like substance is elaborated. Typical growth and maximal mucin production is best maintained in media containing a minimum of growth-stimulating substances. Transformation of synovial cell growths into fibroblastic growth is accompanied by a loss of mucin production. Dying cells apparently do not produce mucin. 5. Amitotic cell division and the formation of macrophage-like cells were observed. 6. Marked tendency to liquefaction of the plasma about the growths was observed and attributed to the elaboration of a proteolytic ferment. 7. The specific designation "synovioblasts" is proposed for these cells.     

急性粒细胞白血病细胞体外长期液体培养中增殖的研究

白血病研究提示人急性粒细胞白血病是发生于多向干细胞水平的肿瘤性变,而另一些可发生在晚些阶段致细胞成熟停滞。本研究为来自11例AML的骨髓或血的单个核细胞的液体培养研究。实验结果表明CFU-AML有2种亚群。一种呈指数性生长并形成集落。另一种是指数性生长不形成集落,以至于甲基纤维素培养中亦同样具有足够的自我更新能力(指数性增殖)一个长时间,这些细胞在适当的体系中不形成BFU-E、CFU-TL、CFU-GEMM和CFU-MK集落。细胞化学染色为阴性,淋巴细胞单抗荧光染色为阴性。两种亚群在PHA依赖性、形态及体外生存时间方面均有区别。我们考虑后者可能为白血病干细胞。