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Extended expression of liver functions of hepatocytes in collagen-contained cell aggregates (cell packs).

The functions of hepatocytes under the collagen-contained cell aggregate (cell pack) conditions were studied using liver-specific protein synthesis. Freshly isolated murine hepatocytes were suspended in the medium containing collagen and centrifuged, and the resultant cell masses were cultured on the porous membranes floating on the medium. In these cultures cells were attached to each other three-dimensionally with collagen present in the intercellular spaces. Cultured hepatocytes in the cell pack maintained high and stable activity in the expression of their functions for more than 2 weeks, even when cultured with the medium lacking any hormones and serum, whereas hepatocytes in monolayer cultures lost their functions within a week. Similarly, when the cell packs of rat hepatocytes were transplanted into rat spleens, they could retain viability in the form of cell aggregate with the expression of liver-specific albumin mRNA at a higher level than in the transplanted cell suspensions. The lifespan and the initial expression level of hepatocellular functions in culture were similar to that of the cell pack in cell aggregates without collagen and in cellular monolayers on the collagen gel respectively. It was concluded that the condition where cells are in contact with each other has an important role in the expression of hepatocellular functions and collagen present in the intercellular spaces enhances the functional levels.

Albumins

[Regulation of age-dependent phenomena. Influence of C6-substituted purines on cell aggregation and cell migration in primary cultures of lense epithelial cells].

The existence of an age dependent latent period of cell emigration has been proved in the primary culture of epithelial cells of bovine lenses. The previously described aggregation phenomenon as well as the latent period of the cell emigration increase with the age of the sponsor animals. Extracellular adenine and other C6-substituted purines, isolated from the cells themselves and added to the medium, act the same way on the lens cells in the primary culture as the increasing age of the sponsor animals. Adenine stimulates cell aggregation and inhibits the adhesion of the cells to the substratum, the cell flattening and the cell migration. The adenine action has been proved down to a concentration of 3 X 10(-6) M. During the primary culture, the lens cells gradually los the adenine sensitivity. The adenine action also occurs on single cells, isolated by trypsination, it differs from the reaction of ouabain and can be removed at low concentration by washing procedures. The results favour the suggestion C6-substituted purines to be involved in cell ageing.

Adenine

Cellular composition of erythropoietic cell populations and aggregate cell cultures derived from early chick blastodiscs.

Light and electron microscopy of suspensions of cells prepared by dispersing chick blastodiscs at primitive-streak and head-fold stages showed the presence of numerous yolk granules, yolk-rich endodermal cells and occasional presumed ecto- and mesodermal cells. Several cell fractions prepared from this suspension by sedimentation through discontinuous Ficoll gradients were of similar composition. No enrichment of any particular cell type which might account for either differential sedimentation or erythropoietic potential of the fractions could be recognized. Two fractions, EP 1, and EP 2, were cultured as cell aggregates on vitelline membranes. EP 1 produced highly organized blood islands containing developing erythrocyte cells, organized endothelium, fibroblasts, thrombocytes and occasional granulocytes. Blood islands derived from EP 2, on the other hand, contained essentially only aggregates of erythroblasts embedded in endoderm. It is tentatively suggested that EP1 contains young multipotential hematopoietic precursors while EP 2 has only older blood-cell precursors committed to erythrocyte development. No cellular basis for the resolution of EP 1 into two complementary subfractions could be recognized.

Animals

Red-cell aggregation and red-cell deformability in diabetes.

The anomaly of the viscosity of human blood is more pronounced in diabetics. This is caused by an increase in plasma viscosity, a more pronounced red-cell aggregation, and a reduction of individual cell deformability. The changes in viscosity and in red-cell aggregation both are the consequence of abnormal plasma proteins, the incidence of which is largely independent of the onset and duration of disease, and actual metabolic state. The presence of complicating infectious diseases further aggravates the pathologic red-cell aggregation. The decreased red-cell deformability is largely independent on onset, duration, and complications but depends critically in the incident metabolic control of the diabetics. The possible role of hemorrheologic factors in the development of microangiopathy is discussed.

Blood Proteins

Inhibition of embryonic cell aggregation by neoplastic cells.

The effects of normal and malignant cells on the aggregation of embryonic cells in gyratory shaker cultures were compared. The addition of 1 times 10-5 simian virus 40 (SV40)-transformed BALB/3T3 (SV40-3T3) cells to 6 times 10-6 embryonic neural retina cells caused a highly significant greater reduct on (22.7 percent) in aggregate diameter than the addition of untransformed BALB/3T3 (3T3) cells. The ratio of the number of single cells to the number of aggregates was significantly higher for cultures containing SV40-3T3 cells than for the cultures containing 3T3 cells. This effect was concentration dependent in the presence of cultured Ehrlich-Lettre hyperdiploid (ELD) ascites cells; however, media from ELD cell cultures or ELD cell sonicates resulted in aggregates of greater diameter and lower ratios of single cells to aggregates. This approach may provide a sensitive assay system for the interactions of tumor and other cells in vitro.

Animals

Differential inhibition of embryonic cell aggregation by cultured human cells with "malignant" of "normal" characteristics.

The effect that cultured human cells have on chick embryonic neural retina cell aggregation was examined. Different types of human cultured cells inhibited aggregation of chick neural retinal cells to differetn degrees when mixed at a human cell:retina cell ration of 1:60. It appeared from the eleven cell lines studied that cells with "malignant" characteristics inhibited retinal cell aggregation to a greater extent than those with more "normal" characteristics. The assay could be used as a further test for abnormality of cell types and also as a method for studying the interactions of malignant cells with cultured cells.

Cell Aggregation

Biochemical and morphological comparison of two tumour-cell-aggregation factors from rat ascites hepatoma cells.

Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.

Animals

Production of plasminogen activator and inhibition of embryonic cell aggregation by cultured human normal and neoplastic cells.

The plasminogen activator (PA) production and the capacity to inhibit embryonic neural retina (NR) cell aggregation by human normal and neoplastic cell lines have been studied. The PA production was detected by both iodinated fibrin and casein lysis assays, and by changes in cell morphology at the presence of activated PA, using dog serum. Since the casein lysis assay and morphological changes proved to be less sensitive than 125I-fibrin lysis assay, a good correlation between these three assays could be observed provided that PA production measured by fibrinolysis exceeded 10--20%. The neoplastic cell lines exhibited the PA production to quite a large extent. The highest fibrinolytic activity (78%) was found in the case of bladder carcinoma cells T24, while the B-5GT cells from giant cell tumor of bone failed to produce any detectable amount of the PA. The cells from synovial sarcoma and both glioma lines exhibited fibrinolytic activity of about 10% and four sarcoma cell lines over the range 20--50%. Out of 13 normal cell lines tested, 7 were negative or exhibited very low fibrinolysis not exceeding 3% of total radioactivity. Four cell lines derived from kidneys, lungs, intestines, and from mixed embryonic tissues showed a marked fibrinolytic activity of about 10--37%, a slightly elevated fibrinolysis being found in embryonic lung cells LEP and cells from fetal skin tissue only at the presence of dog serum. The fibrinolysis detected in the neoplastic cloned cell populations showed considerable differences in the PA production between individual cell clones isolated from the same parental cell line. Unlike the normal fibroblastic cells B-41FB derived from bone, all neoplastic cell lines tested possess the capability to inhibit embryonic NR cell aggregation significantly. The results suggest the effect not to be dependent upon the PA production.

Animals

Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates.

Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...

Animals

[Induced cell aggregation].

Data from literature concerning cell aggregation induced by various substances are considered. The biochemical nature of cell aggregation inducers, participation of membrane receptors in this process, biochemical aspects and practical significance of the cell aggregation process are discussed.

Animals

Nevus cell aggregates associated with lymph nodes: estimated frequency and clinical significance.

Aggregates of cells resembling those of cutaneous nevi have been found in the capsules of lymph nodes. Although seemingly rare, this extraordinary lesion could conceivably occur often enough to be more than a pathological curiosity, and should be differentiated from metastatic tumor. Slides from every axillary lymph node dissection for female mammary carcinoma in the years 1964 and 1974 at Memorial Hospital were reviewed, as were slides from 100 consecutive lymph node dissections performed during 1974 in patients with malignant melanoma. Nevus cell aggregates (NCA) were associated with three of 17,504 lymph nodes (0.017%) obtained from 909 mastectomies, or 0.33% of the cases. Among the 100 lymph node dissections for malignant melanoma, NCA were found associated with three of the 2,607 lymph nodes examined (0.12%), or 3.0% of the cases studied. Since NCA occur in association with lymph nodes more often than previously thought, the possibility that they may be a potential source of malignant melanoma in patients without a demonstrable cutaneous or mucosal primary is discussed.

Axilla

Ultraviolet-induced alterations of beat rate and electrical properties of embryonic chick heart cell aggregates.

Embryonic heart cell aggregates were irradiated with ultraviolet light at wavelengths between 260 and 310 nm. Spontaneous beat rate was monitored with the aid of a closed-circuit TV camera and, in separate experiments, electrophysiological changes were assayed by intracellular recording. The characteristic response of 7-day aggregates was an increase in spontaneous beat rate to a maximum plateau level, followed by a rather abrupt cessation of beating. Intracellular recordings during irradiation showed a marked decline in the maximum rate of rise, overshoot, and repolarization phase of the action potential, and a significant change in threshold toward zero. The action spectrum for the termination of beating peaked between 290 and 295 nm; it fell off sharply at longer wavelengths and more slowly at shorter wavelengths. The maximum increase in beat rate was increasingly greater for shorter wavelengths and exhibited no peak in the wavelength range investigated. The sensitivity of aggregates to 295-nm light, as measured by the inverse of irradiation time required to terminate beating, decreased with increasing aggregate size and external potassium concentration, was relatively independent of temperature, and increased with embryonic age. The ultraviolet-induced increase in beat rate and termination of beating are attributed to separate complementary processes, a depolarization of the membrane, and a decline in "fast" sodium conductance.

Action Potentials

Characterization of tumour cell aggregation promoting factor from rat ascites hepatoma cells: Separation of two factors with different antigenic property.

The previously described glycoprotein that promotes tumour cell aggregation, derived from rat ascites hepatoma cells and capable of partial purification by chromatography, was found to be a mixture of 2 factors with different antigenic property. One was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. The action of the unabsorbed factor was clearly more potent than that of the absorbed factor. Both the factors were found in the serum of tumour bearing rats and the action of the unabsorbed factor was also more potent than that of the absorbed factor; its amount increased with time after i.p. inoculation of the cells. The serum of healthy rats contained the absorbed factor but not the unabsorbed factor. It was thus assumed that the unabsorbed factor was associated with the hepatoma cell surface itself and released into the serum, while the absorbed factor was associated with serum protein coating the cell.

Animals