Anchorage-dependent chick embryo fibroblasts synthesise DNA and proliferate when cultured in multilayered sheets suspended among glass fibres.

Chick embryo fibroblasts (CEFs) spontaneously form multicellular and multilayered sheets suspended on the network of glass fibres which are stabilized by fibronectin containing protein deposits located at cell-to-cell contacts. The cells situated within the sheets are surrounded by the neighbouring cells and their mechanical equilibrium is stabilised by intercellular "parabaric" effects. It was found that CEFs in the sheets retain relatively high mitotic activity corresponding to that observed in sparse monolayer cultures. These cells grew up to much higher local density than in confluent and contact-inhibited monolayer cultures and developed an abundance of microfilament bundles that terminated at vinculin-containing protein complexes. The results presented demonstrate that direct contact with solid substratum, cell-to-cell contacts, local cell density, and intercellular exchange of humoral factors are not directly involved in the density-dependent inhibition of growth observed in monolayer cultures. They also support the concepts concerning the role of mechanical equilibrium of cell membrane and sub-membranous cytoskeleton in the regulation of proliferation of non-transformed cells.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Acute radiation effects on the content and release of plasminogen activator activity in cultured aortic endothelial cells

Confluent monolayers from three lines of bovine aortic endothelial cells were exposed to a single dose of 10 Gy of 60Co gamma rays. Seventy-two hours later, the morphology of the irradiated and sham-irradiated monolayers was examined, and cellular DNA and protein contents were determined. In addition, the release of plasminogen activator (PA) activity into the culture media and PA activity in the cell lysates were assayed. Irradiated monolayers maintained their cobblestone appearance, but individual endothelial cells were enlarged considerably compared to sham-irradiated cells. DNA and protein contents in the irradiated monolayers were reduced to 43-50% and 72-95% of the control levels, respectively. These data indicate that radiation induced cell loss (detachment and/or lysis) from the monolayer, with hypertrophy of surviving (attached) cells to preserve the continuity of the monolayer surface. Total PA activity (lysate plus medium) in the irradiated dishes was reduced to 50-75% of the control level. However, when endothelial PA activity was expressed on the basis of DNA content, the irradiated monolayers from two of the three cell lines contained significantly more PA activity than did sham-irradiated monolayers. Most importantly, the percentage of the total PA activity released into the culture medium by irradiated cells (5-22%) was significantly (P less than 0.001) lower than that released by sham-irradiated cells (23-68%). These data suggest that fibrinolytic defects observed in irradiated tissues in situ may be attributable at least in part to a radiation-induced inhibition of PA release by vascular endothelial cells.     

Enzymically-modified human mammary carcinoma cells: modulators of macrophage functions.

Human mammary carcinoma cells (HMCC), vibrio cholera neuraminidase (VCN) treated HMCC(VCN-HMCC); glutaraldehyde fixed VCN-HMCC(Glut-VCN-HMCC), normal mammary epithelial cells (HMEC), VCN treated HNEC (VCN-HNEC) and glutaraldehyde fixed VCN-HNEC (Glut-VCN HNEC) were prepared and injected into separate groups of mice. Intraperitoneal injection of phytohemagglutinin (PHA) produced a delayed type of inflammatory reaction characterized by an accumulation of macrophages. Injection of HNEC, VCN-HNEC, and Glut-VCN-HNEC had no effect, HMCC inhibited, and VCN-HMCC and Glut-VCN-HMCC increased both the number and the tumoricidal activity of peritoneal macrophages. Cell-free media (CFM) harvested from cultures of HMCC alone or mixed with lymphocytes from patients with mammary carcinoma inhibited, whereas CFM from mixed cultures of HMCC and lymphocytes from normal subjects had no effect on, the chemotactic response and migration of normal peritoneal macrophages. CFM was passes through a series of Amicon membranes, the active principle passed through membrane PM-10 but was retained by PM-5 suggesting a molecular weight of less than 10,000. In culture, HNEC, VCN-HNEC and VCN-HMCC proliferated as sheets of single cell monolayers, whereas HMCC began to spread as a monolayer, but as cellular density increased it formed cell foci, cell colonies and cell clusters packed into solid tissuelike masses.     

Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

Cell-cell contact promotes specific activity of pp60c-src protein kinase in human retinoblastoma cells.

Tyrosine-specific protein kinase activity of pp60c-src was examined in human Y79 retinoblastoma cells cultured in monolayers after clusters in suspension culture had been dissociated. The activity increased five- to six-fold between Days 1 and 7 in the monolayer cultures, with a concomitant increase in numbers of cellular contacts per cell. There was no effect of conditioned medium from high-density cultures in suspension on the activity of cultures with a low degree of contacts. The level of c-src protein in cell lysates was nearly constant irrespective of the degree of cellular contacts. These results suggest that the specific activity of pp60c-src is regulated by cell-cell contact.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

Depletion of NK by cellular immunoadsorption.

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.     

Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing.     

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell–cell and cell–matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or “mesenspheres”, of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Contact inhibition of division: involvement of the electrical transmembrane potential

Measurements of simultaneous mitotic activity, electrical transmembrane potential (E_m), and cell density levels in both 3T3 and Chinese hamster ovary (CHO) cell cultures reveal that a 5- to 6-fold increase in the E_m level is associated with development of mitotic arrest at saturation densities. This rise occurs both in confluent monolayers and in interior areas of isolated colonies, and is independent of the rate at which confluence is attained. The E_m rise is accompanied by a substantial decrease in intracellular Na. Electron microscopy of saturated CHO monolayer sections shows from 46 to 63% of the cell surfaces to be in close apposition (<300 Å spacing). These results for contact inhibited cultures support the hypothesis that mitotic activity may be functionally coupled with the E_m level and associated ionic concentration levels. It is suggested that contact inhibition of mitosis may result from a reduction in synthesis of mitogenically essential RNA following a decrease in intracellular Na produced by contact-induced alteration of surface ion-transport activity.     

12-O-tetradecanoylphorbol-13-acetate induces an immediate, but transient increase in mitotic activity uncoupled from DNA synthesis in the monolayer cultures of rat keratinocyte

A single wave of mitotic activity was observed in a monolayer culture of rat keratinocytes immediately after exposure to 12-O-tetradecanoylphorbol-13-acetate. A peak for cells in prophase, observed at 10 min after the exposure, was followed by a peak for metaphase at 20 min, for anaphase at 25 min and telophase at 30 min after the exposure. Thereafter, the mitotic activity began to subside. This transient stimulation of mitotic activity resulted in an increase of population density in the monolayer culture. There was neither a stimulation of DNA synthesis during this period nor a change of the DNA content after the mitotic activity was completed. This single burst of synchronous mitotic activity which did not require a substantial stimulation of DNA synthesis suggests that the effect was on the initiation process of mitosis among a subpopulation of cells, presumably cells delayed in the G2 phase of the cell cycle.     

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.     

A mild procedure for the rapid release of cytoplasmic enzymes from cultured animal cells.

Rapid release of cytosolic enzymes from animal cells in monolayer culture is achieved by treatment of dispersed cells with digitonin. This mild procedure causes efficient cell lysis (>95%) with minimal organelle disruption. A second procedure suitable for isolation of cytosolic enzymes by direct digitonin treatment of cell monolayers has also been developed.     

Shedding of mitotic cells from the surface of multicell spheroids during growth

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exonential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytoflurometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. I. Kinetics

An in vitro culture method giving a high cell recovery rate of 25–38% has been developed to generate a relatively pure population of T cell progeny reacting to xenogeneic cell antigen. The method is basically a modification of the Ginsburg method and utilizes mosaic monolayers that are comprised of syngeneic Lewis and xenogeneic BALB/c fibroblasts. Thoracic duct lymphocytes of Lewis rats were cultured for 6 days on these monolayers and the resulting lymphocyte population, rich in blast cells, was collected and separated free of contaminating fibroblasts by an additional 6 hr incubation without monolayers. Cytotoxic activity of these harvested lymphocytes was assessed by a modification of the Hellström method in which embryonic target fibroblasts, Lewis or BALB/c, were plated at a critical density of 2200 cells/cm² of culture area. This, coupled with the use of a newly developed culture medium, allowed the demonstration of specific as well as nonspecific activity of up to 100% suppression at 48 hr of test incubation. The specific activity was clarified and determined critically by comparing the activity of these lymphocytes to that of lymphocytes grown on syngeneic monolayers, though rich in blast cells, reacting only to the horse serum in the medium. The nonspecific activity was not due to the deterioration of test cultures and needed the continued presence of fully active blast cells for complete suppression of the target cell growth in the later phase of test cultures. By changing the ratio of lymphocytes to target cells and following the kinetics of target cell destruction, it was shown that specific activity effects an active killing of target cells, whereas the nonspecific activity was primarily the suppression of target cell growth. The specific activity was effective with far less lymphocytes per target cell; was nearly complete by 12 hr of test cultures while the nonspecific activity was complete at 48 hr. Yet, the increase in the specific activity was nearly proportional to that of nonspecific activity and even the nonspecific activity seemed to kill sensitive Lewis target cells at its full intensity. These observations are in accordance with an interpretation of the mechanism of the specific target cell destruction as involving contact and recognition followed by the release of lymphotoxin into or onto target cells.