Untreated Dishes Research Articles

Juxtacrine Activation of Epidermal Growth Factor (EGF) Receptor by Membrane-anchored Heparin-binding EGF-like Growth Factor Protects Epithelial Cells from Anoikis While Maintaining an Epithelial Phenotype

Loss of cell-matrix adhesion is often associated with acute epithelial injury, suggesting that "anoikis" may be an important contributor to cell death. Resistance against anoikis is a key characteristic of transformed cells. When nontransformed epithelia are injured, activation of the epidermal growth factor (EGF) receptor (EGFR) by paracrine/autocrine release of soluble ligands can induce a prosurvival program, but there is generally evidence for concomitant dedifferentiation. The EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), is synthesized as a membrane-anchored precursor that can activate the EGFR via juxtacrine signaling or can be released and act as a soluble growth factor. In Madin-Darby canine kidney cells, expression of membrane-anchored HB-EGF increases cell-cell and cell-matrix adhesion. Therefore, these studies were designed to test the effects of juxtacrine HB-EGF signaling upon cell survival and epithelial integrity when cells are denied proper cell-matrix interactions. Cells expressing a noncleavable mutated form of membrane-anchored HB-EGF demonstrated increased survival from anoikis, formed larger cell aggregates, and maintained epithelial characteristics even following prolonged detachment from the substratum. Physical association between membrane-anchored HB-EGF and EGFR was observed. Signaling studies indicated synergistic effects of EGFR activation and phosphatidylinositol 3-kinase signaling to regulate apoptotic and survival pathways. In contrast, although administration of exogenous EGF partially suppressed anoikis in wild type cells, it also led to an increased expression of mesenchymal markers, suggesting dedifferentiation. Taken together, we propose a novel role for membrane-anchored HB-EGF in the cytoprotection of epithelial cells.

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, approximately 63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.

CAMP Has Distinct Acute and Chronic Effects on Aquaporin-5 in Lung Epithelial Cells

Aquaporin-5 (AQP5) is present on the apical membrane of epithelial cells in various secretory glands as well as on the apical membrane of the airway epithelium, airway submucosal glands, and type 1 pneumocytes, where it can participate in respiratory tract water homeostasis. We examined the effects of cAMP on AQP5 distribution and abundance. When AQP5-expressing mouse lung epithelial cells were treated with cAMP or the beta-adrenergic agonist terbutaline, a biphasic AQP5 response was observed. Short term (minutes) exposure to cAMP produced internalization of AQP5 off of the membrane and a decrease in protein abundance. Both of these responses were blocked by inhibition of protein kinase A and the decrease in abundance was blocked by chloroquine, indicating lysosome-mediated degradation. Sustained cAMP exposure (hours) produced an increase in membrane localization and increased abundance; these effects were also blocked by protein kinase A inhibition. The beta-adrenergic agonist terbutaline produced changes in AQP5 abundance in mouse trachea and lung, consistent with our findings in cultured epithelial cells. Purified AQP5 protein was phosphorylated by protein kinase A but not protein kinase C or casein kinase II, and aquaporin-5 was phosphorylated in cultured cells after long term (but not short term) exposure to cAMP. These studies indicate that cAMP and beta-adrenergic agonists produce distinct short and long term effects on AQP5 distribution and abundance that may contribute to regulation of lung water homeostasis.

Identification of extracellular matrix components and their integrin receptors in the human fetal adrenal gland.

The development of the human fetal adrenal gland is characterized by a gradient of mitotic activity, cell migration, and cell apoptosis, all of which dictate its particular function. Such plasticity may possibly be under the control of the extracellular environment. The goal of this study was to identify components of the extracellular matrix in second-trimester fetal adrenal glands. Whereas collagen IV was expressed evenly throughout the gland, both fibronectin and laminin demonstrated a mirror-imaged distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. The integrin subunit alpha1 was found mainly in the definitive zone and the alpha2-subunit mainly in the transitional zone, whereas integrin alpha3 (which binds both fibronectin and laminin) was detected only in the fetal zone. The beta2-subunit was observed solely in chromaffin cells. Such specific gradients of integrin and MEC component expression suggest that the extracellular environment does play a definite role during adrenal gland development. Indeed, compared with that in untreated plastic dishes, ACTH stimulation of dehydroepiandrosterone sulfate and cortisol was enhanced by collagen IV. In addition, fibronectin enhanced dehydroepiandrosterone sulfate but decreased cortisol secretion, compared with collagen IV substrates. These results provide fundamental insight into the contribution of the microenvironment in cellular processes leading to fetal adrenal gland development.

負イオン注入によるポリスチレン表面の神経細胞接着特性の改質とパターン化処理

Modification of polystyrene surface by negative ion implantation for improving and patterning attachment properties of neurons was investigated. This modification technique is required for artificial formation of neural network with neuron cells and for development of “bio-interface” between vital neural system and external silicon circuit. Silver-negative ions were implanted into various kinds of polystyrene surfaces through a mask with slit array of 60μm in width and 4 mm in length with 60μm spacing. In the cell culture of PC-12h (rat adrenal pheochromocytoma) cells with the ion-implanted polystyrenes, PC-12h cells attached and neurites extended only on the ion-implanted regions of untreated polystyrene dishes (NTPS) and spin-coated polystyrene (SCPS). This means that pattering ion implantation is able to make patterning treatment both of cell attachment and neurites extension properties.

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture.

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells.

Surface modification by silver-negative-ion implantation for controlling cell-adhesion properties of polystyrene

Silver-negative ions were implanted into polystyrene (Corning, untreated polystyrene dish). Negative ions were used for implantation to avoid charging problems on the insulator surface. Polystyrene dishes were implanted with Ag − at various ion energies of 5, 10, 20, and 30 keV, with a constant ion dose of 3.0×10 15 ions cm −2 at a current density of about 100 nA cm −2 in a residual gas pressure under 1×10 −3 Pa. Human umbilical vascular endothelial cells (HUVEC) were cultured on these polystyrene dishes in a culture medium based on Medium 199 with 5% CO 2 in air after sterilization. Although there was no observation of cell-adhesion on unimplanted polystyrene, HUVEC attached and grew on Ag-implanted polystyrene dishes by spreading pseudopodia from the cell body. Excellent results were obtained at 20 keV. Among these samples, the greater the ion energy for implantation, the greater the cell-adhesion property, except for at 30 keV. The implantation-energy dependence of the cell-adhesion property of polystyrene corresponded well to that of the contact angle.

Oviposition of resistant and susceptible strains of Drosophila melanogaster in the presence of deltamethrin

AbstractOviposition of three strains of Drosophila melanogaster in the presence of deltamethrin was observed. These strains had different levels of physiological susceptibility to deltamethrin. Two‐choice tests were conducted with couples of flies in petri‐dish arenas containing two oviposition dishes. On the first day of the experiment, females were given a choice between a treated oviposition dish and an untreated control dish. On the second day of the experiment, two control oviposition dishes were given to females. Although individual females showed a tendency to aggregate their eggs in one of the dishes, control experiments demonstrated an overall equal distribution of eggs between the dishes. When one of the two oviposition dishes in the arena was treated with deltamethrin, the percentage of females ovipositing and the mean number of eggs laid by females were reduced, compared with control arenas. Females avoided the treated oviposition dish and laid significantly more eggs on the control dish. Furthermore, when the deltamethrin concentration was increased on the first day, female flies postponed their oviposition and laid significantly more eggs on the second day. The resistant strain, SR, demonstrated the same capacity to select the untreated site for oviposition as the susceptible strain, but it showed a smaller oviposition reduction and egg retention. The relationship between physiological and behavioural susceptibility to deltamethrin is discussed.

A New Simple Method for Isolation of Microvascular Endothelial Cells Avoiding Both Chemical and Mechanical Injuries

Our study indicates that when small pieces of lung or muscles of chest wall are cultured, erythrocytes and leukocytes (PMNs) leave the tissues first, followed by vascular endothelial cells (ECs). Fibroblasts and other mixed cells grow after 72 hr culture. The ECs can then be isolated avoiding mechanical and chemical injuries. The lung tissue is obtained from the peripheral surface and muscles from the chest. It is then cut into pieces and cultured with DMEM containing 20% fetal bovine serum. After 60 hr culture, the tissues are discarded. The flask contains only ECs and blood cells. Blood cells can be cleared out after the cells are subcultured once or twice. The primary cells and the subcultured cells cultured on gelatinized culture dish give the capillary-like structure. Cells cultured on untreated dishes have regular cobblestone morphology and junctional contacts. The isolated cells were not mesothelial cells because the cells did not react to antibody against cytokeratin 18, while mesothelial cells reacted strongly to the antibody. The cells can be isolated from the lung tissue without pleura. The primary microvascular ECs are also cultured on microcarriers (cytodex 3). Because both mechanical and proteolytic injuries are avoided, the cells may be more similar to cells in the in vivo state. There are no significant differences in PMN-endothelium adherence and monolayer responses to second messengers, platelet activating factor, and phospholipase A2 when pulmonary and muscular microvascular endothelial cells are compared.

Larval secretions and food odors affect orientation in female Plodia interpunctella

AbstractSubstrates contaminated by wandering fifth instar larvae of Plodia interpunctella (Hübner)(Lepidoptera: Pyralidae) elicit oviposition by conspecific female moths, and larval rearing diet enhances oviposition and also induces upwind flight. Two‐choice oviposition assays determined that four‐day‐old gravid female P. interpunctella preferred to lay eggs on dishes containing cornmeal‐based rearing diet compared to empty dishes. Pieces of cheesecloth contaminated by fifth instar larvae elicited more oviposition than untreated cheesecloth or dishes with food. The combination of larval contamination and food was preferred over food only or larval contamination only in both two‐ and four‐choice experiments. The factor(s) in larval contamination responsible for eliciting oviposition in female moths was extracted in hexane, confirming that organic semiochemicals are responsible for the effect. The oviposition‐eliciting activity of larval contamination was retained on cheesecloth for up to 30 days following treatment with larvae, suggesting the active component(s) is stable and of low relative volatility. In two‐choice windtunnel bioassays female moths initiated flight only when rearing food was present in one of the treatments, and they displayed the highest landing responses to a combination of larval contamination and food. Earlier work on P. interpunctella and related pyralid species found that larval contamination due to secretions from the mandibular glands acted as both a spacing pheromone for wandering larvae and as a kairomone for host‐seeking parasitoid wasps. The present study suggests that the same or a similar secretion acts as an oviposition‐eliciting pheromone for conspecific females.

Adhesion, growth, and matrix production by osteoblasts on collagen substrata.

A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel. Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

The different effects of type I and IV collagens on the survival and proliferation of cultured BSC-1 cells.

The effects of type I and IV collagens on the survival and proliferation of cells were investigated to clarify a possible involvement of the substratum in the regulation of cell function. BSC-1 cells attached, spread and sustained their viability in the absence of calf serum on culture dishes coated with type IV collagen, but were unable to spread and survive on untreated culture dishes. The effects of adding type IV collagen in solution were similar to those of type IV coating. The fraction of the solution of type IV collagen with molecular mass of more than 100 kDa enhanced spreading and survival of cells, but the fraction of less than 100 kDa did not. Type I collagen did not support cell viability in the absence of calf serum. Moreover, coating of culture dishes with type I collagen, but not with type IV collagen, inhibited DNA synthesis and cell proliferation in the presence of calf serum. The cells grown on type I collagen were long, thin and spindle-shaped, and their stress fibers were not well developed, but the cells grown on type IV collagen, as well as those grown on untreated culture dishes, were polygonal in shape with well-developed stress fibers. These results indicate that the interactions of BSC-1 cells with the substratum, when it is derived from type I and IV collagens, differentially modulate the survival and proliferation of BSC-1 cells.

Response to Capture 2 EC, 1988

Abstract Edelson & Magaro (1988; Response of spider mites to Capture, 1987, Insecticide & Acaricide Tests, Vol. 13, p. 265) indicated that the LC50 for Tetranychus spp. collected from cotton in south Texas during 1987 was 15.4 ppm. Two source populations of carmine spider mite were compared in the 1988 test. One population was collected from a commercial corn field near Mercedes, Tex., in July and the other was collected from a cotton field in Mercedes in August. The corn field had not been treated previously with pesticides. The cotton field had been treated 6 times with cypermethrin (Ammo, 0.5 lb [AI]/acre) prior to collection of the mites. Disposable 50- by 9-mm Petri dishes were treated with a 15.4-ppm solution of Capture 2 EC according to methods discussed by Edelson & Magaro (1988). Twenty to thirty adult female mites were placed in each of 6 replicates treated with the LC50 dose and 20-30 adult females were placed in 6 untreated control dishes. The dishes were held at 25°C for 24 h and then the mites were examined to determine mortality. The mites were considered dead if they could not move more than one body length when prodded. The same techniques were used to test populations from each field.

Long-term effects of verapamil on aortic smooth muscle cells cultured in the presence of hypercholesterolemic serum.

Smooth muscle cells derived from rabbit and bovine aorta were cultured for up to 5 weeks in the presence of d less than 1.019 g/ml fraction of hypercholesterolemic rabbit serum. When this fraction was added to serum containing culture medium, there was a significant increase in DNA, protein, and cholesteryl ester per dish. Addition of 50 microM verapamil markedly reduced the stimulatory effect of the d less than 1.019 g/ml fraction on both DNA and protein content per dish. The effect of verapamil on cholesteryl ester content was more complex: there was an increase within the first week, but later the net accumulation of cholesteryl ester per dish was lower than in untreated dishes. The recovery of less DNA in verapamil-treated dishes was not due to increased cell loss, as evidenced by retention of a residualizing marker, 3H-cholesteryl linoleyl ether. Moreover, verapamil did reduce incorporation of 3H-thymidine into DNA. In verapamil-treated dishes, there was flattening and a cobblestone appearance of the cells. A hypothesis is proposed to explain the inhibitory effect of verapamil on the development of atheroma formation in cholesterol-fed rabbits: Assuming that macrophages play an active role in cholesteryl ester removal from atheroma, verapamil, which reduces lysosomal cholesteryl ester hydrolysis in macrophages, would permit the lipid-laden macrophage to remove more cholesteryl ester per cell from the arterial wall. In addition, the presently reported results support the possibility that verapamil may impede the development of atheroma formation by reduction of smooth muscle cell proliferation.

Initiation of Primary Cell Cultures from Human Intracranial Tumors on Extracellular Matrix from Bovine Corneal Endothelial Cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.

Comparative Toxicity of Atracurium and Metocurine in Isolated Rat Hepatocytes

Primary cultures of liver cells isolated from seven rats were used to study the possible toxicity of atracurium and metocurine. The muscle relaxants were separately added to the culture medium and the cells then incubated for 4 hr. The amount of lactic dehydrogenase (LDH) that leaked into the culture medium was determined at the end of incubation. The customary assumption was made that the exudation of LDH reflects the toxic effects of the relaxants. In untreated dishes, approximately 11% of the total intracellular LDH leaked out during the incubation. The net leakage of LDH produced by the relaxants was obtained by subtracting this amount from the LDH activity determined in the media of dishes with the relaxants added. On this basis, metocurine, in concentrations of 12-850 X 10(-6)M, did not cause a net leak of LDH. On the other hand, atracurium, in similar molar concentrations, caused a statistically significant and concentration-dependent leak of LDH that, at its maximum, amounted to more than one half of the intracellular LDH. The results are interpreted in terms of damage to cellular membranes produced by atracurium or its metabolites. Although the exact biochemical process was not identified, we hypothesize that acrylates--produced by Hofmann elimination from atracurium--might be the likely toxic species.

Saliva and salivary sulphated glycoprotein inhibit adhesion and locomotion of human gingival fibroblast‐like cells in vitro

The purpose of this study was to determine the influence of clarified human whole saliva on the attachment and locomotion of human gingival fibroblast‐like cells to plastic substrata in vitro. Saliva was subjected to gel filtration on Sepharose CLAB to isolate a high molecular weight sulphated glycoprotein fraction (SGP, Hogg & Embery 1979, 1982) from the residual fractions (RSF) which were pooled. 25 cm2 plastic culture dishes were coated with either sterile whole saliva, SGP (0.3 mg/ml) or RSF (0.6 mg/ml) in Ringer‐Locke saline. They were then seeded with 2.50 × 103 fibroblasts suspended in Ringer‐Locke saline and numbers of adhering cells determined at times up to 60 min.Cell adherence in both saliva and glycoprotein‐treated dishes was significantly less than that in untreated control dishes (p < 0.01, p < 0.05, respectively), but RSF treatment had no effect on attachment. Incubating the cells with saliva or SGP prior to plating did not significantly suppress their subsequent attachment behavior when cell death caused by the incubation procedure was taken into account. Migration of fibroblasts from confluent monolayers on glass coverslips onto the surface of SGP‐ or saliva‐treated dishes was significantly delayed compared with controls, for periods up to 5 d (p < 0.01, p < 0.0 respectively). It was concluded that whole saliva inhibits the attachment or locomotion of gingival fibroblast‐like cells on plastic substrata in vitro, and that this property is primarily mediated by its glycoprotein component.

Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes

Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.

Melanoma cells growing in aggregates on a non-adhesive poly(HEMA) substrate exhibit polykaryocytosis but do not develop an increased metastatic capability.

B16-F1 melanoma cells were plated onto plastic tissue-culture dishes rendered non-adhesive for cells by coating with 0.12 per cent poly(2-hydroxyethyl methylacrylate), poly(HEMA). These growth conditions caused the normally flat, adherent B16-F1 cells to grow as single cells in suspension. Within 24 hours, the rounded cells formed aggregates and grew at a slower rate than control cells grown at the same density on untreated plastic dishes. Microscopic observations provided evidence that polykaryocytosis was occurring among the aggregates. Following replating onto standard adhesive tissue-culture plastic, 20-30 per cent of the aggregates were observed to contain varying numbers of multinucleated giant cells (polykaryocytes). The study has revealed a previously undescribed propensity of certain B16-F1 cells cultivated as aggregates in suspension to develop into polykaryocytes, most probably as a result of spontaneous tumor cell-tumor cell fusion. The possible relevance of this behavior in vitro to events in tumor progression is discussed. This study, however, does not support the findings of others that the metastatic capability of B16-F1 cells is increased by such non-adherent culture conditions. No increase in metastatic potential was observed for B16-F1 cells, or for a low metastatic clone (F1-7) derived from it, grown for 72 or 96 hours in a spherical configuration compared to control cells grown in a flat, adherent monolayer.

Role of serum in maintenance of functional hepatocytes in primary culture.

Serum at 5 to 10% is required for maintenance of functional adult rat hepatocytes in primary culture. One effect of the serum is to induce attachment and spreading of hepatocytes on plates as monolayers. Another role is to maintain cell viability for over 2 days. For the first effect, serum could be replaced completely by fibronectin (Fn). The effects of Fn on attachment and spreading of cells were dose-dependent and maximum at 10 micrograms/ml. Cells in serum-free medium on Fn-coated dishes showed similar activities of glycogenolysis and glycogenesis to cells cultured in medium containing 5% calf serum on untreated dishes in response to glucagon, dibutyryl cyclic AMP (bt2c AMP), isoproterenol and insulin. The increase in alkaline phosphatase [EC 3.1.3.1] activity and induction of tyrosine aminotransferase [EC 2.6.1.5] by dexamethasone (Dex) in cells under the two conditions were also similar. However, the inductions of tryptophan oxygenase [EC 1.13.11.11] by Dex, glucagon, and bt2cAMP were 4-7 times higher in cells cultured in serum-free medium. The inductions by Dex plus glucagon in the two types of cultures were inhibited similarly by insulin. In serum-free medium containing Dex and insulin in Fn-coated dishes, the cells survived as monolayers for about 50 h without detachment from the dishes, but for longer survival it was necessary to add 5% serum to the medium. A fraction with a molecular weight of over 50,000 from serum was separated by ultrafiltration and this fraction showed a similar effect to serum in increasing survival. A similar factor, but with about 70 times higher specific activity, was found in an extract of bovine pituitary gland.