Monolayer Tissue Culture Research Articles

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

Plasmin induces the formation of multicellular spheroids of breast cancer cells.

MCF7 and ZR75-1 breast cancer cells grow as adherent monolayers in tissue culture. Treatment with the serum serine protease plasmin causes them to detach and to grow as floating multicellular spheroids. Two plasmin activators, urokinase plasminogen activator and streptokinase, induce the same growth pattern changes in the presence of plasminogen. Serum contains also plasminogen activator inhibitors. Aged serum, deficient in plasminogen activator inhibitors, converts spontaneously monolayer breast cancer cells into multicellular spheroids which readily revert to monolayer growth after addition of fresh serum. Urokinase blocks the reversion. The formation of multicellular spheroids does not affect the proliferative rate of breast tumor cells but endows tumor cells with increased resistance to the chemotherapeutic drugs, doxorubicin and paclitaxel.

INVASION OF BRAIN MICROVASCULAR ENDOTHELIAL CELLS BY GROUP B STREPTOCOCCI• 1332

Group B streptococci (GBS) are the leading cause of meningitis in newborns. Although meningitis develops following bacteremia, the precise mechanism or mechanisms whereby GBS leave the bloodstream and gain access to the central nervous system (CNS) are not known. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells (BMEC), the single-cell layer which constitutes the blood-brain barrier. In order to test this hypothesis, we developed an in vitro model with BMEC isolated from a human, immortalized by simian virus 40 transformation, and propagated in tissue culture monolayers. GBS invasion of BMEC monolayers was demonstrated by electron microscopy. Intracellular GBS were found within membrane-bound vacuoles, suggesting the organism induced its own endocytic uptake. GBS invasion of BMEC was quantified with a gentamicin protection assay. Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently than strains from other common GBS serotypes. GBS survived within BMEC for up to 20 h without significant intracellular replication. GBS invasion of BMEC required active bacterial DNA, RNA, and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. The polysaccharide capsule of GBS attenuated the invasive ability of the organism. At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury; this cytotoxicity was correlated to beta-hemolysin production by the bacterium. Finally, GBS demonstrated transcytosis across intact, polar BMEC monolayers grown on Transwell membranes. GBS invasion of BMEC may be a primary step in the pathogenesis of meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial blood-brain barrier.

Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal VirusReproduced from Proc. Natl Acad. Sci 38, 747-752 (1952) with kind permission of Proceedings of the National Academy of Sciences, USA.

Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal VirusReproduced from Proc. Natl Acad. Sci 38, 747-752 (1952) with kind permission of Proceedings of the National Academy of Sciences, USA.

Progesterone receptors in tumor fragment spheroids of human meningiomas.

Progesterone receptors (PgR) are detectable in about 60-70% of tissue specimens of human meningiomas. Despite these data, PgR are hardly to be found in monolayer tissue culture of meningiomas. Aim of this study was to elucidate whether PgR might be preserved in tumor fragment spheroids of meningiomas maintained in organ culture since the morphological appearance of the original tumor is preserved by this culture technique. Aliquots of meningioma specimens of 25 patients (17 females) were snap frozen in liquid nitrogen immediately after removal. Additionally, monolayer tissue cultures of the same specimen were obtained as primary culture and passage #3. Tumor fragment spheroids were kept on medium-agar with liquid medium overlay and harvested after 1 and 3 weeks in culture. PgR were detected by immunohistochemistry using a rat monoclonal antibody. 18/25 meningioma tissue specimens were positive for PgR. In 8 out of 15 PgR-positive tumors which formed spheroids we could detect PgR in fragment spheroids after 1 and 3 weeks in culture. In contrast, none of the monolayers depicted PgR. PgR is preserved in a considerable amount of tumor fragment spheroids of PgR-positive meningiomas. They remain detectable after 3 weeks of culture whereas monolayer tissue cultures are PgR-negative. Thus, tumor fragment spheroids seem to be a suitable tool to investigate progesterone/antiprogesterone effects in vitro.

Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF.

Seeding of intracoronary stents with immortalized human microvascular endothelial cells

Intracoronary stents are effective in decreasing the complications associated with acute closure during coronary angioplasty. A major complication associated with the use of coronary stents is acute thrombotic occlusion. It has been postulated that the stent loses its thrombogenic potential after it becomes covered with a layer of endothelial cells. Human dermal microvascular endothelial cells were transfected with a plasmid containing the simian virus 40 large T-antigen gene. Stents were placed in culture media with cells for 2 weeks. Seeding efficiency of the stent with the endothelial cells was assessed by scanning electron microscopy. Balloon-expandable coronary stents placed in cell culture with immortalized human microvascular endothelial cells showed near-complete coverage after 2 weeks. After balloon inflation, persistence of cells on the stent was noted only on the lateral aspect of the balloon-expanded stents. If these stents were placed in culture, complete recovery of the monolayer was noted after 3 days. Stents were then covered with endothelial cells and frozen for 4 days. After thawing, the cells adhered to the devices and divided to form a monolayer in tissue culture. Seeded balloon-expandable stents were frozen for 4 months, thawed, and then implanted in a pig coronary artery. Human endothelial cells were identified on the stent 4 hours after deployment. These studies demonstrate the feasibility of using a human microvascular endothelial cell line to seed an uncoated metal stent. The cells remain adherent to the stent, are functional after freezing, and remain on the stent at least 3 hours after intracoronary implantation.

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Automating the quantitative analysis of 2-D neural dendritic trees

Neurons in the central and peripheral nervous system vary widely in their dendritic branching patterns. Quantification of the morphological characteristics used to identify different classes of neurons and relate neural structure to function requires that accurate metric and non-metric data be obtained from neural images obtained by camera-lucida drawing or from digitized video images made with transmitted, fluorescence or confocal microscopy. This paper describes a largely automated procedure for determining the dendritic tree structure of largely planar cells (such as retinal ganglion cells or cells in tissue culture monolayers) from an initial pictorial representation or digitized image. From this structure, non-metric data (such as the ordered ‘tree’ of branches) and metric information (such as total dendritic length and dendritic field area) can be automatically computed. The use of this method is specifically illustrated in the capture of the dendritic tree structure of retinal ganglion cells from the rabbit retina.

Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins

Syncytia formation in either CV-1 or HeLa T4 + cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers.

A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems.

Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.

Growth advantage ("clonal dominance") of metastatically competent tumor cell variants expressed under selective two- or three-dimensional tissue culture conditions.

In previous experiments it was shown that injection into syngeneic CBA/J mice of cell mixtures containing an excess of non-metastatic SP1 mouse mammary carcinoma cells with a ras transfected metastatic variant of SP1 called C1, always resulted in the eventual dominance of the C1 subpopulation at the site of inoculation. This occurred despite the growth rates of the two cell populations being identical in vivo when grown separately. The means by which the C1 subpopulation achieved "clonal dominance" is thought to involve its responsiveness to stimulatory paracrine growth factors liberated by the non-metastatic SP1 population. The clonal dominance process, however, could not be recapitulated in conventional monolayer tissue culture conditions in which SP1 and C1 cells were grown together in high concentrations of serum, i.e. under non-limiting culture conditions. We now show that clonal dominance of C1 cells can be observed when the cell mixture is maintained in tissue culture for extended periods, or when the cells are grown under selective, limiting conditions, some of which may mimic growth conditions in vivo more accurately. These conditions were a) growth in low (limiting) serum concentrations; and b) growth as three-dimensional multicellular aggregates, i.e. as "tumor spheroids". Under all of these conditions dominance of the C1 subpopulation always took place, but with an efficiency 6- to 40-fold less than generally observed in vivo. C1 cells were also able to form more stable (compact) spheroids compared to SP1 cells. Entrapment of the latter in mixed C1/SP1 spheroids increased the recovery of the SP1 cells suggesting some kind of "rescue" mechanism in which cells are protected from physical forces by three-dimensional structure. The relevance of these in vitro interactions for clonal dominance in primary tumors and metastasis in vivo are discussed.

Calcium and potassium currents in porcine granulosa cells maintained in follicular or monolayer tissue culture.

We studied membrane currents in granulosa cells (GC), immediately after collection or after variable culture time in the everted-follicle wall or in the monolayer. GC in both systems express an inward calcium current (ICa) with T-type kinetics and voltage dependence. GC in the everted-follicle culture express an outward potassium current (IK) kinetics, which remains unchanged during three days in culture. IK has delayed-rectifier kinetics, but is insensitive to TEA, 4-AP and apamine. GC in monolayer culture develop a new, inactivating delayed-rectifier potassium current (InK), which progressively dominates as cells advance from day one to day three in culture. A similar InK was recorded in large luteal cells. A possible link between luteinization and the appearance of InK is hypothesized.

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti- Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

A filamentous-like mutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellular protein invades and grows in 3T6 and Caco-2 cells.

We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.

Characterization of 20 entamoeba histolytica strains isolated from patients with HIV infection

Twenty Entamoeba histolytica strains from patients with HIV-1 infection were isolated and compared with E. histolytica strains from patients without HIV infection. The isoenzyme pattern of the hexokinase as well as the hybridization with known DNA-probes were used as markers for pathogenicity. According to these markers, all 20 strains could be regarded as being nonpathogenic. Direct measurements of the virulence were carried out: destruction of monolayer tissue culture cells, capacity of phagocytosis and the ability to induce liver abscesses in the hamster. The virulence of strains from HIV patients was comparable to that of E. histolytica strains which had been isolated from HIV-negative asymptomatic carriers. In agreement with this, none of the HIV-positive patients showed symptoms of an invasive amebiasis. By PRC, no HIV-1 proviral DNA could be evidenced in the E. histolytica strains which had been isolated from HIV patients.

Mammalian neurons in dissociated cultures form clusters in the presence of retinal pigment epithelium

The objective of this study was to investigate the cellular processes involved in the formation of the cytoarchitectonics of the retina. Neurons derived from the retina, spinal cord, cerebral cortex and hippocampus were grown in dissociated monolayer tissue culture using standard techniques. The cultures of retina were unique in that the neurons actively formed into cell clusters. On the other hand, cultures of neurons from the other regions of the CNS grew without forming any obvious histotypical pattern. Cell clusters consisted of an apparent monolayer of neurons above a population of flat cells and clusters were observed in retinal cultures derived from all species studied (mouse, cat and guinea pig). Each cluster was surrounded by whorls of fibroblasts; astrocytes (GFAP-positive cells) were often closely associated with clusters. Formation of clusters appeared to depend strongly upon the presence of cells derived from the retinal pigment epithelium (RPE) because the ability of retinal cells to form clusters was markedly impaired when the RPE was omitted from the cultures. Interestingly, monolayer cultures of neurons from other regions of the CNS could be induced to form clusters, but only when cells of the RPE layer were included at the time of plating. In cultures grown without the RPE layer, clusters did not form when media taken from cultures expressing clusters was used, indicating that the formation of clusters was not caused by a media-bourne factor. On the other hand, clusters did form when neurons without RPE were grown on feeder plates in which clusters had previously been expressed and the neurons subsequently killed by prolonged culturing or by treatment with kainic acid. Hence, physical contact between neurons and cells derived from the RPE appears critical for the formation of clusters. Our results suggest that the cellular processes underlying the formation of clusters may reflect those in the development of the retina in vivo. Thus, cluster formation may be a useful model for investigating the initial stages in the development of retinal cytoarchitecture.

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10 −6 M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Expression of somatostatin and GABA immunoreactivity in cultures of rat hippocampus

Somatostatin (SOM) synthesis and release were studied with radioimmunoassay and immunocytochemical techniques in rat fetal hippocampal neurons maintained in monolayer tissue culture. SOM immunoreactivity increased from undetectable to over 4,000 pg/ml in media and over 2,500 pg/culture in neurons by 3 to 5 weeks. After 3 weeks, approximately 11% of the neurons stained for SOM. Gamma-aminobutyric (GABA) immunoreactivity was present in hippocampal neurons from 1 day to 5 weeks with 40–50% of the neurons staining for GABA by 5 weeks in vitro. Costaining neurons for SOM and GABA revealed that 63% which were positive for SOM also stained for GABA.

Medulloblastoma cell line secretes platelet-derived growth factor

Medulloblastoma is the most common malignant childhood brain tumor in which aggressive growth produces recurrence in approximately 50% of appropriately treated cases and metastases along the neuraxis in 30%. To date, no studies exist concerning the production of autocrine growth factors by this brain tumor type. Malignant brain tumors in adults often produce platelet-derived growth factor (PDGF). A medulloblastoma cell line, TE-671, has been used for many years in pediatric neuro-oncologic studies. We assayed this medulloblastoma cell line for the production of PDGF. PDGF is produced by medulloblastoma cells grown in monolayer tissue culture and stimulates PDGF-sensitive 3T3 fibroblasts to incorporate tritiated thymidine in a dose-dependent fashion. This biologic activity is blocked by PDGF antibodies in a dose-dependent relationship. We postulate that PDGF produced by medulloblastoma cells plays a role in the growth of this tumor by stimulating mitogenic activity.