Biomedical subjects

R Bjerkvig

Publications and source records attributed to R Bjerkvig.

At least 91 records · Page 5Linked to original sources

PubMed1986

Reaggregation of fetal rat brain cells in a stationary culture system. I: Methodology and cell identification.

A stationary tissue culture system for reaggregation cultures of rat brain cells is described. Aggregates were formed by placing cells at high concentration in liquid overlay cultures on a nonadherent nutrient agar surface. No physical stress in the form of rotation or shaking was applied to the aggregating cell population. Transmission electron microscopy and immunohistochemistry showed that the cells developed from homogeneously dispersed, immature cells in Day 4 aggregates, to mature astrocytes, oligodendrocytes, and neurons in Day 20 aggregates. Twenty days older aggregates had a tightly packed neuropil which was most prominent in a cell-sparse outer layer of the aggregates. When the aggregates were allowed to adhere to a substrate, both glial fibrillary acidic protein (GFAP) positive and negative cells were observed migrating out from the aggregates. Cells giving a positive reaction for neuron specific enolase (NSE) were also present. This reaggregation procedure, with transfer of selected brain cell aggregates into agar-coated multiwells is an alternative three-dimensional culture system which can be potentially useful in the study of morphogenesis and cell interactions in the nervous system.

Animals↗

PubMed1986

Reaggregation of fetal rat brain cells in a stationary culture system. II: Ultrastructural characterization.

Ultrastructural characteristics of fetal rat brain cell aggregates in a three-dimensional stationary culture system are described. Transmission electron microscopy showed immature cells which developed into mature astrocytes, oligodendrocytes, and neurons during 20 d in culture. This was accompanied by the development of a neuropil where myelinated axons and synaptic complexes were observed. In addition to confirming earlier ultrastructural investigations on fetal rat brain cell aggregates, the stationary culture system also showed the presence of histiotypic regions within the aggregates. These regions consisted of ependymal cells where cilia were observed on the cell surfaces. Structures resembling subependymal basement membrane labyrinths were also observed. Macrophages seemed to be more numerous in the stationary cultures as compared to other culture systems. The stationary culture system may provide aggregates that are ultrastructurally more complex than those obtained by rotation mediated systems.

Animals↗

PubMed1986

Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo.

Two malignant rat neurogenic cell lines have been confronted with reaggregation cultures of fetal rat brain in a stationary culture system. In this organ culture system several morphological aspects of the developing brain are imitated. The malignant cell lines showed invasive and progressive growth into the brain tissue, ending with a completely destroyed aggregate within 10 days of coculture. One cell line (BT5C) showed solid invasion with groups of cells replacing different layers of the brain tissue. A considerable degree of normal cell lysis was seen, both at the edge of invasion and at distant parts of the aggregate. This cell line seemed to activate macrophages in the aggregates. In addition, conditioned medium from these cultures caused degeneration of the brain aggregates. The other cell line (BT4Cn) showed invasion by single cells where the replacement and destruction of the tissue was not accompanied by distant lysis. Both cell lines showed the same pattern of invasion in vitro as was seen in the brains of isogeneic animals in vivo, showing that the individual tumor characteristics were also reflected during invasive growth in organ culture.

Animals↗

PubMed1985

Cell and tissue culture of the central nervous system: recent developments and current applications.

A survey of methods for cell and tissue culture of the central nervous system (CNS) is given. This includes a brief historical outline and description of methods in current use. Recent methodological improvements are emphasized, and it is shown how these are applied in modern neurobiological research. Both monolayer cell cultures and three-dimensional organ culture systems are widely used, each having advantages and limitations. In recent years, there has been considerable improvement of culture for prolonged periods in chemically defined media. Brain tissue from a wide spectrum of species have been used, including different types of human brain cells which can be propagated for several months. At present, these culture systems are employed for dynamic studies of the developing, the adult and ageing brain. It is possible to select neurons and the different classes of glial cells for culture purposes. Cell culture of the CNS has given new insights into the biology of brain tumours. Culture systems for experimental tumour therapy in vitro are also available. Recently, it has been shown that organ cultures of brain tissue can be used as targets for invasive glioma cells, enabling a direct study of the interactions between tumour cells and normal tissue to take place.

Aging↗

PubMed1985

DNA content and chromosomal composition of malignant human gliomas.

A short review is given on DNA aberrations and chromosomal composition of malignant human gliomas. By flow cytometric DNA analysis, a wide range of different ploidies has been reported in biopsied gliomas, from diploid to strongly aneuploid nuclear DNA. However, with the preparation and analysis methods used so far, no clear relationship between the type of ploidy and histology or prognosis has been established. A high proportion of glioblastomas is near-diploid, indicating a high degree of biologic malignancy is not necessarily connected to aberration of the nuclear DNA content. It is possible that improved methods giving a higher degree of resolution will allow separation of the near-diploid populations of malignant human gliomas from normal diploid cells and permit the detection of subpopulations with small differences from the dominant DNA mode. Chromosomal studies of malignant gliomas have confirmed that the majority of them have near-diploid stemlines. These populations are seldom normal diploid, however, as both numerical and structural abnormalities are usually present. In addition, chromosomal analyses have shown that when gliomas are bimodal, the polyploid populations are usually doubled versions of the near-diploid ones. In contrast to the near-diploid populations that characterize biopsied malignant gliomas, both FCM studies and karyotyping have demonstrated that permanent cultured cell lines derived from malignant gliomas are usually near-triploid or near-tetraploid. Sequential karyotypic studies of these tumors from biopsy through establishment in vitro have shown an evolutionary pattern consisting of doubling of the original stemline, followed by gains or losses of individual chromosomes with new marker formation in late culture. Evaluation of biopsied malignant gliomas by karyotyping has also demonstrated that subgroups of them are characterized by specific numerical and structural deviations. These groupings may prove useful in predicting prognosis or responsiveness to specific therapeutic regimens. The specific chromosomal abnormalities observed in malignant human glioma may provide clues as to the genes important in glial transformation. As chromosomal loci for production of structural proteins and enzymes involved in glial metabolism are mapped and patterns of oncogene activation and amplification are determined for human gliomas, the meaning of the nonrandom chromosomal changes seen in these tumors may become clear.

Adult↗

PubMed1985

Interaction between rat glioma cells and normal rat brain tissue in organ culture.

A system for coculture between normal rat brain fragments and multicellular spheroids of rat glioma cells is described. The tumor cells were derived from fetal BD IX rats treated transplacentally with the carcinogen N-ethyl-N-nitrosourea (CAS: 759-73-9). Brain fragments were obtained from fetal BD IX rats and precultured for 20 days before confrontation with multicellular tumor spheroids. The cocultures were grown in nonadherent stationary organ culture for 30 days. Due to morphologic similarities between normal brain cells and tumor cells, the tumor cells were labeled with tritiated thymidine, which made them easily recognizable in autoradiographs. The two structures adhered to each other, and glioma cells progressively invaded and replaced the normal brain tissue. Invasion and replacement are characteristic features of brain tumors in vivo. Therefore, this organotypic and syngeneic model may be useful for investigation of these phenomena outside the body.

Animals↗

PubMed1984

Invasiveness of primary brain tumors.

Primary malignant neoplasms of the nervous system differ from other types of malignancy in several ways. Clinical progression is due to local invasive growth, while metastases outside the skull are rare. The tumors show no sharp delimitation from the surrounding normal tissue. At the edge, an ill-defined area of invasive tumor cells, reacting glial cells and inflammatory cells is present. At the same time the primary brain tumors are biologically heterogeneous. In this review, a short survey of markers for malignancy in primary brain tumors is given, and some properties of importance for invasive behavior, are listed. These include different cellular enzymes, phagocytotic property, locomotive and proliferative characteristics. Studies of primary brain tumors in situ show invasive growth into the surrounding brain tissue, often followed by hemorrhage and necrosis. In addition spread of tumor cells takes place along preexisting intracranial structures. Recently, several systems for the study of brain tumor invasiveness in culture have been elaborated. Both experimental and human gliomas have been tested. The target tissues include organ culture of embryonic chick heart muscle, chorioallantoic membrane, fetal rat brain tissue and reconstructed vessel walls. It has been shown that glioma cells are able to split junctions between normal cells. They destroy and phagocytose the normal cells and penetrate the normal tissue. The use of brain tissue and reaggregated brain cell cultures as target for glioma cells in culture opens the possibility for an elucidation of invasiveness as one of the most important properties of malignancy in the nervous system.

Animals↗

PubMed1983

The relative resistance of non-cycling cells in 9L multicellular spheroids to spirohydantoin mustard.

Surviving fractions of 9L spheroid cells treated with 1.5, 3.0 and 6.0 micrograms/ml of spirohydantoin mustard (SHM) decreased when assayed 6 hr after treatment but increased thereafter. Flow cytometric analysis showed that exponentially growing 9L monolayer cells treated with SHM accumulated at the G2/M border within 24 hr. Cells dissociated from spheroids treated with 3 and 6.0 micrograms/ml of SHM accumulated at the G2/M border during the first 24 hr after treatment and remained there for the next 12 hr. However, 50% of the cells remained at the 2C DNA peak. Spheroid cells with 2C DNA content 24 hr after treatment were assumed to be non-cycling cells at the time of treatment, and cells that accumulated at the G2/M peak appeared to be cycling cells at the time of treatment; approximately 50% of cells in untreated 9L spheroids are in the noncycling pool. G1 and/or early S phase 9L cells in exponential growth elutriated immediately after treatment with SHM had significantly lower (P greater than 0.001) plating efficiencies than 9L cells in S and G2/M phases. When spheroids were dissociated, elutriated and plated for colony-forming efficiency 24 hr after treatment with 3 micrograms/ml of SHM, fractions enriched in 2C DNA content had significantly higher (P greater than 0.001) plating efficiencies than elutriated cells enriched in 4C DNA. These results indicate that SHM is less effective against non-cycling 9L spheroid cells with 2C DNA content than against cycling 9L spheroids cells.

Animals↗

PubMed1983

Cell cycle age response of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea and modification by alpha-difluoromethylornithine.

We have determined the cell cycle age response of 9L rat brain tumor cells to 1,3-bis(2-chloroethyl)-1-nitrosourea using centrifugal elutriation to obtain populations of cells enriched in G1, S, and G2-M phases. While cells in all phases of the cell cycle were killed by 20 or 40 microM 1,3-bis(2-chloroethyl)-1-nitrosourea, cells in G1 and G2-M phases were more sensitive than cells in S phase. The differential sensitivity was more pronounced at the higher dose, which will markedly alter the distribution of cells through the cell cycle. In a clinical setting, this factor could affect the efficacy of either fractionated or multimodality protocols. Treatment with alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, potentiated the cytotoxic effects of 20 microM 1,3-bis(2-chloroethyl)-1-nitrosourea against G1- and G2-M- but not against S-phase cells; however, at a higher dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM), the cytotoxicity was potentiated for cells in all phases of the cell cycle. In alpha-difluoromethylornithine-treated cells, the phenomenon could be reversed by adding 1 mM putrescine 24 hr before treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea. Therefore, the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity appears to be related to polyamine depletion.

Animals↗

PubMedDate not supplied

Retroviral transfection of the lacZ gene from Liz-9 packaging cells to glioma spheroids.

Despite the development of numerous vectors for gene transfection to gliomas, patient survival length remains unaffected in clinical trials. For glioma gene therapy to be successful, the extent of gene transfer to the solid tumor tissue has to be high. In the present work we review some of the vector types and strategies so far utilized in experimental and clinical glioma gene therapy. Since gene transfer efficacy into solid glioma tissue is unknown for many vectors, we studied the gene transfer efficacy into multicellular spheroids derived from a human glioma cell line GaMg as well as into spheroids derived from human glioma biopsies (glioblastoma multiforme, GBM). A replication deficient retroviral vector from the Liz 9 packaging cell line was used for transfer of the bacterial beta-galactosidase lacZ gene into the target tissue. Gene transfer was obtained by adding medium containing virus from the producer cells to the target tissue. The experiments were also conducted with EGF (epidermal growth factor) added to the medium. The data show that the transfection rate ranged from 0-4.5% where the transfection efficacy was higher in spheroids after the addition of EGF. Most of the transfected cells were found at the surface, but transfected cells could also be observed in the center of the spheroids. We conclude that using this vector system, the transfection efficacy was low, even if the number of replicating cells was increased by adding EGF. The findings are consistent, and may partly explain, the lack of effect using this vector system during in vivo studies.

Brain Neoplasms↗

PubMedDate not supplied

Cells encapsulated in alginate: a potential system for delivery of recombinant proteins to malignant brain tumours.

Growth and progression of malignant brain tumours occurs in a micromilieu consisting of both tumour and normal cells. Several proteins have been identified with the potential of interfering directly with tumour cells or with the neovascularisation process, thereby inhibiting tumour growth. A continuous delivery of such inhibitory proteins to the tumour microenvironment by genetically engineered cells could theoretically be of considerable therapeutic importance. In this study we have investigated the growth characteristics of cells encapsulated in alginate, which represents a potential delivery system for recombinant proteins that may have antitumour effects. Three different cell lines, NHI 3T3, 293 and BT4C were encapsulated in alginate, which is an immuno-isolating substance extracted from brown seaweed. The encapsulated cells were observed at specific intervals during a 4-month period after in vitro propagation and as transplants into the cortex of BD-IX rats. Morphological studies showed that encapsulated cells proliferated and formed spheroids within the alginate in the in vitro cultures and after implantation into the brain. Even after 4 months in vivo a substantial amount of living cells were observed within the alginate beads. A vigorous infiltration of mononuclear cells was observed in the brain bordering the alginate beads, one week after implantation. However, there was a gradual decrease of mononuclear cells at the border zone beyond the first week of implantation. The majority of inflammatory cells were reactive microglia and invading monocytes, as verified by immunohistochemistry. The data further shows that alginate encapsulated cells can be frozen in liquid N2 and will retain their viability and proliferative capacity.

3T3 Cells↗

PubMedDate not supplied

Laminin expression by glial fibrillary acidic protein positive cells in human gliomas.

Extracellular matrix components are regarded as important substrates for invasive tumor cells. The present work focuses on the expression of laminin in the brain in response to invading brain tumors. Biopsies obtained from tissue macroscopically evaluated as the border zone between tumor and normal brain, in 5 patients undergoing surgery for glioblastoma multiforme, were examined by immunocytochemistry and scanning confocal microscopy for the expression of laminin and glial fibrillary acidic protein. Laminin was mainly found in all the specimens associated with the basal lamina of blood vessels, but a variable degree of punctate laminin deposits were also observed in the parenchyma not associated with blood vessels. In the specimens with substantial deposits, scanning confocal microscopy showed that some of the laminin co-localized with intracellular glial fibrillary acidic protein. Punctate deposits of laminin were also seen in an intracranial BT4C rat glioma model, where it was particularly abundant in the brain/tumor confrontation zone. Previous in vitro studies have shown that laminin, among several extracellular matrix components, represent a highly permissive substrate for glioma cell migration. The presented results indicate that laminin can be produced by glial fibrillary acidic protein positive cells during glioma cell invasion in humans. This glycoprotein may thus represent one important substrate among many, which contribute to the invasive phenotype of gliomas.

Animals↗

PubMedDate not supplied

Bromelain reversibly inhibits invasive properties of glioma cells.

Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that alpha3 and beta1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a transactivating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation.

Animals↗

PubMedDate not supplied

Alginate-encapsulated producer cells: a potential new approach for the treatment of malignant brain tumors.

In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells. However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells. These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate. This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation. This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells. The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry. The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture. The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks. The production of the bacterial E. coli beta-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged beta-galactosidase activity. H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined. The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro. To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells. The migration in vitro was totally inhibited in the presence of H528 encapsulated cells. Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry. It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments. Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors. Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors.

3T3 Cells↗

PubMedDate not supplied

Effects of high and low single dose irradiation on glioma spheroid invasion into normal rat brain tissue in vitro.

The effects of radiation on direction on directional migration in monolayer cultures and brain tissue invasion by two glioblastoma cell lines (D-54 MG, D-247 MG) were investigated. The Leksell Gamma Unit was the radiation source and invasion was registered in an in vitro invasion assay developed in our laboratory. As tumor spheroids and brain tissue aggregates were treated simultaneously in cocultures; the effects of radiation on the interaction between the two tissues could be investigated. Tumor spheroids from both cell lines retained their ability to invade and destroy normal brain tissue, even after irradiation with 47.6 Gy. However, while the D-54 MG tumor spheroids showed a dose-dependent reduction of invasion, tumor spheroids from the D-247 MG cell line did not. In addition, radiation produced a dose dependent inhibition of directional migration of cells from D-54 MG spheroids. A similar significant inhibition of directional migration was found in D-247 MG, but it was not dose-dependent. Transmission electron microscopy revealed a loosening of the neuropil in the brain tissue of irradiated cocultures. However, this structural change did not seem to affect the invasiveness of the tumor. In this preliminary study, irradiation could not prevent invasion of two different glioblastoma cell lines into fetal rat brain tissue. Further studies using the same technique may help to understand the influence of ionizing radiation upon the invasion process in gliomas.

Animals↗

PubMedDate not supplied

A metalloproteinase, capable of destroying cultured brain tissue isolated from rat glioma cells.

A continuous rat glioma cell line, BT5C, which causes severe invasion and tissue destruction when cocultured with fetal rat brain aggregates, secreted into the culture medium a metalloproteinase in a hMr latent (86000) and an lMr active (76000) isoform. Purified preparations of the enzyme were added to cultures of fetal rat brain aggregates, which were then examined by light and by scanning electron microscopy. After 48h of enzyme exposure, destruction of the neural tissue was observed. This was morphologically similar to the tissue destruction seen when BT5C spheroids were cocultured with brain aggregates. The protease had no effect on the morphology of BT 5C tumour spheroids. The report suggests a possible role of metalloproteinases in tissue degradation during brain tumour invasion.

Animals↗

PubMedDate not supplied

Invasive glioma cells in tissue culture.

Malignant human glioma is characterized by an uncontrolled cell proliferation and infiltrative growth into the brain. The mechanisms by which invasion occurs are poorly understood. Due to recent development in tissue culture methods, it is possible to study invasion in organotypic coculture systems consisting of glioma spheroids and reaggregated fetal brain cells. Spheroids from well-characterized continuous human glioma cell lines have been tested for invasiveness in this model, which also allows studies of the invasive capacity of glioma cells derived from biopsy material within a week after surgery. Invasion may furthermore be studied in chemically defined media. The methods of studying in vitro glioma invasiveness are reviewed, together with recent results which may throw light upon important mechanisms related to glioma invasion, at the peri- and extracellular level. Mechanisms of glioma cell invasion are discussed with emphasis on the interactive process between cells, growth factors, proteolytic enzymes and the extracellular matrix.

Animals↗

PubMedDate not supplied

Stereotactic biopsies as a model for studying the interaction between gliomas and normal brain tissue in vitro.

In order to study tumor invasiveness, the authors used a model system which allows the direct interaction between glioma cells and normal brain tissue to be studied. The spiral needle biopsy technique provides a tissue core with a completely preserved architecture from brain tumors. These biopsies, once cultured, produce three-dimensional multicellular tumor spheroids; they display morphological features similar to those of the original tumor in vivo. As a target fetal rat brain cell aggregates were used, in order to study the invasive process in the microscope. Not surprisingly, different tumors show different patterns of invasion; this variability is not yet clearly correlated with the clinical course of the patient.

Animals↗