sepharose and Glioma

Treatment of glioblastoma, the most common and aggressive type of primary brain tumors, is a major medical challenge and the development of new alternatives requires simple yet realistic models for these tumors. In vitro spheroid models offer attractive platforms to mimic the tumor behavior in vivo and have thus, been increasingly applied for assessment of drug efficacy in various tumors. The aim of this study was to produce and characterize size-controlled U251 glioma spheroids towards application in glioma drug evaluation studies. To this end, we fabricated agarose hydrogel microwells with cylindrical shape and diameters of 70-700 μm and applied these wells without any surface modification for glioma spheroid formation. The resultant spheroids were homogeneous in size and shape, exhibited high cell viability (> 90%), and had a similar growth rate to that of natural brain tumors. The final size of spheroids depended on cell seeding density and microwell size. The spheroids' volume increased linearly with the cell seeding density and the rate of this change increased with the well size. Lastly, we tested the therapeutic effect of an anti-cancer drug, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) on the resultant glioma spheroids and demonstrated the applicability of this spheroid model for drug efficacy studies.

Topics: Cell Culture Techniques; Cell Line, Tumor; Glioma; Humans; Hydrogels; Sepharose; Spheroids, Cellular

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Topics: Algorithms; Aminolevulinic Acid; Brain Neoplasms; Diagnostic Imaging; Disease-Free Survival; Glioma; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Optics and Photonics; Phantoms, Imaging; Protoporphyrins; Sepharose; Signal-To-Noise Ratio

In the present study, we have explored porphyran as a reducing agent for one pot size controlled green synthesis of gold nanoparticles (AuNps) and further investigated its application as a carrier for the delivery of an anticancer drug. The prepared AuNps showed surface plasmon resonance centered at 520 nm with average particle size of 13±5 nm. FTIR spectra suggested that the sulfate moiety is mainly responsible for reduction of chloroauric acid. The capping of the AuNps with porphyran was evident from the negative zeta potential value responsible for the electrostatic stability. Thus, porphyran acts as reducing as well as capping agent. These AuNps are highly stable in a wide range of pH and electrolyte concentration. Porphyran capped AuNps exhibited enhanced cytotoxicity on human glioma cell line (LN-229) as compared to native porphyran. Consequently, these AuNps have been utilized as a carrier for delivery of the anticancer drug doxorubicin hydrochloride (DOX). Spectroscopic examination revealed that DOX conjugated onto AuNps via hydrogen bonding. The release of DOX from DOX loaded AuNps was found to be sixfold higher in acetate buffer (pH 4.5) as compared to physiological buffer (pH 7.4). Further, the DOX loaded AuNps demonstrated higher cytotoxicity on LN-229 cell line as compared with an equal dose of native DOX solution. This established the potential of these AuNps as a carrier for anticancer drug delivery.

Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; Chlorides; Doxorubicin; Drug Carriers; Drug Delivery Systems; Glioma; Gold; Gold Compounds; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Metal Nanoparticles; Particle Size; Sepharose; Spectroscopy, Fourier Transform Infrared; Static Electricity; Surface Plasmon Resonance

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Topics: Biomechanical Phenomena; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Elasticity; Extracellular Matrix; Fibrillar Collagens; Gels; Glioma; Humans; Mesoderm; Microscopy, Electron, Scanning; Neoplasm Invasiveness; Porosity; Sepharose; Stress, Mechanical; Time Factors

The indications for stereotactic biopsies or implantation of probes for local chemotherapy in diffuse brainstem tumors have recently come under debate. The quality of performing these procedures significantly depends on the precision of the probes' placement in the brainstem. The authors evaluated the precision of brainstem probe positioning using a navigated frameless stereotactic system in an experimental setting.. Using the VarioGuide stereotactic system, 33 probes were placed into a specially designed model filled with agarose. In a second experimental series, 8 anatomical specimens were implanted with a total of 32 catheters into the pontine brainstem using either a suboccipital or a precoronal entry point. Before intervention in both experimental settings, a thin-sliced CT scan for planning was obtained and fused to volumetric T1-weighted MR imaging data. After the probe positioning procedures, another CT scan and an MR image were obtained to compare the course of the catheters versus the planned trajectory. The deviation between the planned and the actual locations was measured to evaluate the precision of the navigated intervention.. Using the VarioGuide system, mean total target deviations of 2.8 +/- 1.2 mm on CT scanning and 3.1 +/- 1.2 mm on MR imaging were detected with a mean catheter length of 151 +/- 6.1 mm in the agarose model. The catheter placement in the anatomical specimens revealed mean total deviations of 1.95 +/- 0.6 mm on CT scanning and 1.8 +/- 0.7 mm on MR imaging for the suboccipital approach and a mean catheter length of 59.5 +/- 4.1 mm. For the precoronal approach, deviations of 2.2 +/- 1.2 mm on CT scanning and 2.1 +/- 1.1 mm on MR imaging were measured (mean catheter length 85.9 +/- 4.7 mm).. The system-based deviation of frameless stereotaxy using the VarioGuide system reveals good probe placement in deep-seated locations such as the brainstem. Therefore, the authors believe that the system can be accurately used to conduct biopsies and place probes in patients with brainstem lesions.

Topics: Biopsy; Brain Neoplasms; Brain Stem; Catheterization; Child; Glioma; Humans; Magnetic Resonance Imaging; Models, Anatomic; Neuronavigation; Reproducibility of Results; Sepharose; Tomography, X-Ray Computed

PTEN is a candidate tumor suppressor gene identified on human chromosome 10q23.3 that is frequently mutated or deleted in 30% to 44% of glioblastomas. Transient expression study of PTEN in glioma cells indicates that PTEN plays an important role in cellular proliferation, tumorigenicity, cell migration, and focal adhesions. In this study, we examined the biological consequences on U87MG glioma cells after stable gene transfer of wild-type PTEN. Cells stably expressing wild-type PTEN protein were found to have suppressed proliferation, as determined by cell counting and Ki-67 staining, as well as inhibited anchorage-independent growth. The PTEN-expressing cells also showed higher expression of glial fibrillary acidic protein and changed morphologically from spindle-shaped to elongated cell bodies with multiple slender processes, suggesting that these cells have undergone differentiation. In addition, telomerase activity decreased more than 10-fold in PTEN-expressing cells when compared with control cells. More importantly, apoptosis was detected in about 5% of PTEN-expressing cells, representing a 17-fold (p < 0.01) increase over the control cells. Taken together, these results suggest that PTEN plays an important role in regulation of cell homeostasis by maintaining a balance between proliferation, differentiation, and apoptosis.

Topics: Apoptosis; Cell Differentiation; Cell Division; DNA Primers; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioma; Humans; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Telomerase; Transformation, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Brain invasion prevents complete surgical extirpation of malignant gliomas; however, invasive cells from distant, histologically normal brain previously have not been isolated, cultured, and characterized. To evaluate invasive human malignant glioma cells, the authors established cultures from gross tumor and histologically normal brain. Three men and one woman, with a mean age of 67 years, underwent two frontal and two temporal lobectomies for tumors, which yielded specimens of both gross tumor and histologically normal brain. Each specimen was acquired a minimum of 4 cm from the gross tumor. The specimens were split: a portion was sent for neuropathological evaluation (three glioblastomas multiforme and one oligodendroglioma) and a portion was used to establish cell lines. Morphologically, the specimens of gross tumor and histologically normal brain were identical in three of the four cell culture pairs. Histochemical staining characteristics were consistent both within each pair and when compared with the specimens sent for neuropathological evaluation. Cultures demonstrated anchorage-independent growth in soft agarose and neoplastic karyotypes. Growth rates in culture were greater for histologically normal brain than for gross tumor in three of the four culture pairs. Although the observed increases in growth rates of histologically normal brain cultures do not correlate with in vivo behavior, these findings corroborate the previously reported stem cell potential of invasive glioma cells. Using the radial dish assay, no significant differences in motility between cultures of gross tumor and histologically normal brain were found. In summary, tumor cells were cultured from histologically normal brain acquired from a distance greater than 4 cm from the gross tumor, indicating the relative insensitivity of standard histopathological identification of invasive glioma cells (and hence the inadequacy of frozen-section evaluation of resection margins). Cell lines derived from gross tumor and histologically normal brain were usually histologically identical and demonstrated equivalent motility, but had different growth rates.

Topics: Aged; Brain; Brain Neoplasms; Cell Adhesion; Cell Division; Cell Movement; Cells, Cultured; Coloring Agents; Culture Media; Female; Frontal Lobe; Glioblastoma; Glioma; Histocytochemistry; Humans; Karyotyping; Male; Neoplasm Invasiveness; Oligodendroglioma; Sepharose; Stem Cells; Temporal Lobe; Tumor Cells, Cultured

A rapid and simple two-step procedure for the quantitative analysis of GDP-mannose (GDP-Man) recovered in ethanol extracts of cultured mammalian cells is described. GDP-Man is initially separated from water-soluble metabolites and other nucleotide sugars, including UDP-glucose (UDP-Glc) and GDP-fucose (GDP-Fuc), due to a weak, alpha-mannoside-specific interaction with concanavalin A (Con A)-Sepharose at pH 3.5. The specificity and pH dependence of the GDP-Man-Con A interaction have been characterized. The partially purified fraction from Con A-Sepharose can be further purified by high-performance anion-exchange chromatography on a Partisil-10 SAX silica gel column, and the concentration of GDP-Man was determined by monitoring the HPLC column eluate for absorbance at 254 nm. This procedure provides a simple means of calculating the specific activity of cellular GDP-[3H]Man pools, metabolically labeled with [2-3H]mannose. Using this new procedure, the relative rates of Glc3Man9Glc-NAc2-P-P-dolichol (Oligo-P-P-Dol) biosynthesis and protein N-glycosylation were assayed in C 6 rat glial tumor cells, COS P6 cells, Chinese hamster ovary (CHO) cells, and mouse L929 cells by metabolic labeling with [2-3H]mannose. A comparison of the relative rates of incorporation of [2-3H]mannose into Oligo-P-P-Dol and N-linked oligosaccharides in four different cultured cell lines demonstrates that misleading results can be obtained if the calculation of the biosynthetic rates is not based on the specific activity of the nucleotide sugar pools.

Topics: Animals; B-Lymphocytes; Carbohydrate Metabolism; Carbohydrate Sequence; Cell Line; CHO Cells; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cricetinae; Female; Glioma; Glycoproteins; Guanosine Diphosphate Mannose; Hydrogen-Ion Concentration; Lymphocyte Activation; Mannose; Mice; Mice, Inbred DBA; Molecular Sequence Data; Nucleotides; Polyisoprenyl Phosphate Sugars; Rats; Sensitivity and Specificity; Sepharose; Tritium

Type beta transforming growth factor (beta-TGF) is a potent regulator of cell growth and differentiation. The human glioblastoma cell line, T-MGI, was growth inhibited by beta-TGF under anchorage independent conditions. The antiproliferative effect of beta-TGF was potentiated to nearly total arrest by low doses of retinoic acid (RA) or tumor necrosis factor (TNF), while epidermal growth factor, platelet-derived growth factor, interleukin-2, and gamma interferon did not have this potentiating effect. The potentiation of the beta-TGF effect by RA and TNF could not be explained by modulation of the epidermal growth factor receptor, the beta-TGF receptor, or the TNF receptor. beta-TGF alone and in combination with RA or TNF were further tested on primary cultures from freshly resected human glioma biopsies (n = 13). There was great individual variation in sensitivity to beta-TGF, RA, or TNF. The astrocytoma and oligodendroglioma cells were inhibited to various degrees by beta-TGF or TNF, while most of the glioblastomas were not sensitive to these agents. Most of the biopsies were stimulated by RA. RA or TNF did not potentiate the growth inhibitory effect of beta-TGF on biopsy cells. We therefore think it unlikely that beta-TGF in combination with RA or TNF will be effective agents in the treatment of gliomas.

Topics: Adjuvants, Immunologic; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line; Clone Cells; Drug Screening Assays, Antitumor; ErbB Receptors; Glioma; Growth Inhibitors; Humans; Iodine Radioisotopes; Peptides; Sepharose; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Type beta transforming growth factor (B-TGF) is a potent regulator of cell growth and differentiation. Growth of many human tumour cell lines are inhibited by B-TGF. The effect of B-TGF on proliferation of clonogenic cells from 12 human glioma biopsies was registrated in a thymidine incorporation assay. B-TGF appeared to be a potent growth inhibitor for some gliomas, while it had no effect on others. Maximum inhibition was about 60%. Though not significant, glioblastomas appeared to be less sensitive to inhibition by B-TGF than astrocytomas and oligodendrogliomas. Very little is known about the growth inhibitory action of B-TGF. The negative autocrine growth theory for cancer cells postulates that a reduced production, or production of a defective growth inhibitor normally found in the cell, may account for the autonomous nature of some cancer cells. In view of this theory, we searched for B-TGF in protein extracts from a glioblastoma cell line, T-MGl, whose growth was inhibited by B-TGF. Protein extract from T-MGl cells was analysed for B-TGF activity using a soft agar colony formation assay with normal rat kidney fibroblasts. B-TGF was not found in the extract. Since, according to the literature B-TGF has been found in all other cell lines examined, we believe that there may be a lack of B-TGF or an altered B-TGF in the gliomas whose growth are inhibited by B-TGF. This problem will be studied further.

Topics: Brain Neoplasms; Cell Division; Cell Line; Clone Cells; Culture Media; Glioma; Humans; Sepharose; Transforming Growth Factors

Plastic dishes were coated with an agarose layer. The layer was modified by covalently binding proteins to it, using the CNBr-method. Cells were seeded on the dishes and the number of attached cells was evaluated. The specificity of the attachment was demonstrated by showing that cells, carrying specific membrane-bound immunoglobulins, attached only to the corresponding anti-immunoglobulins. This indicated that the method could be used for cell sorting. The attachment of cells to proteins was influenced by the amount of bound protein, incubation time, temperature and the degree of trypsinization. Most attached cells were viable for several days and when dying they detached. Detailed morphological and cytochemical analyses of the dynamics of attachment and cytoplasmic spreading on the chemically well-defined surfaces were possible using the new method.

Topics: Burkitt Lymphoma; Cell Adhesion; Cell Line; Concanavalin A; Glioma; Humans; Leukemia; Lymphoma, Non-Hodgkin; Neuroglia; Polylysine; Polysaccharides; Protamines; Protein Binding; Proteins; Sepharose

The growth of human glioma cells, cultured as spherical colonies in agarose gel, stopped after about 10 days for both large and small colonies apparently due to an increased osmolality in the gel. When osmolality was kept under control by addition of distilled water, growth continued. However, a continuous increase in the population-doubling period, similar for both large and small colonies, then was observed. The increase persisted although excess amounts of nutrition were added. When the cells were cultured in liquid suspension above a thin layer of agarose gel and most of the medium was repeatedly changed, the colonies continued to grow beyond the limits in gel cultured. HeLa and hamster embryonic lung cell colonies showed a growth pattern in agarose gel similar to the glioma cells. The results imply that the osmolality must be kept under precise control to prevent growth inhibition. However, it seems difficult to ascertain optimal growth in gel culture for more than about 2 weeks probably because of the accumulation of toxic products.

Topics: Animals; Cell Division; Cricetinae; Culture Media; Culture Techniques; Gels; Glioma; Humans; Osmolar Concentration; Sepharose

Human glioma cells, growing as spherical colonies in agarose gel, or as monolayers on glass or plastic, were studied with time-lapse cinematography and electron microscopy. The cells in the agarose cultured colonies often had ruffling-like membrane structures which were similar in for, although smaller in size, than those observed on monolayer cultured cells. The ruffling-like structures were more frequent at the periphery than in the central regions of the colonies which was in parallel to the proliferative pattern. In time-lapse cinematography it was seen that pinocytotic vacuoles were formed from ruffling membranes in the monolayer cultures. In the transmission electron microscope, such vacuoles were also found near the ruffling-like structures in the agarose cultured cells. In dense monolayer cultures, ruffling and associated pinocytosis were to a large extent transferred from the margin to the upper surface of the cells. This capacity may be an important property for the ability of the malignant cells to attain extreme high cell densities in monolayer cultures and to grow as colonies in agarose cultures. As has been previously shown, the normal counterpart of the gliomas, the glia cells, cannot grow to high densities; they do not ruffle on their upper cell surface and are unable to grow in suspension or agarose culture.

Topics: Autoradiography; Cell Division; Cell Line; Cell Membrane; Cells, Cultured; Cineradiography; Glioma; Humans; Methods; Microscopy, Electron; Sepharose

The fine structure of human glioma cells cultivated as three-dimensional colonies in agarose was investigated during exponential growth. The colonies did not show central degeneration, although they reached diameters of up to 600 mum. Large extra-cellular spaces extended throughout the colonies. The mean volumetric fraction of the spaces increased from about 20 per cent at the periphery up to nearly 40 per cent in the central regions. The quotient between nuclei and cytoplasm showed a slight decrease with depth. A proliferative gradient existed in the colonies, i.e. the mitotic index decreased almost exponentially with the distance from the surface. The distance at which the mitotic index changes by a factor of 2 was about 90 mum, corresponding to nearly five cell diameters. Cytoplasmic extensions, with a ruffling-like appearance, occurred both at the periphery and in the centre of the colonies but were larger and more frequent at the periphery. The fractions of mitochondria and vacuoles in the cytoplasm showed rather large local variations. However, the mean number of mitochondria decreased somewhat towards the centre and the number of vacuoles containing highly electron-absorbing substance increased in the most central regions.

Topics: Cell Aggregation; Cell Line; Cell Nucleus; Culture Media; Cytoplasm; Glioma; Humans; Microscopy, Electron; Mitochondria; Mitosis; Sepharose; Subcellular Fractions; Vacuoles

Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The sufaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [3H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests.

Topics: Bacteria; Cell Line; Cells, Cultured; Culture Media; DNA, Neoplasm; Glioma; Glycoside Hydrolases; Humans; Mitotic Index; Polysaccharides; Sepharose; Thymidine