sepharose and Neuroblastoma

Three-dimensional bioprinting continues to advance as an attractive biofabrication technique to employ cell-laden hydrogel scaffolds in the creation of precise, user-defined constructs that can recapitulate the native tissue environment. Development and characterisation of new bioinks to expand the existing library helps to open avenues that can support a diversity of tissue engineering purposes and fulfil requirements in terms of both printability and supporting cell attachment. In this paper, we report the development and characterisation of agarose-gelatin (AG-Gel) hydrogel blends as a bioink for extrusion-based bioprinting. Four different AG-Gel hydrogel blend formulations with varying gelatin concentration were systematically characterised to evaluate suitability as a potential bioink for extrusion-based bioprinting. Additionally, autoclave and filter sterilisation methods were compared to evaluate their effect on bioink properties. Finally, the ability of the AG-Gel bioink to support cell viability and culture after printing was evaluated using SH-SY5Y cells encapsulated in bioprinted droplets of the AG-Gel. All bioink formulations demonstrate rheological, mechanical and swelling properties suitable for bioprinting and cell encapsulation. Autoclave sterilisation significantly affected the rheological properties of the AG-Gel bioinks compared to filter sterilisation. SH-SY5Y cells printed and differentiated into neuronal-like cells using the developed AG-Gel bioinks demonstrated high viability (>90%) after 23 d in culture. This study demonstrates the properties of AG-Gel as a printable and biocompatible material applicable for use as a bioink.

Topics: Bioprinting; Cell Encapsulation; Gelatin; Humans; Hydrogels; Neuroblastoma; Printing, Three-Dimensional; Sepharose; Tissue Engineering; Tissue Scaffolds

In vitro studies have been popularly used to determine the cellular and molecular mechanisms for many decades. However, the traditional two-dimension (2D) cell culture which grows cells on a flat surface does not fully recapitulate the pathological phenotypes. Alternatively, the three-dimension (3D) cell culture provides cell-cell and cell-ECM interaction that better mimics tissue-like structure. Thus, it has gained increasing attention recently. Yet, the expenses, time-consuming, and complications of cellular and biomolecular analysis are still major limitations of 3D culture. Herein, we describe a cost-effective and simplified workflow of the 3D neuronal cell-laden agarose-laminin preparation and the isolation of cells, RNAs, and proteins from the scaffold. To study the effects of the amyloidogenic condition in neurons, we utilized a neuron-like cell line, SH-SY5Y, and induced the amyloidogenic condition by using an amyloid forty-two inducer (Aftin-4). The effectiveness of RNAs, proteins and cells isolation from 3D scaffold enables us to investigate the cellular and molecular mechanisms underlying amyloidogenic cascade in neuronal cells. The results show that SH-SY5Y cultured in agarose-laminin scaffold differentiated to a mature TUJ1-expressing neuron cell on day 7. Furthermore, the gene expression profile from the Aftin-4-induced amyloidogenic condition revealed the expression of relevant gene-encoding proteins in the amyloidogenic pathway, including APP, BACE1, PS1, and PS2. This platform could induce the amyloid-beta 42 secretion and entrap secreted proteins in the scaffold. The induction of amyloidogenic conditions in a 3D culture facilitates the interaction between secreted amyloid-beta and neurons, which makes it resembles the pathological environment in Alzheimer's brain. Together, this workflow is applicable for studying the cellular and molecular analysis of amyloid-induced neuronal toxicity, such as those occurred in Alzheimer's disease progression. Importantly, our method is cost-effective, reproducible, and easy to manipulate.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Humans; Hydrogels; Laminin; Neuroblastoma; Neurons; Sepharose; Workflow

beta-Amyloid peptide (Abeta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. Abeta is toxic only when aggregated, however, the size and structure of the aggregated species associated with toxicity is unknown. In the present study, we developed a diffusion-based method to simultaneously separate and detect the biological activity of toxic Abeta oligomers and used the method to examine the relationship between size of aggregated protein and toxicity to SH-SY5Y cells. From these measurements, the effective diffusivity and hydrodynamic radius of the toxic oligomeric species of Abeta could be determined. A sensitivity analysis was performed to examine the effects of model assumptions used in data analysis on the effective diffusivity calculated. The method provides a new estimate of the size of small toxic Abeta species associated with fibril formation. This work contributes to our understanding of the relationship between Abeta structure and toxicity and with further refinements may aid in our ability to design agents which alter the Abeta aggregation/dissociation processes associated with neurotoxicity.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Survival; Diffusion; Equipment Design; Flow Cytometry; Humans; Macromolecular Substances; Models, Biological; Molecular Weight; Neuroblastoma; Protein Interaction Mapping; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Sepharose; Species Specificity; Toxicity Tests; Tumor Cells, Cultured

Retinoic acid (RA) decreased growth and increased morphologic differentiation of human neuroblastoma LA-N-1 cells. These phenomena correlated with a specific enhancement of PHA-E lectin binding to a glycoprotein of MW 67 kDa (gp67). Gp67 was found susceptible to N-glycanase and displayed BSA binding by affinity chromatography analysis. The chemotherapeutic agent methotrexate (MTX) also reduced growth and induced differentiation of LA-N-1 cells. In addition, the cells responded to MTX as well as to doxorubicin by a marked increase in PHA-E binding to gp67. We conclude that reduced growth and induction of morphological differentiation of LA-N-1 cells correlates with increased binding of PHA-E to gp67.

Topics: Amidohydrolases; Antibodies; Cell Differentiation; Cell Division; Chromatography, Affinity; Humans; Lectins; Membrane Glycoproteins; Methotrexate; Neoplasm Proteins; Neuroblastoma; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Phytohemagglutinins; Protein Binding; Sensitivity and Specificity; Sepharose; Serum Albumin, Bovine; Staining and Labeling; Tretinoin; Tumor Cells, Cultured

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity-isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects.

Topics: Animals; Antibody Specificity; Antigen-Antibody Reactions; Autoantibodies; Autoimmune Diseases; Blotting, Western; Brain Neoplasms; Cell Membrane; Cerebral Cortex; Chromatography, Affinity; Disease Models, Animal; Female; Fibroblasts; Fluorescent Antibody Technique; Gels; Immunosorbent Techniques; Kidney; Liver; Liver Neoplasms, Experimental; Lupus Erythematosus, Systemic; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Molecular Weight; Nerve Tissue Proteins; Neuroblastoma; Neurons; Organ Specificity; Sepharose

The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (AngII) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT2 subtype. However, displacement of 125I-AngII with the AT2 nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2 receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound 125I-AngII with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented approximately 80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCl for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2 receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT1-selective antagonist Losartan. However, whereas the nonpeptidic AT2-selective antagonist PD-123319 completely displaced the binding of 125I-AngII from peak I in a monophasic fashion (IC50 = 9.1 +/- 4.1 nM; mean +/- SEM; n = 3), PD-123319 was much less effective in displacing 125I-AngII from peak III (IC50 = 196 +/- 27 nM; mean +/- SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125I-AngII specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTP gamma S significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125I-AngII binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populat

Topics: Angiotensin II; Animals; Cell Membrane; Chromatography, Agarose; Dithiothreitol; Guanine Nucleotides; Immunoblotting; Mice; Neuroblastoma; Receptors, Angiotensin; Sepharose; Solubility; Tumor Cells, Cultured

The presence of suppressive antibody activity in sera from patients spontaneously recovered from the Guillain-Barré syndrome was investigated by analyzing the ability of postrecovery serum to inhibit anti-neuroblastoma cell line antibody binding in sera from seven patients in the prerecovery phase or with a chronic form of the disease. All 12 recovered patients analyzed were found to have inhibitory IgG antibodies in their postrecovery sera, of which the F(ab')2 fragments mediated the inhibitory effect. The pattern of inhibition suggests that about half of the patients share cross-reactive idiotypes of high affinity. The efficiency of the inhibition mediated by anti-idiotypic antibodies in spontaneously recovered patients was twice as high as that mediated by anti-idiotypes present in therapeutical preparations of polyclonal immunoglobulins for intravenous use (IVIG). Affinity chromatography of IVIG and serum from a recovered Guillain-Barré syndrome patient on autoantibody-containing F(ab')2 fragments revealed, first, that inhibitory anti-idiotypic antibodies are specifically retained on autoantibodies and, second, that these antibodies constitute less than 1% of the total IgG antibody content.

Topics: Antibodies; Antibodies, Anti-Idiotypic; Antibody Formation; Cross Reactions; Humans; Immunoglobulin Fab Fragments; Immunoglobulins, Intravenous; Neuroblastoma; Polyradiculoneuropathy; Sepharose; Tumor Cells, Cultured

Our aim was to determine if the selection of human tumor cells with enhanced anchorage-independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK-N-MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose-selected variants were completely unable to proliferate in methionine-free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5-methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine-free medium. These variants were unable to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S-adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S-adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5-methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.

Topics: Cell Line; Cell Transformation, Neoplastic; Genetic Variation; Homocysteine; Humans; Melanoma; Methionine; Neuroblastoma; Nucleic Acids; S-Adenosylmethionine; Sepharose; Tumor Cells, Cultured

Two new neuroblastoma (NB) cell lines, NUB-6 and NUB-7, were established from recurrent and primary NB tumours respectively and identified conclusively as NB by their phenotypic characteristics, catecholamine production and N-myc amplification. The cell lines could be distinguished on the bases of distinctive growth patterns in monolayer culture and semi-solid media (collagen gel and agarose), neurite formation and their response to four classes of growth and differentiation modulators. The NUB-6 cell line consisted of two distinct cell subtypes, small typical neuroblasts and larger spheroid-forming cells, while NUB-7 was homogeneously neuroblastic. Class-I agents (dibutyrl cyclic AMP [dbcAMP], butyrate, and papaverine) inhibited growth of both cell lines, while only dbcAMP stimulated the formation of short neurites by NUB-6 neuroblast cells in monolayer culture and collagen. Of the class-II agents (vitamins), retinoic acid inhibited growth of both cell lines and stimulated formation of long neurites by NUB-6 cells and NUB-7 cells in later passages. In contrast, vitamin E inhibited growth of NUB-6 and late-passage NUB-7, but stimulated early passage NUB-7. The class III agent (nerve growth factor) resembled vitamin E. The class-IV agents (interferons; rIFN-alpha 2a and rIFN-gamma 1) inhibited growth of both cell lines in monolayer culture and agarose, but stimulated NUB-6 neuroblasts and early passage NUB-7 cells to form long neurites. Thus phenotypically distinct NB cell lines were established in vitro and shown to be differentially influenced by various growth and differentiation modulators. The potent effect of IFN suggests a role for these modulators in NB behaviour in vivo.

Topics: Cell Aggregation; Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Collagen; Flow Cytometry; Gels; Humans; Neuroblastoma; Phenotype; Sepharose; Tumor Cells, Cultured

We have used three continuous human neuroblastoma cell lines to establish patterns of in vitro drug sensitivities, as judged by clonogenic assay. We evaluated 12 'standard' antitumor drugs already in clinical usage, and tested four newer analogues, one of cisplatin and three of doxorubicin, and the investigational agent desferrioxamine. A certain heterogeneity of drug sensitivities was noted amongst these three cell lines, but a few general conclusions can be drawn. Responses of all lines tested were similar for actinomycin D, dibromodulcitol, doxorubicin, 5-fluorouracil, melphalan and VM 26. However, line CHP 100 proved hypersensitive to amsacrine, bleomycin, methotrexate and vincristine yet refractory to cisplatin, carboplatin and VP-16, compared with the other two lines. This emphasizes the necessity for using a panel of cell lines for this type of drug screening programme. A comparison of IC50 drug concentrations, derived from these in vitro tests, with plasma levels achievable clinically, indicate that VP-16, VM 26, doxorubicin and cisplatin appear to be the most effective agents in this tumor type. This finding is consistent with clinical experience. The newer doxorubicin analogues proved 2-5 fold more cytotoxic than doxorubicin itself. However, these differences also appear to be reflected in the lower dose ranges now being tested in phase I/II clinical trials. Desferrioxamine, which proved cytotoxic against all three neuroblastoma cell lines, exerted comparable cytotoxicity against two of the three non-neuroblastoma human tumor cell lines evaluated. Therefore we suggest that attempts to identify any specific antineuroblastoma activities by new investigational agents using this type of model systems require evaluation against panels of both neuroblastoma and non-neuroblastoma lines.

Topics: Antineoplastic Agents; Deferoxamine; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Sepharose; Tumor Cells, Cultured

High activity of angiotensin-converting enzyme was demonstrated in human neuroblastoma tissue. This activity required the presence of chloride ion and was almost completely inhibited by a specific converting enzyme inhibitor captopril (10 nM), indicating that the activity measured is indeed angiotensin-converting enzyme. Furthermore, the biochemical features of the enzyme were closely similar to the well-known properties of human lung converting enzyme, such as molecular weight (290,000), optimum pH (8.0-8.5), the presence of glycoprotein residues, and dependence on chloride ion concentration. These results provide definitive evidence for the presence of true angiotensin-converting enzyme in human neuroblastoma tissue.

Topics: Angiotensin-Converting Enzyme Inhibitors; Captopril; Child, Preschool; Chromatography, Ion Exchange; Female; Humans; Hydrogen-Ion Concentration; Infant; Lung; Molecular Weight; Neuroblastoma; Peptidyl-Dipeptidase A; Proteins; Sepharose

Serum ferritin is present in two forms--a glycosylated form that results from active secretion by cells and a nonglycosylated form that is directly released by damaged cells. Glycosylated ferritin binds to concanavalin A (Con A) through the glucose and/or mannose residues of the molecule. Patients with neuroblastoma frequently present at diagnosis with abnormally elevated levels of serum ferritin. The ferritin levels will, however, return to normal with clinical remission, suggesting that the tumor is the origin of the elevated ferritin. With the use of a Con A binding assay, an investigation was made as to whether the increased levels of serum ferritin at diagnosis in neuroblastoma patients resulted from active secretion by the tumor or were the consequence of direct release of ferritin from damaged tissue. Serum samples were collected at diagnosis from 36 children with neuroblastoma and from 16 normal healthy subjects. Tissue ferritins were purified from normal human liver, placenta, HeLa cells, human neuroblastoma, and hepatoma cells grown in culture. Serum and tissue ferritins were measured before and after binding with Con A. Sixty-three percent of serum ferritin from neuroblastoma patients and 66% of serum ferritin from normal subjects were bound to Con A, suggesting that they were glycosylated and were likely to have been secreted. On the other hand, only 28% of tissue ferritin were bound to Con A. Furthermore, most patients showed abnormally elevated levels of serum ferritin, and 63% of these ferritins were bound to Con A. These results are compatible with the hypothesis that much of the elevated ferritin in sera of patients with neuroblastoma seen at diagnosis is the result of secretion of ferritin by the tumor.

Topics: Concanavalin A; Ferritins; Humans; Neuroblastoma; Prognosis; Sepharose