ADVANCED ENGINEERING MATERIALS, Issue 4 2010
Yuan-Min Lin
Functional groups on a material surface affect the response of many cell types. As part of our strategy aimed at engineering lung tissue, we introduced functional groups into the surface of Poly(D,L -lactide) (PDLLA) films to improve its suitability for the culture of mature pulmonary epithelial cells (A549 line) using two different methods. The first method, aminolysis, can introduce primary amines into PDLLA films by transesterification using 1,15% of ethylenediamine in isopropanol. The second method, a branching modification, can generate amine-terminated or carboxylic acid-terminated tree-like branched architectures. All modified PDLLA surfaces exhibited lower water contact angles, i.e. are more hydrophilic than unmodified PDLLA. PDLLA treated with 15% ethylenediamine exhibited a rougher surface than the control, and PDLLA with branching modification had a droplet-like surface topography as visualized by atomic force microscopy (AFM). PDLLA treated with 15% ethylenediamine and branching modification with two and three generations enhanced the attachment of pulmonary epithelial cells measured using Hoechst dye. Immunostaining demonsatrated that amine-terminated branched architectures allowed for better focal adhesion point formation than the control 24,h after cell seeding. Furthermore, they also induced higher A549 cell populations and levels of activity after 4 days in culture measured using Hoechst dye and WST1 cell proliferation reagents, respectively. In contrast, carboxylic acid-terminated branching architectures were found to reduce the cell population size after 4 days. It was concluded that the concentration, type and distribution of surface functional groups can affect significantly the behavior of pulmonary epithelial cells growing on a PDLLA surface, and PDLLA film modified with two or three generations of amine-terminated branched architectures is a suitable 2D scaffold for the culture of of pulmonary epithelial cells. [source]

Potential and Bottlenecks of Bioreactors in 3D Cell Culture and Tissue Manufacturing

ADVANCED MATERIALS, Issue 32-33 2009
David Wendt
Abstract Over the last decade, we have witnessed an increased recognition of the importance of 3D culture models to study various aspects of cell physiology and pathology, as well as to engineer implantable tissues. As compared to well-established 2D cell-culture systems, cell/tissue culture within 3D porous biomaterials has introduced new scientific and technical challenges associated with complex transport phenomena, physical forces, and cell,microenvironment interactions. While bioreactor-based 3D model systems have begun to play a crucial role in addressing fundamental scientific questions, numerous hurdles currently impede the most efficient utilization of these systems. We describe how computational modeling and innovative sensor technologies, in conjunction with well-defined and controlled bioreactor-based 3D culture systems, will be key to gain further insight into cell behavior and the complexity of tissue development. These model systems will lay a solid foundation to further develop, optimize, and effectively streamline the essential bioprocesses to safely and reproducibly produce appropriately scaled tissue grafts for clinical studies. [source]

Chitosan-Based Inverse Opals: Three-Dimensional Scaffolds with Uniform Pore Structures for Cell Culture

ADVANCED MATERIALS, Issue 29 2009
Sung-Wook Choi
Chitosan inverse opal scaffolds: Three-dimensional chitosan scaffolds with an inverse opal structure have been fabricated using a cubic close packed lattice of polymer beads as the template. The scaffolds have a uniform and interconnected pore structure as well as a fibrous morphology on the wall. They can be used as a model system for in vitro studies of cell culture and as clinically practical scaffolds for tissue engineering. [source]

Detection of Viable Rotaviruses in Shellfish by means of Cell Culture and Immunofluorescence Assay

JOURNAL OF FOOD SCIENCE, Issue 5 2002
C. S. Santos
ABSTRACT: The goal of this work was to examine the use of cell culture and immunofluorescence assays to detect viable rotaviruses in artificially seeded oyster meat and to determine if the method for oyster extracts preparation affects the viability of the viruses. Oyster tissues were seeded with rotavirus SA11, followed by tissue extract preparation. For cytopathogenicity assays (CPE), non-cytotoxic dilutions of seeded oyster extracts were inoculated in MA 104 cells. For immunofluorescence assays (IFA) the monoclonal antibody Mab M60 anti-VP7 capsid glycoprotein of rotavirus was used. For CPE tests, no significant difference between the oyster extracts inoculated with rotavirus before and after extract preparation was detected. Similar results have been observed in IFA assays and the recoveries of viable rotavirus obtained were close to 100%. [source]

Electrospun, Biofunctionalized Fibers as Tailored in vitro Substrates for Keratinocyte Cell Culture

MACROMOLECULAR BIOSCIENCE, Issue 9 2010
Dirk Grafahrend
Abstract Cell adhesion preventing fiber surfaces were tailored differently with bioactive peptides (a fibronectin fragment (GRGDS), a collagen IV fragment (GEFYFDLRLKGDK) and a combination of both) to provide an artificial extracellular matrix as a substrate for HaCaT keratinocyte cell culture. Therefore, a polymer blend containing a six-arm star-shaped statistical copolymer of ethylene oxide and propylene oxide in the ratio 80:20 (NCO- sP[EO- co -PO]) and poly-[D,L -(lactide- co -glycolide)] (PLGA) was electrospun. The resulting fibers were biofunctionalized and investigated as in vitro substrates using the HaCaT kerationcyte cell line. Appropriate surface chemistry on these electrospun fibers proved to prevent adhesion of keratinocytes, while additional immobilization of certain peptide sequences induced cell adhesion. These specific fibers enable investigation of immobilized active molecules and the subsequent cellular response to the scaffold. HaCaT keratinocytes were found to selectively adhere to those fibers modified with either collagen IV segment GEFYFDLRLKGDK or a mixture of the two peptide sequences GEFYFDLRLKGDK and GRGDS (1:1). However, the synergistic effects of both (the fibronectin fragment and the collagen IV fragment) seem to significantly increase the numbers of adherent keratinocytes. [source]

A Metal,Collagen Peptide Framework for Three-Dimensional Cell Culture,

ANGEWANDTE CHEMIE, Issue 42 2009
Marcos
Unter physiologischen Bedingungen bewirken Metallionen, dass sich tripelhelicale Collagenpeptide mit Liganden an ihren Enden und ihrer Mitte schnell zu dreidimensionalen Netzwerken organisieren (siehe Bild). Für Anwendungen in der regenerativen Medizin könnte interessant sein, dass ein mildes Chelatisierungsmittel die Netze rasch wieder auflösen kann. Humane Endothelzellen ließen sich in dem Collagenpeptid-Netzwerk bereitwillig einschließen und kultivieren. [source]

Alterations in Taxol Production in Plant Cell Culture via Manipulation of the Phenylalanine Ammonia Lyase Pathway

BIOTECHNOLOGY PROGRESS, Issue 6 2002
Michelle C. Brincat
One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of l -phenylalanine to trans -cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40,50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor ,-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas l -,-aminooxy-,-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 ,M but having no effect at 10 ,M. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production. [source]

Utilization of an Alternative Carbon Source for Efficient Production of Human ,1 -Antitrypsin by Genetically Engineered Rice Cell Culture

BIOTECHNOLOGY PROGRESS, Issue 3 2001
Masaaki Terashima
Human ,1 -antitrypsin was produced by genetically engineered rice cells using promoter and signal peptide of a rice ,-amylase isozyme. Batch and continuous cultures were employed to investigate the effects of alternative carbon sources on the ,1 -antitrypsin production. While this expression system is inducible by sugar depletion, we have found that the productivity of ,1 -antitrypsin increased 2.4- to 3.4-fold, compared with the control medium without carbon source, in medium containing an alternative carbon source, such as pyruvic acid and glyoxylic acid. The accumulated ,1 -antitrypsin in the medium containing pyruvic acid reached 18.2,24.2 mg/g-dry cell in 50,70 h by batch culture. [source]

Osmoprotective Effect of Glycine Betaine on Thrombopoietin Production in Hyperosmotic Chinese Hamster Ovary Cell Culture: Clonal Variations

BIOTECHNOLOGY PROGRESS, Issue 5 2000
Tae Kyung Kim
When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (qTPO) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced qTPO. However, the relative decrease in qTPO varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production. [source]

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

JOURNAL OF PHYTOPATHOLOGY, Issue 10 2001
E. Ku
Generation of O2, and H2O2 as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid , a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O2, production. The H2O2 level was not significantly affected. In toxin-treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid-induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro- and anti-oxidant systems are involved in response to fusaric acid treatment although differential response of H2O2 and its metabolism-related enzymes between the whole leaf and cell culture assays was found. [source]

UVB Irradiation of Normal Human Skin Favors the Development of Type-2 T-cells In Vivo and in Primary Dermal Cell Cultures,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2002
Sergio Di Nuzzo
ABSTRACT To determine the effect of UVB exposure on the balance of type-1 or type-2 T-cells in skin, we examined the expression of key markers interferon (IFN)-, and interleukin (IL)-4 in cryostat sections. IFN-, mRNA was clearly detectable in nonirradiated control skin, and IFN-, protein was found in 2% of the dermal CD3pos T-cells, whereas IL-4 mRNA was hardly detectable, and no IL-4 protein was found. In contrast, IL-4 mRNA expression increased upon irradiation, and IL-4 was found in 2% of the T-cells at day 2 after UVB-exposure. Concomitantly, IFN-, mRNA expression decreased, and IFN-, protein became absent. We also analyzed T-cells present in primary dermal cell cultures, which were used as an in vitro equivalent of the in vivo situation. As compared with T-cells from control skin, T-cells in dermal cell cultures from UVB-exposed skin displayed an increased IL-4 and decreased IFN-, expression. No such skewing occurred when the T-cells from irradiated skin were cloned in the absence of a dermal microenvironment. Except for an occasional positive T-cell, type-1,associated cell-surface markers (CCR5, CXCR3) or type-2 markers (CCR3, CD30, CRTH2) were undetectable in situ. But these markers were expressed on cultured dermal T-cells from UVB-exposed and control skin at a comparable level, but did not correlate with the IFN-, and IL-4 production. Altogether, UVB-induced changes of the dermal microenvironment favor the development of type-2 T-cells. [source]

Defined Protein-Free NS0 Myeloma Cell Cultures: Stimulation of Proliferation by Conditioned Medium Factors

BIOTECHNOLOGY PROGRESS, Issue 1 2005
Erika Spens
A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, ,-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 × 106 cells mL,1 in this medium. The antibody yield was 195 mg L,1 in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 × 106 cells mL,1, had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20,25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes. [source]

Growth Behavior in Plant Cell Cultures Based on Emissions Detected by a Multisensor Array

BIOTECHNOLOGY PROGRESS, Issue 4 2004
Palle Komaraiah
The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line. [source]

Bioreactor Production of Human ,1 -Antitrypsin Using Metabolically Regulated Plant Cell Cultures

BIOTECHNOLOGY PROGRESS, Issue 3 2002
Melody M. Trexler
Transgenic rice cell cultures, capable of producing recombinant human ,1 -antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (,max) observed from two bioreactor runs were 0.40 day,1 (doubling time of 1.7 days) and 0.47 day,1 (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O2/(g dw h). Using a metabolically regulated rice ,-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots. [source]

Influence of Fungal Elicitors on Production of Ajmalicine by Cell Cultures of Catharanthus roseus

BIOTECHNOLOGY PROGRESS, Issue 1 2002
Ajay Namdeo
Suspension cultures of Catharanthus roseus ( C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A.niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T.viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A.niger, F. moniliforme, and T. viride. The maximum ajmalicine production (75 ,g g,1 dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 ,g g,1 DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 ,g g,1 DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 ,g g,1 DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis. [source]

Ammonia Removal Using Hepatoma Cells in Mammalian Cell Cultures

BIOTECHNOLOGY PROGRESS, Issue 5 2000
Yeon Sook Choi
It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2,3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells. [source]

ChemInform Abstract: Synthesis of Styrenes Through the Biocatalytic Decarboxylation of trans-Cinnamic Acids by Plant Cell Cultures.

CHEMINFORM, Issue 46 2001
Masumi Takemoto
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Imprinting Status of G,S, NESP55, and XL,s in Cell Cultures Derived from Human Embryonic Germ Cells: GNAS Imprinting in Human Embryonic Germ Cells

CLINICAL AND TRANSLATIONAL SCIENCE, Issue 5 2009
Janet L. Crane M.D.
Abstract GNAS is a complex gene that through use of alternative first exons encodes signaling proteins G,s and XL,s plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of G,s, NESP55, and XL,s prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of G,s was biallelic (maternal allele: 52.6%± 2.5%; paternal allele: 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%± 10%; paternal allele: 18.1%± 10%; p= 0.002) and XL,s was preferentially expressed from the paternal allele (maternal allele: 2.7%± 0.3%; paternal allele: 97.3%± 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5,11 weeks postfertilization, and that imprinting of XL,s occurs very early postfertilization. By contrast, mprinting of G,s most likely occurs after 11 weeks postfertilization and after tissue differentiation. [source]

Cell culture,produced hepatitis C virus does not infect peripheral blood mononuclear cells,

HEPATOLOGY, Issue 6 2008
Svetlana Marukian
Hepatitis C virus (HCV) replicates primarily in the liver, but HCV RNA has been observed in association with other tissues and cells including B and T lymphocytes, monocytes, and dendritic cells. We have taken advantage of a recently described, robust system that fully recapitulates HCV entry, replication and virus production in vitro to re-examine the issue of HCV infection of blood cell subsets. The HCV replicase inhibitor 2,C-methyl adenosine was used to distinguish HCV RNA replication from RNA persistence. Whereas cell culture,grown HCV replicated in Huh-7.5 hepatoma cells, no HCV replication was detected in B or T lymphocytes, monocytes, macrophages, or dendritic cells from healthy donors. No blood cell subset tested expressed significant levels of Claudin-1, a tight junction protein needed for HCV infection of Huh-7.5 cells. A B cell line expressing high levels of Claudin-1, CD81, and scavenger receptor BI remained resistant to HCV pseudoparticle infection. We bypassed the block in HCV entry by transfecting HCV RNA into blood cell subsets. Transfected RNA was not detectably translated and induced high levels of interferon-,. Supernatants from HCV RNA,transfected macrophages inhibited HCV replication in Huh-7.5 cells. Conclusion: We conclude that multiple blocks prevent blood cells from supporting HCV infection. (HEPATOLOGY 2008;48:1843-1850.) [source]

Cancer stem cells: Cell culture, markers, and targets for new therapies

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009
Candace A. Gilbert
Abstract A cancer stem cell (CSC) is defined as an undifferentiated cell with the ability to self-renew, differentiate to multiple lineages and initiate tumors that mimic the parent tumor. In this review, we focus on glioblastomas, describing recent progress and problems in characterizing these cells. There have been advances in CSC culture, but tumor cell heterogeneity has made purification of CSCs difficult. Indeed, it may be that CSCs significantly vary from tumor to tumor. We also discuss the proposal that CSCs are resistant to radiotherapy and chemotherapy and play a major role in repopulating tumors following treatment. To overcome their resistance to conventional therapies, we may be able to use our extensive knowledge of the signaling pathways essential for stem cells during development. These pathways have potential as targets for new glioblastoma therapies. Hence, although there is an ongoing debate on the nature of CSCs, the theory continues to suggest new ideas for both the lab and the clinic. J. Cell. Biochem. 108: 1031,1038, 2009. © 2009 Wiley-Liss, Inc. [source]

Self-hardening calcium phosphate composite scaffold for bone tissue engineering,

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2004
Hockin H. K. Xu
Abstract Calcium phosphate cement (CPC) sets in situ to form solid hydroxyapatite, can conform to complex cavity shapes without machining, has excellent osteoconductivity, and is able to be resorbed and replaced by new bone. Therefore, CPC is promising for craniofacial and orthopaedic repairs. However, its low strength and lack of macroporosity limit its use. This study investigated CPC reinforcement with absorbable fibers, the effects of fiber volume fraction on mechanical properties and macroporosity, and the cytotoxicity of CPC,fiber composite. The rationale was that large-diameter absorbable fibers would initially strengthen the CPC graft, then dissolve to form long cylindrical macropores for colonization by osteoblasts. Flexural strength, work-of-fracture (toughness), and elastic modulus were measured vs. fiber volume fraction from 0% (CPC Control without fibers) to 60%. Cell culture was performed with osteoblast-like cells, and cell viability was quantified using an enzymatic assay. Flexural strength (mean ± SD; n == 6) of CPC with 60% fibers was 13.5 ± 4.4 MPa, three times higher than 3.9 ± 0.5 MPa of CPC Control. Work-of-fracture was increased by 182 times. Long cylindrical macropores 293 ± 46 ,m in diameter were created in CPC after fiber dissolution, and the CPC,fiber scaffold reached a macroporosity of 55% and a total porosity of 81%. The new CPC,fiber formulation supported cell adhesion, proliferation and viability. The method of using large-diameter absorbable fibers in bone graft for mechanical properties and formation of long cylindrical macropores for bone ingrowth may be applicable to other tissue engineering materials. Published by Elsevier Ltd. on behalf of Orthopuedic Research Society. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source]

The minotaur proteome: Avoiding cross-species identifications deriving from bovine serum in cell culture models

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010
Jakob Bunkenborg
Abstract Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5,15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented. [source]

Biotransformation of n -hexadecane by cell suspension cultures of Cinchona robusta and Dioscorea composita

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2001
Carolina Vega-Jarquin
Abstract This manuscript evaluates the phytotoxicity and biotransformation of n -hexadecane as well as peroxidase activity and cytochrome P450 concentration in microsomes for cell suspension cultures of Cinchona robusta and Dioscorea composita. Phytotoxicity was evaluated based on viability and growth. Cell cultures were exposed to a 2 and 4% (v/v) dose of n -hexadecane. The biotransformation of n -hexadecane was determined based on labeled recovery in polar, nonpolar, and cell residue fractions after cell culture extraction during exponential cell growth phase and stationary phase. Differences were observed in accumulation of label during cell growth phase and stationary phase for the cells of the two plants. Differences also were observed between phases for label in polar and nonpolar fractions. Thin-layer chromatography determined labeled intermediates and some were identified. The activity of peroxidase and concentration of cytochrome P450 was lower in C. robusta than in controls and greater in D. composita than in controls. In vitro biotransformation was not successful. [source]

TGF-,1 alone and in combination with calcium hydroxide is synergistic to TGF-,1 production by osteoblasts in vitro

INTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2000
A. Jaunberzins
Abstract Aim To examine the effects of calcium hydroxide (Ca(OH)2), transforming growth factor-beta (TGF-,1), and Ca(OH)2/TGF-,1 coadministration on TGF-,1 and interleukin-6 (IL-6) synthesis by early (subculture 1) and late (subculture 5) osteoblast cultures. Methodology Early and late cultures were established using bone cells harvested from 21-day-old fetal rat calvaria. Cell cultures of both early and late osteoblasts were divided into four groups: group 1, control; group 2, cells challenged with Ca(OH)2; group 3, cells challenged with TGF-,1; and group 4, cells challenged with Ca(OH)2 and TGF-,1 in combination. TGF-,1 and IL-6 levels for all groups were determined using ELISA methodology. Results anova and Tukey HS analyses revealed that osteoblasts of groups 3 and 4 significantly increased (P < 0.001) TGF-,1 synthesis in both early and late cultures of osteoblasts. IL-6 was not detected in any of the groups considered in this study. Conclusions Exogenous TGF-,1 has an autocrine effect on cell cultures of osteoblasts. Administration of TGF-,1 alone or in combination with Ca(OH)2 increases the synthesis of TGF-,1 in osteoblast cultures. Ca(OH)2 and TGF-,1 are compatible when placed in a culture of osteoblasts. Ca(OH)2 provides a favourable environment for the anabolic effects of TGF-,1. [source]

Expression of phospholipase C beta family isoenzymes in C2C12 myoblasts during terminal differentiation,

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004
Irene Faenza
In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLC,) family in muscle differentiation, given that nuclear PLC,1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLC,1, ,2, ,3, ,4 have been assessed. As further characterization, we investigated the localization of PLC, isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLC,4 was the most expressed isoform in the cytoplasm, whereas PLC,1 and ,3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLC,1 isoform was expressed at the highest extent. A marked decrease of PLC,4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLC,3 at both cytoplasmatic and nuclear level, whilst PLC,2 expression was almost undetactable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLC,1 and the decrease of cytoplasmatic PLC,4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLC,s. © 2004 Wiley-Liss, Inc. [source]

Elastin in oral connective tissue modulates the keratinization of overlying epithelium

JOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2010
Po-Chen Hsieh
Hsieh P-C, Jin Y-T, Chang C-W, Huang C-C, Liao S-C, Yuan K. Elastin in oral connective tissue modulates the keratinization of overlying epithelium. J Clin Periodontol 2010; 37: 705-711 doi: 10.1111/j.1600-051X.2010.01542.x Abstract Aim: One of the most discernable differences between keratinized and non-keratinized oral mucosas is the quantity of elastin they contain in the connective tissues. Whether elastin modulates the keratin expression of oral epithelial cells is unknown. Methods: Four specimens containing both keratinized and non-keratinized mucosas were processed for immunohistochemical (IHC) stainings for elastin and four keratins. Six keratinized and non-keratinized portions of oral mucosas were dissected and cultured on an organ culture system. Purified elastin and elastase were added separately to the media. After 14 days, the mucosas were examined for four keratin expressions. Cell cultures of keratinized and non-keratinized gingival fibroblasts were established and tested for elastin expression. Oral mucosa equivalents were then engineered and tested for keratin expression. Results: Keratinized epithelium exclusively expressed keratin-1 and -10 (K1/10), while non-keratinized epithelium expressed keratin-4 and -13 (K4/13). Only non-keratinized fibroblasts expressed elastin in cell culture. Both the native and the engineered keratinized gingiva changed phenotypes and expressed K4/13 when treated with exogenous elastin. On the contrary, the native non-keratinized mucosa started to express K1/10 when elastase eradicated inherent elastin. Conclusions: Our study demonstrated that the elastin in the oral connective tissue is important for the non-keratinized phenotypes of overlaying epithelium. [source]

Uptake of nicotine from suspension culture of Nicotiana tabacum by molecularly imprinted polymers

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2010
Mohamed Salaheldin A. Abdelkader
Abstract Objectives The aim was to use molecularly imprinted polymers (MIPs) for the selective recovery of nicotine in plant cell cultures. MIPs can selectively uptake nicotine from suspension cultures of N. tabacum, and therefore may be useful for improving levels of secondary metabolites in plant cell cultures. Methods Suspension cultures of N. tabacum were initiated from callus and maintained in liquid Murashige and Skoog (MS) media containing 3% w/v sucrose, 0.1 mg/l ,-naphthaleneacetic acid acid (NAA) and 0.25 mg/l kinetin. Tween 80 at 1% was used for permeabilisation of cell cultures. Pre-weighed XAD-2 and two types of synthesized polymers, MIPs (A and B with one and two functional monomers, respectively) and corresponding non-imprinted polymers (NIPs), A and B, were introduced aseptically into the permeabilised suspension cultures of N. tabacum, the nicotine contents of polymers were determined by gas chromatography and the adsorption yield of polymers were determined. Key findings Cell cultures of N. tabacum accumulated nicotine alkaloid intracellularly in varying levels, 6.8,14.9 mg/l fresh weight. MIPs were able to uptake 50,70% of released nicotine in suspension cultures of N. tabacum, whereas XAD-2 recovered only 30,40%. The total levels of accumulated nicotine were enhanced up to 20 mg/l by simultaneous use of Tween 80 and MIPs. Conclusions The findings indicate the potential use of MIPs to uptake nicotine from suspension cultures of N. tabacum, and increase productivity of secondary metabolites in plant cell cultures. [source]

Ethanol Effects on Nitric Oxide Production in Cerebral Pial Cultures

ALCOHOLISM, Issue 4 2001
Chin-Lung Shih
Background: Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these changes are associated with ethanol (EtOH) action on nitric oxide (NO) production in the cerebrovascular cells. The purpose of this study was to examine the effects of EtOH treatment on basal and cytokine-induced NO production in cortical pial cultures. Methods: Cell cultures for this study included murine primary pial vascular cells, primary glial cells and cortical neurons. These cells were exposed to cytokines or EtOH for 24 to 48 hr. The culture media were used for measurement of nitrite, as an indication for NO release, and lactate dehydrogenase (LDH), as an index of cell membrane integrity. In addition, immunocytochemical determinations were carried out to identify cell types and to assess inducible nitric oxide synthase (iNOS). Results: Exposure of primary pial vascular cultures to cytokines that consisted of interleukin-1, (IL-1,; 250 pg/mL) and interferon-, (IFN,; 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly elevated NO production. NO production could be attenuated by N -nitro-L-arginine (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS inhibitor. Increased iNOS immunoreactivity was observed in cytokines- or EtOH-treated pial cells. When pial cells were cocultured with cortical neurons, prolonged EtOH exposure led to a large increase in NO production as well as LDH release. However, this increase was not observed in pial culture alone or in mixed cortical culture. Nevertheless, inhibition of NO production with N-arg or AG did not alter the EtOH-induced LDH release in the pial cells cocultured with cortical neurons. Conclusion: These results show that EtOH exposure led to increased production of NO in primary pial cell culture. In mixed culture that contained cortical neurons and pial cells, EtOH induced increase in NO as well as LDH release, which is an indication of loss of cell membrane integrity. However, EtOH-mediated LDH release in mixed cortical pial cultures was not a consequence of the increase in NO production by these cells. Studies that use mixed cortical-pial cultures may provide a unique in vitro system for examining the interactions among glial cells, neurons, and cerebrovascular cells. [source]

Mitochondrial Responses of Normal and Injured Human Skin Fibroblasts Following Low Level Laser Irradiation,An In Vitro Study

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009
Innocent L. Zungu
Laser irradiation has proved to be very efficient in speeding and improving the quality of healing in pathological conditions of diverse etiologies. However, the mechanisms by which the beneficial effects are attained are not clear. Mitochondria are the primary phototargets during irradiation. The study aimed to establish if laser irradiation had an effect on hypoxic and acidotic cells. The study also aimed to use existing information regarding the possible mechanism of action (established in wounded cells) and apply these principles to acidic and hypoxic irradiated cells to determine whether laser has a stimulatory or inhibitory effect. Cell cultures were modified to simulate conditions of hypoxia (hypoxic gas mixture 95% N2 and 5% O2) and acidosis (pH 6.7) whereas the central scratch model was used to simulate a wound. Cells were irradiated with a helium,neon (632.8 nm, 3 mW cm,2) laser using 5 or 16 J cm,2 on days 1 and 4. Mitochondrial responses were measured 1 or 24 h after laser irradiation by assessing changes in mitochondrial membrane potential (MMP), cyclic AMP, intracellular Ca2+ and adenosine triphosphate (ATP) cell viability. Hypoxia and acidosis significantly reduced MMP when compared with normal nonirradiated control cells. Wounded, hypoxic and acidotic cells irradiated with 5 J cm,2 showed an increase in mitochondrial responses when compared with nonirradiated cells while 16 J cm,2 showed a significant decrease. The study confirmed that laser irradiation with 5 J cm,2 stimulated an increase in intracellular Ca2+ which resulted in an increase in MMP, ATP and cAMP, which ultimately results in photobiomodulation to restore homeostasis of injured cells. [source]

Effects of Mitomycin-C on Normal Dermal Fibroblasts,

THE LARYNGOSCOPE, Issue 4 2006
Theodore Chen MD
Abstract Objectives: To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. Study Design: In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Methods: Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-,1. Results: Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-,1. Conclusions: A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro. [source]