Showing papers on "Tissue culture published in 2009"

Monolayer and Spheroid Culture of Human Liver Hepatocellular Carcinoma Cell Line Cells Demonstrate Distinct Global Gene Expression Patterns and Functional Phenotypes

Abstract: Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.

Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: Enhanced development of mineralized bone-like tissue in culture

Abstract: Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.

Henrietta Lacks, HeLa Cells, and Cell Culture Contamination

Abstract: Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.

Generation and transplantation of an autologous vascularized bioartificial human tissue.

Abstract: Background The lack of transplant vascularization forecloses the generation and clinical implementation of bioartificial tissues. We developed techniques to generate a bioartificial human tissue with an innate vascularization. The tissue was implanted clinically as proof of concept to evaluate vascular network thrombogenicity and tissue viability after transplantation. Methods A porcine small bowl segment was decellularized in a two-step procedure, preserving its vascular structures. The extracellular matrix was characterized quantitatively for DNA residues and protein composition. The vascular remainings were reseeded with human endothelial cells in a dynamic tissue culture. The engineered tissue was characterized by (1) histology, (2) immune-histology, (3) life-dead assay, and (4) metabolic activity. To evaluate the tissue capabilities, it was implanted clinically and recovered after 1 week. Results Tissue preparation with sodium desoxycholate monohydrate solution resulted in an incomplete decellularization. Cell residues were removed by additional tissue incubation with DNAse. The human endothelial cells formed a viable endothelium inside the primarily porcine extracellular matrix, expressing CD31, Flk-1, and vascular endothelium-cadherin. The metabolic activity of the bioartificial tissue increased continuously over time in vitro. Clinical tissue transplantation confirmed vessel patency and tissue viability for 1 week. Conclusions The feasibility to bioengineer a human tissue with an innate vascularization has been shown in vitro and the clinical setting. These results may open the door for the clinical application of various sophisticated bioartificial tissue substitutes and organ replacements.

Characterization of Matrigel interfaces during defined human embryonic stem cell culture

Abstract: Differences in attachment, proliferation, and differentiation were measured for human embryonic stem (hES) cells cultured on various substrata coated with Matrigeltm, a blend of extracellular matrix proteins derived from murine tumor cells. The authors observed that hES cells attach and grow poorly on Matrigel adsorbed onto polystyrene, while they proliferate when exposed to Matrigel adsorbed onto glass or oxygen plasma treated polystyrene (e.g., “tissue culture” treated polystyrene). Furthermore, hES cells grown on the Matrigel-coated tissue culture polystyrene are less likely to differentiate than those grown on the Matrigel-coated glass. To assess the mechanism for these observations, they replicated the cell culture interface in a quartz crystal microbalance with dissipation monitoring. In addition, they used ellipsometry and scanning electron microscopy to determine the thickness and topography of Matrigel on the varying surfaces. Matrigel formed a viscoelastic multilayer with similar thickness on al...

In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

Abstract: Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).

Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis.

Abstract: Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

Review: Seaweed tissue culture as applied to biotechnology: Problems, achievements and prospects

Abstract: SUMMARY Advances have been made in cell and tissue culture of seaweeds to define a unique branch of in vitro techniques; however, they are lagging far behind those of land plants and have limited applications. Explants can be cultivated axenically in enriched or artificial seawater culture media, and regeneration and even callus formation are achieved. In this state of the art technique, seaweed tissue culture may be already useful for certain biotechnological applications, such as clonal propagation of seed material for mariculture. Nevertheless, the absolute control of growth and development as it is exerted in higher plant tissue culture is lacking, and it is required for more complex biotechnological applications in seaweeds. Definitively, we need appropriate cells (competent cells) to induce growth with the most effective chemical regulators in culture medium adjusted towards the addition of carbon sources. Still, free cells and protoplast isolation and regeneration in marine seaweeds constitute the most developed topic in seaweed tissue culture. The regulation of growth and development of seaweed free cell and protoplast cultures may sustain a purposeful use of techniques in the era of genomic applications.

Plant Cell and Tissue Culture - A Tool in Biotechnology: Basics and Application

Abstract: 1 Introduction 2 Historical Developments of Cell and Tissue Culture Techniques 3 Callus Cultures 3.1 Establishment of a Primary Culture from Explants of the Secondary Phloem of the Carrot Root 3.2 Fermenter Cultures 3.3 Immobilized Cell Cultures 3.4 Nutrient Media 3.5 Evaluation of Experiments 3.6 Maintenance of Strains, Cryopreservation 3.7 Some Physiological, Biochemical, and Histological Aspects 4 Cell Suspension Cultures 4.1 Methods to Establish a Cell Suspension 4.2 Cell Population Dynamics 5 Protoplast Cultures 5.1 Production of Protoplasts 5.2 Protoplast Fusion 6 Haploid Techniques 6.1 Application Possibilities 6.2 Physiological and Histological Background 6.3 Methods for Practical Application 6.4 Haploid Plants 7 Plant Propagation-Meristem Cultures, Somatic Embryogenesis 7.1 General Remarks, and Meristem Cultures 7.2 Protocols of Some Propagation Systems 7.2.1 In Vitro Propagation of Cymbidium 7.2.2 Meristem Cultures of Raspberries 7.2.3 In Vitro Propagation of Anthurium 7.3 Somatic Embryogenesis 7.3.1 Basics of Somatic Embryogenesis 7.3.2 Ontogenesis of Competent Cells 7.3.3 Genetic Aspects-DNA Organization 7.3.4 The Phytohormone System 7.3.5 The Protein System 7.3.6 Cell Cycle Studies 7.4 Practical Application of Somatic Embryogenesis 7.5 Artificial Seeds 7.6 Embryo Rescue 8 Some Endogenous and Exogenous Factors in Cell Culture Systems 8.1 Endogenous Factors 8.1.1 Genetic Influences 8.1.2 Physiological Status of 'Mother Tissue' 8.1.3 Growth Conditions of the 'Mother Plant' 8.2 Exogenous Factors 8.2.1 Growth Regulators 8.2.2 Nutritional Factors 8.2.2.1 Improvement of Nutrient Uptake by Transgenic Carrot Cultures 8.3 Physical Factors 9 Primary Metabolism 9.1 Carbon Metabolism 9.2Nitrogen Metabolism 10 Secondary Metabolism 10.1 Introduction 10.2 Mechanism of Production of Secondary Metabolites 10.3 Historical Background 10.4 Plant Cell Cultures and Pharmaceuticals, and Other Biologically Active Compounds 10.4.1 Antitumor Compounds 10.4.2 Anthocyanin Production 10.5 Strategies for Improvement of Metabolite Production 10.5.1 Addition of Precursors, and Biotransformations 10.5.2 Immobilization of Cells 10.5.3 Differentiation and Secondary Metabolite Production 10.5.4 Elicitation 10.5.4.1 Jasmonic Acid 10.5.4.2 Effect of UV on Production of Secondary Metabolites in Cultured Tissues 10.6 Organ Cultures 10.6.1 Shoot Cultures 10.6.2 Root Cultures 10.7 Genetic Engineering of Secondary Metabolites 10.8 Membrane Transport and Accumulation of Secondary Metabolites 10.9 Bioreactors 10.9.1 Technical Aspects of Bioreactor Systems 10.10 Prospects 11 Phytohormones and Growth Regulators 12 Cell Division, Cell Growth, Cell Differentiation 13 Genetic Problems and Gene Technology 13.1 Somaclonal Variations 13.1.1 Ploidy Stability 13.1.2 Some More Somaclonal Variations 13.2 Gene Technology 13.2.1 Transformation Techniques 13.2.1.1 Direct Gene Transfer 13.2.1.2 Agrobacterium-Mediated Gene Transformation 13.2.2 Selectable Marker Genes 13.2.2.1 Reporter Genes 13.2.2.2 Variants of GFP 13.2.3 ss-Glucuronidase (GUS) 13.2.3.1 Procedures for Assay of GUS Gene Expression 13.2.4 Antibiotics Resistance Genes 13.2.5 Elimination of Marker Genes 13.2.5.1 Cre-lox Recombination-Based Systems 13.2.5.2 Ac/Ds System 13.2.5.3 Double Cassette System 13.2.6 Agrobacterium-Mediated Transformation in Dicotyledonous Plants 13.2.6.1 Transgenic Carrot: Potential Source of Edible Vaccines 13.2.6.2 Cell Culture and Transformation Procedures 13.2.6.3 Uses of Transgenes to Increase Host Plant

Factors contributing to in vitro shoot-tip necrosis and their physiological interactions

Abstract: Plant tissue culture plays an important role in the production and conservation of plant species. Its application, however, is hindered by some growth abnormalities such as shoot-tip necrosis (STN) caused by the culture conditions. This review article summarizes the literature published on the causes of in vitro STN in plants such as medium type, plant growth regulators, calcium, boron, medium additives, the culture environment, their interaction and physiological effects.

Tissue culture as a plant production technique for horticultural crops

Abstract: Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture. Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments of plant tissues. At the peak of the plant tissue culture era in the 1980s, in a relatively short time, many commercial laboratories were established around the world to capitalize on the potential of micropropagation for mass production of clonal plants for the horticulture industry. Today plant tissue culture applications encompass much more than clonal propagation. The range of routine technologies has expanded to include somatic embryogenesis, somatic hybridization, virus elimination as well as the application of bioreactors to mass propagation. Perhaps the greatest value of these tissue culture technologies lies not so much in their application to mass clonal propagation but rather in their role underpinning developments and applications in plant improvement, molecular biology and bioprocessing, as well as being a basic research tool. Plant tissue culture technique though an underutilized tool in Nigeria, it can be extensively applied in horticulture to increase crop production. This paper highlights some of the applications of plant tissue culture to horticulture, the achievements and limitations of tissue culture and some insights into current and possible future developments. With rapid population growth, the total acreages of fruits, vegetables and various ornamental plants have not been able to meet the needs of people in the developing countries.

DAS181 Inhibits H5N1 Influenza Virus Infection of Human Lung Tissues

Abstract: DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both α2-6-linked and α2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1.

Tissue culture as a plant production technique for horticultural crops

Abstract: Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments of plant tissues At the peak of the plant tissue culture era in the 1980s, in a relatively short time, many commercial laboratories were established around the world to capitalize on the potential of micropropagation for mass production of clonal plants for the horticulture industry Today plant tissue culture applications encompass much more than clonal propagation The range of routine technologies has expanded to include somatic embryogenesis, somatic hybridization, virus elimination as well as the application of bioreactors to mass propagation Perhaps the greatest value of these tissue culture technologies lies not so much in their application to mass clonal propagation but rather in their role underpinning developments and applications in plant improvement, molecular biology and bioprocessing, as well as being a basic research tool Plant tissue culture technique though an underutilized tool in Nigeria, it can be extensively applied in horticulture to increase crop production This paper highlights some of the applications of plant tissue culture to horticulture, the achievements and limitations of tissue culture and some insights into current and possible future developments With rapid population growth, the total acreages of fruits, vegetables and various ornamental plants have not been able to meet the needs of people in the developing countries Key words: Bioprocessing, clone, micropropagation, somatic embryogenesis, tissue culture

Lettuce line PS06519080

Abstract: The invention provides seed and plants of the lettuce line designated PS06519080. The invention thus relates to the plants, seeds and tissue cultures of lettuce line PS06519080, and to methods for producing a lettuce plant produced by crossing a plant of lettuce line PS06519080 with itself or with another lettuce plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of lettuce line PS06519080, including the gametes of such plants.

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method.

Abstract: Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid-gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ-induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor-derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin-containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.

Development of an in vitro culture method for cells and tissues from the zebra mussel (Dreissena polymorpha).

Abstract: Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.

Plant Cell and Tissue Culture - A Tool in Biotechnology

Abstract: 1 Introduction 2 Historical Developments of Cell and Tissue Culture Techniques 3 Callus Cultures 3.1 Establishment of a Primary Culture from Explants of the Secondary Phloem of the Carrot Root 3.2 Fermenter Cultures 3.3 Immobilized Cell Cultures 3.4 Nutrient Media 3.5 Evaluation of Experiments 3.6 Maintenance of Strains, Cryopreservation 3.7 Some Physiological, Biochemical, and Histological Aspects 4 Cell Suspension Cultures 4.1 Methods to Establish a Cell Suspension 4.2 Cell Population Dynamics 5 Protoplast Cultures 5.1 Production of Protoplasts 5.2 Protoplast Fusion 6 Haploid Techniques 6.1 Application Possibilities 6.2 Physiological and Histological Background 6.3 Methods for Practical Application 6.4 Haploid Plants 7 Plant Propagation-Meristem Cultures, Somatic Embryogenesis 7.1 General Remarks, and Meristem Cultures 7.2 Protocols of Some Propagation Systems 7.2.1 In Vitro Propagation of Cymbidium 7.2.2 Meristem Cultures of Raspberries 7.2.3 In Vitro Propagation of Anthurium 7.3 Somatic Embryogenesis 7.3.1 Basics of Somatic Embryogenesis 7.3.2 Ontogenesis of Competent Cells 7.3.3 Genetic Aspects-DNA Organization 7.3.4 The Phytohormone System 7.3.5 The Protein System 7.3.6 Cell Cycle Studies 7.4 Practical Application of Somatic Embryogenesis 7.5 Artificial Seeds 7.6 Embryo Rescue 8 Some Endogenous and Exogenous Factors in Cell Culture Systems 8.1 Endogenous Factors 8.1.1 Genetic Influences 8.1.2 Physiological Status of 'Mother Tissue' 8.1.3 Growth Conditions of the 'Mother Plant' 8.2 Exogenous Factors 8.2.1 Growth Regulators 8.2.2 Nutritional Factors 8.2.2.1 Improvement of Nutrient Uptake by Transgenic Carrot Cultures 8.3 Physical Factors 9 Primary Metabolism 9.1 Carbon Metabolism 9.2Nitrogen Metabolism 10 Secondary Metabolism 10.1 Introduction 10.2 Mechanism of Production of Secondary Metabolites 10.3 Historical Background 10.4 Plant Cell Cultures and Pharmaceuticals, and Other Biologically Active Compounds 10.4.1 Antitumor Compounds 10.4.2 Anthocyanin Production 10.5 Strategies for Improvement of Metabolite Production 10.5.1 Addition of Precursors, and Biotransformations 10.5.2 Immobilization of Cells 10.5.3 Differentiation and Secondary Metabolite Production 10.5.4 Elicitation 10.5.4.1 Jasmonic Acid 10.5.4.2 Effect of UV on Production of Secondary Metabolites in Cultured Tissues 10.6 Organ Cultures 10.6.1 Shoot Cultures 10.6.2 Root Cultures 10.7 Genetic Engineering of Secondary Metabolites 10.8 Membrane Transport and Accumulation of Secondary Metabolites 10.9 Bioreactors 10.9.1 Technical Aspects of Bioreactor Systems 10.10 Prospects 11 Phytohormones and Growth Regulators 12 Cell Division, Cell Growth, Cell Differentiation 13 Genetic Problems and Gene Technology 13.1 Somaclonal Variations 13.1.1 Ploidy Stability 13.1.2 Some More Somaclonal Variations 13.2 Gene Technology 13.2.1 Transformation Techniques 13.2.1.1 Direct Gene Transfer 13.2.1.2 Agrobacterium-Mediated Gene Transformation 13.2.2 Selectable Marker Genes 13.2.2.1 Reporter Genes 13.2.2.2 Variants of GFP 13.2.3 ss-Glucuronidase (GUS) 13.2.3.1 Procedures for Assay of GUS Gene Expression 13.2.4 Antibiotics Resistance Genes 13.2.5 Elimination of Marker Genes 13.2.5.1 Cre-lox Recombination-Based Systems 13.2.5.2 Ac/Ds System 13.2.5.3 Double Cassette System 13.2.6 Agrobacterium-Mediated Transformation in Dicotyledonous Plants 13.2.6.1 Transgenic Carrot: Potential Source of Edible Vaccines 13.2.6.2 Cell Culture and Transformation Procedures 13.2.6.3 Uses of Transgenes to Increase Host Plant

Enterohemorrhagic Escherichia coli O157∶H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia

Abstract: Background The pathogenesis of enterohemorrhagic Escherichia coli (EHEC) O157∶H7 infection is attributed to virulence factors encoded on multiple pathogenicity islands. Previous studies have shown that EHEC O157∶H7 modulates host cell signal transduction cascades, independent of toxins and rearrangement of the cytoskeleton. However, the virulence factors and mechanisms responsible for EHEC-mediated subversion of signal transduction remain to be determined. Therefore, the purpose of this study was to first identify differentially regulated genes in response to EHEC O157∶H7 grown in the presence of epithelial cells, compared to growth in the absence of epithelial cells (that is, growth in minimal essential tissue culture medium alone, minimal essential tissue culture medium in the presence of 5% CO2, and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction.

Antileukemia activity from root cultures of Vernonia amygdalina.

Abstract: Vernonia amygdalina, Del (Compositae) is an African medicinal plant well known for producing the anticancer agents’ vernodaline and vernolide. It grows wild under severe anthropogenic and environmental pressures. The roots are the principle material for herbal medicine. It is collected from the wild there is great variability in the quality and effectiveness of the root extracts. It is necessary to establish a fast-growing root culture and to test the extracts of cultured roots for activity against leukemia cells in vitro. Leaves were cultured on half-strength MS medium supplemented with different auxin types and concentrations. Basal medium supplemented with indole-3-butyric acid (IBA) at 2.0 mg/l favored induction of the highest number of roots/explant (38.3 ± 1.1). After six weeks well-established roots were separated. About 100 mg of fresh root tissue was cultured in 80 ml full-strength MS liquid medium supplemented with 2.0 mg/l IBA and under continuous agitation (80 rpm). The biomass of root cultures increases by 21 fold after 5 weeks of culture. Cold water, hot water and ethanol extracts from the in vitro cultured roots were prepared and tested for their antioxidant activity and efficacy against leukemia cells. All of the extracts showed significant antioxidant activity. All the extracts could kill the majority (50-75%) of abnormal cells among primary cells harvested from 3 patients with acute lymphoblastic leukemia (ALL) and 3 with acute myeloid leukemia (AML). DNA fragmentation patterns were detected within treated cells and inferred targeted cell death by apoptosis. The metabolites within the extracts may act as tumor inhibitors that promote apoptosis. Therefore in vitro root culture can be an alternative to collection from the wild, cultivation in the field or to chemical synthesis of anticancer agents. In addition the plant extracts may be used to supplement or replace established drugs treatments. Key words: Anticancer, natural products, plant extracts

Osteoblasts interact with MTA surfaces and express Runx2

Abstract: Objective Periradicular healing involves osteoblasts that are dependent on the Runt-related transcription factor 2 (Runx2). The purpose of this study was to determine if mineral trioxide aggregate (MTA) root-end filling materials support Runx2 expression in osteoblasts. Study design Human alveolar bone cells were grown on alternative formulations of MTA. Cell-surface interactions were visualized by scanning electron microscopy. Gene expression was examined by reverse-transcription polymerase chain reaction and Western blot analysis. Results Cells attached to and spread out on MTA surfaces within 24 hours and formed a collagenous matrix overlay within 1 week of growth. Runx2 expression increased from low levels in the 24-hour cultures to an abundance during 2 weeks of growth and differentiation on MTA surfaces and on tissue culture plastic controls. The cells responded similarly to ProRoot, Tooth-Colored MTA, and MTA mixed with local anesthetic solution. Conclusion Mineral trioxide aggregate materials support cell attachment and Runx2 expression in osteoblasts.

Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies

Abstract: For a more individualized therapeutic approach we explored a protease-free method to culture primary cells from breast cancer biopsies. Tumor tissue from breast cancer patients after surgery was cultured ex vivo without enzymatic digestion for more than one year and revealed the continuous outgrowth of adherent and proliferating primary cell populations. Immunofluorescence staining of these human breast cancer-derived epithelial cells (HBCEC) and quantification by flow cytometry revealed nearly exclusively cytokeratin-expressing cells. Analysis of surface markers during long term tumor culture of primary HBCEC (more than 476d) demonstrated a prominent expression of CD24, CD44 and MUC1 (CD227). According to aging markers, expression of senescence-associated β-galactosidase revealed little if any positive staining in a primary tumor-derived HBCEC population after 722d in culture, whereas the majority of normal human mammary epithelial cells (HMEC) demonstrated senescent cells already after a culture period of 32d. In this context, HBCEC populations derived from a tumor culture after 152d and 308d, respectively, exhibited a significant telomerase activity, suggesting continuous proliferative capacity. Treatment with several chemotherapeutic compounds and their combinations revealed distinct cytotoxic effects among HBCEC from different breast cancer patients, indicating an individualized response of these tumor-derived primary cells. The protease-free outgrowth of primary HBCEC offers a patient-specific approach to optimize an individually-designed cancer therapy. Moreover, HBCEC from long term breast tumor tissue cultures resemble tumor cell-like properties by an intact ECM formation and a stable cell surface protein expression providing a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumor samples.

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

Abstract: 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.

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions.

Abstract: Object The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O2 concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change. Methods The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. Results Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly...