Showing papers on "Tissue culture published in 2007"

In vitro cell culture infectivity assay for human noroviruses.

Abstract: Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24–48 hours. Because of the lack of suitable tissue culture or animal models, the true nature of norovirus pathogenesis remains unknown. We show that noroviruses can infect and replicate in a 3-dimensional (3-D), organoid model of human small intestinal epithelium. Cells grown on porous collage-coated beads under fluid shear conditions in rotating wall vessel bioreactors differentiate into 3-D architectures resembling both the morphologic and physiologic function of in vivo tissues. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Cytopathic effect and norovirus RNA were detected at each of the 5 cell passages for genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed.

Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment

Abstract: BACKGROUND: Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation. METHODS: Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen–thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy. RESULTS: No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage. CONCLUSIONS: Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.

History of plant tissue culture

Abstract: Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the begining of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter.

Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin?

Abstract: Benzyladenine (BA) is the most widely used cytokinin in the micropropagation industry due to its effectiveness and affordability. It, however, has disadvantages such as genetic alteration and abnormal growth in some plants. Naturally occurring zeatin on the other hand is not as widely used as BA and is far more expensive. The use of meta-topolin and its derivatives as alternatives to BA and zeatin, both of which frequently have negative effects in tissue culture was investigated. In vitro grown Aloe polyphylla (an endangered medicinal and ornamental aloe) were cultured on full strength Murashige and Skoog basal medium with different concentrations of cytokinins and solidified with 1% Bacteriological Agar (Oxoid No. 1). mT was the preferred cytokinin both in terms of multiplication rate and rooting. The optimum concentration that induced regeneration and rooting is 5.0 μM. The problem of hyperhydricity was totally controlled. Plants rooted spontaneously in multiplication medium, thus avoiding the extra rooting step of the protocol. More than 91% of the plants transferred to ex vitro conditions were successfully acclimatized.

Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture.

Abstract: Objective: The aim of the present study was to investigate whether Ga-As laser irradiation can enhance adenosine triphosphate (ATP) production in normal human neural progenitor (NHNP) cells in culture. Methods: NHNP were grown in tissue culture and were treated by Ga-As laser (808 nm, 50 mW/cm2, 0.05 J/cm2), and ATP was determined at 10 min after laser application. Results: The quantity of ATP in laser-treated cells was 7513 ± 970 units, which was significantly higher (p < 0.05) than the non-treated cells, which comprised 3808 ± 539 ATP units. Conclusion: Laser application to NHNP cells significantly increases ATP production in these cells. These findings may explain the beneficial effects of low-level laser therapy (LLLT) in stroked rats. Tissue culture of NHNP cells might offer a good model to study the mechanisms associated with promotion of ATP production in the nervous system by LLLT.

Angiotensin-(1-7) Inhibits Growth of Human Lung Adenocarcinoma Xenografts in Nude Mice through a Reduction in Cyclooxygenase-2

Abstract: Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((1-7)) receptor. The current study investigates the effect of Ang-(1-7) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(1-7), delivered by implanted osmotic mini-pumps. Treatment with Ang-(1-7) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(1-7)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(1-7) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-1 mRNA in xenograft tumors or A549 cells. Because Ang-(1-7), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((1-7)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.

Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro

Abstract: Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.

Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

Abstract: It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, β-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering. Copyright © 2007 John Wiley & Sons, Ltd.

Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.).

Abstract: Background When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.

Gene expression by fibroblasts seeded on small intestinal submucosa and subjected to cyclic stretching.

Abstract: Extracellular matrix scaffolds derived from porcine small intestinal submucosa (SIS-ECM) have been shown to promote the formation of site-specific tissue in a number of preclinical animal studies. However, this constructive remodeling process requires that the scaffold be subjected to a site-specific mechanical environment. The specific quantitative effects of mechanical loading on the gene expression patterns of fibroblasts seeded on SIS-ECM are unknown and yet very important in the tissue remodeling process. The objective of the present study was to evaluate the expression of collagen type I (Col I), collagen type III (Col III), smooth muscle actin (SMA), tenascin-C (TN-C), matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-beta1), and transforming growth factor-beta3 (TGF-beta3) by fibroblasts subjected to various magnitudes (0%, 5%, 10%, and 15%) and frequencies (0.1 Hz, 0.3 Hz, and 0.5 Hz) of stretch. A new cyclic-stretching tissue culture (CSTC) system was developed. This system consists of eight independently controlled culture chambers that can be operated in a sterile incubator. Each chamber includes a load cell so that the load in each scaffold can be monitored. It was found that different stretching regimens led to complex and distinctive patterns of gene expression by fibroblasts seeded onto SIS-ECM. In general, the fibroblasts increased expression of Col I up to 5-fold and decreased that of Col III with increased frequency of stretch. In addition, the fibroblasts exhibited a contractile phenotype with increased expression of SMA, TN-C, and TGF-beta1. These findings support the concept that the mechanical environment of a remodeling ECM scaffold may have substantial effects on the behavior of cells within the scaffold and contribute to the site-specific tissue remodeling that has been observed in in vivo studies.

Gene Expression Profile of Metastatic Human Pancreatic Cancer Cells Depends on the Organ Microenvironment

Abstract: To determine the influence of the microenvironment on changes in gene expression, we did microarray analysis on three variant lines of a human pancreatic cancer (FG, L3.3, and L3.6pl) with different metastatic potentials. The variant lines were grown in tissue culture in the subcutis (ectopic) or pancreas (orthotopic) of nude mice. Compared with tissue culture, the number of genes of which the expression was affected by the microenvironment was up-regulated in tumors growing in the subcutis and pancreas. In addition, highly metastatic L3.6pl cells growing in the pancreas expressed significantly higher levels of 226 genes than did the L3.3 or FG variant cells. Growth of the variant lines in the subcutis did not yield similar results, indicating that the orthotopic microenvironment significantly influences gene expression in pancreatic cancer cells. These data suggest that investigations of the functional consequence of gene expression require accounting for experimental growth conditions.

Engineered liver-like tissue on a capillarized matrix for applied research.

Abstract: Liver tissue that is functional and viable for several weeks in vitro represents an auspicious test system for basic and applied research. In this study, a coculture system for hepatocytes (HCs) and microvascular endothelial cells (mECs) was generated applying tissue-engineering techniques, establishing the basis for a new bioartificial liver in vitro model. Porcine mECs were seeded on a decellularized porcine jejunal segment with preserved vascular structures. Porcine HCs were seeded onto this vascularized scaffold, and the resulting coculture was maintained for 3 weeks in vitro. Tissue morphology and differentiation was monitored using histology and immunohistochemistry. Tissue metabolism was monitored using daily assessment of urea and lactate production. HC monolayer cultures served as controls. The 2-stage seeding procedure resulted in a 3-dimensional coculture system harboring HC cell clusters in multiple cell layers lining the generated mEC-seeded capillary structures. It was viable for 3 weeks, and HCs maintained their morphology and differentiation. Biochemical testing revealed stable metabolic activity of the tissue culture. In contrast, HCs cultured in monolayer showed morphological dedifferentiation and an unfavorable metabolic state. Our mEC-HC coculture represents a new approach toward a functional bioartificial liver-like tissue applicable as a test system for basic and applied research.

Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion).

Abstract: The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine.

Herpes simplex virus type 1 infection induces oxidative stress and the release of bioactive lipid peroxidation by-products in mouse P19N neural cell cultures

Abstract: To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1 Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (pi) and ROS levels remained elevated at 24 h pi This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h pi, but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h pi HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system

Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro–regenerated plants and seedlings

Abstract: An efficient protocol has been developed for high-frequency shoot regeneration and plant establishment of Clitoria ternatea - a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), kinetin, α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxy acetic acid (2,4-D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μ m BA and 2.0 μ m NAA. Organogenic calli were produced on a medium containing 2,4-D (10 or 20 μ m) and BA (5.0 μ m). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5-10 μ m BA and 2.0 μ m NAA. The microshoots were rooted on half-strength MS medium supplemented with 5.0 μ m indole-3-butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro-regenerated and in vivo-grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).

Effects of flavonoid on calcium content in femoral tissue culture and parathyroid hormone-stimulated osteoclastogenesis in bone marrow culture in vitro.

Abstract: The effect of various flavonoids, which are present in food and plants, on bone calcium content and osteoclastogenesis were investigated to compare action of flavonoid on bone formation and bone resorption in vitro Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with antibiotics and bovine serum albumin Amoung quercetin, myricetin, kaempferol, isorhamnetin, curcumin, hesperidin, or astaxanthin in the range of 10−7–10−5 M, culture with quercetin (10−6 or 10−5 M) caused a significant increase in diaphyseal calcium content Such an effect was not seen in other compounds Mouse bone marrow cells were cultured for 7 days in the presence of parathyroid hormone (PTH; 10−7 M), a bone-resorbing factor, in vitro Culture with PTH caused a significant increase in osteoclast-like cell formation This increase was significantly inhibited in the presence of quercetin, myricetin, kaempferol, isorhamnetin, or curcumin in the range of 10−8–10−6 M Such an effect was not seen in the case of hesperidin or astaxanthin In addition, culture with PTH (10−7 M) caused a significant decrease in diaphyseal calcium content This decrease was completely prevented in the presence of quercetin, myricetin, kaempferal, or isorhamnetin of 10−6 M This study demonstrates that various flavonoids have a potent inhibitory effect on osteoclastogenesis and bone resorption rather than bone formation in vitro Among various flavonoids, quercetin had a stimulatory effect on bone formation and an inhibitory effect on bone resorption in vitro

Novel intra-tissue perfusion system for culturing thick liver tissue.

Abstract: Innovative scaffold fabrication, angiogenesis promotion, and dynamic tissue culture techniques have been utilized to improve delivery of media into the core of large tissue constructs in tissue engineering. We have developed here an intra-tissue perfusion (ITP) system, which incorporates an array of seven micron-sized needles as a delivery conduit, to improve mass transfer into the core of thick liver tissues slices (>>300 μm mass transport limit). The ITP system improves the uniformity and distribution of media throughout the tissue, resulting in improved cell viability over the static-cultured controls. The ITP-cultured thick liver slices also exhibit improved phase I and phase II metabolic functions and albumin and urea synthetic functions after 3-day culture, which is the minimal period required by the U.S. Food and Drug Administration (FDA) for studying drug–drug interaction. This ITP system can also be used for culturing other thick tissue constructs of larger dimensions for various in vitro and in ...

Cell Culture Models of Biological Barriers: In vitro Test Systems for Drug Absorption and Delivery

Abstract: General Aspects of Epithelial Cell and Tissue Culture. Basic Aspects of Cell Growth and Cell Cycle in Culture. Cell Culture Media: Selection and Standardization. Bioelectrical Characterization of Epithelial Cell (Mono)Layers and Tissues. Characterization of Transport Over Epithelial Barriers. Studying Cellular Binding and Uptake of Bioadhesive Lectins. High-throughput Epithelial Cell Culture Systems for Screening Drug Intestinal Permeability. Good Cell Culture Practice (GCCP). Regulatory Acceptance of in vitro Test Systems as an Alternative to Safety Testing in Animals. Regulatory Acceptance of in vitro Permeability Studies in the Context of the Biopharmaceutics Classification System. Models of Specific Epithelial and Endothelial Barriers Relevant to Drug Delivery. Caco-2 Cell Monolayers as a Model for Studies of Drug Transport Across Human Intestinal Epithelium. Transport Studies Using Intestinal Tissue ex vivo. Models of Alveolar Epithelium. Bronchial Epithelial Cell Cultures. In vitro Methodologies to Study Nasal Delivery Using Excised Mucosa. Cell Culture Models of the Corneal and Conjunctival Epithelium. Cell Cultures of the Retinal Pigment Epithelium to Model the Blood-retina Barrier for Retinal Drug and Gene Delivery. Human Skin and Skin Equivalents to Study Dermal Penetration and Permeation. In vitro Models of the Human Buccal Epithelium: the TR146 Cell Culture Model and the Porcine in vitro Model. Drug Transport Across the Blood-brain Barrier: A Molecular and Functional Perspective. BeWo Cells: An in vitro System Representing The Blood-placental Barrier. Emerging Tools for Studying Biological Barriers and Drug Transport. Predicting Drug Absorption by Computational Methods. Confocal and Two-photon Fluorescence Microscopy. On the Application of Scanning Force Microscopy in Cell Biology. Fluorescence Correlation Spectroscopy.

Plasma polymer coated surfaces for serum-free culture of limbal epithelium for ocular surface disease.

Abstract: The potential use of plasma polymer coatings as substrates for serum-free expansion of limbal epithelial cells was investigated. Preliminary studies using a human corneal epithelial cell line showed that acrylic acid-coated surfaces performed better than allyl amine and allyl alcohol coated surfaces in terms of cell metabolic activity and confluence as assessed using the MTT assay. Subsequently, the proliferation and maturity of primary human limbal epithelial cells in co-culture with growth arrested 3T3 fibroblasts on a range of acrylic acid plasma coated surfaces, octadiene plasma coated surfaces and tissue culture plastic was investigated using MTT and cytokeratin 3 immunostaining. The cells performed better in the presence of serum on all surfaces. However, the acrylic acid coated surfaces successfully sustained a serum-free fibroblast/epithelial cell co-culture. The metabolic activity of the epithelial cells was superior on the acrylic acid coated surfaces than on tissue culture plastic in serum-free conditions and their levels of differentiation were not significantly higher than in the presence of serum. These results suggest that these surfaces can be used successfully for the serum-free expansion of human limbal epithelial cells.

Effectiveness of auxin induced in vitro root culture in chicory

Abstract: An efficient protocol has been developed for the root culture of (Cichorium intybus L. cv. Focus), the leaf and hypocotyl explants from 25 days old in vitro raised seedlings were cultured on half-strength Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), α-Napthalenacetic acid (NAA). 0.5 mg/l NAA and 0.1 mg/l IBA induced highest percentage of rooting from matured leaf explants, under total dark condition. After three weeks well established roots were separated. Fresh root tissue, in amount of 0.5 was subcultured in half-strength MS liquid medium supplemented with 0.2 mg/l NAA and 0.5 mg/l IBA, under continuous agitation at 110 rpm and total dark condition. The biomass of root culture was increased to 5.820 g after 6 weeks of culture. The root culture was maintained up to the 8 weeks.

Highly efficient gas permeable devices and methods for culturing cells

Abstract: This invention relates to methods and devices that improve cell culture efficiency. They include the use of gas permeable culture compartments that reduce the use of space while maintaining uniform culture conditions, and are more suitable for automated liquid handling. They include the integration of gas permeable materials into the traditional multiple shelf format to resolve the problem of non-uniform culture conditions. They include culture devices that use surfaces comprised of gas permeable, plasma charged silicone and can integrate traditional attachment surfaces, such as those comprised of traditional tissue culture treated polystyrene. They include culture devices that integrate gas permeable, liquid permeable membranes. A variety of benefits accrue, including more optimal culture conditions during scale up and more efficient use of inventory space, incubator space, and disposal space. Furthermore, labor and contamination risk are reduced.

Plant regeneration through callus initiation from mature embryo of Triticum

Abstract: The behaviour of diverse Triticum genotypes in the tissue culture response of mature embryo callus was compared, and factors affecting tissue culture response were studied in this paper. Significant differences were detected in callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency when mature embryos of 31 plants of different Triticum species were compared. These were the main wheat cultivars of the Chinese northern winter-type wheat region and breeding lines (Triticum aestivum L.), durum wheat (Triticum durum Desf.), cultivable emmer wheat (Triticum dicoccum Schuble) and the common wheat progenitors Triticum dicoccoides and Triticum aegilopides. The genotype dependency was particularly high in tissue culture of mature embryos of these Triticum genotypes. The efficiency of induction, differentiation and regeneration of mature embryos callus was high in genotypes selected out. Mature embryo-derived callus of HB341, TS021, SN2618, T. dicoccum, HB188, and T9817 showed better tissue culture response than the other genotypes. Plantlets can be regenerated from mature embryo-derived callus of 31 genotypes, saving on growth facility resources and time required for the collection of other explants, and providing a solid basis for the genetic transformation and molecular plant breeding of Triticum plants.

Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression.

Abstract: During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.

Agronomic performance of locally adapted sweet potato ( Ipomoea batatas (L) Lam.) cultivars derived from tissue culture regenerated plants

Abstract: Tissue culture techniques have opened a new frontier in agricultural science by addressing food security and agricultural production issues. A study was conducted to compare growth and yield characteristics between the tissue culture regenerated and conventionally propagated sweet potato cultivars. Five locally adapted sweet potato cultivars Mugande, SPK004, Kemb10, Japon tresmesino and Zapallo were regenerated in vitro by the methods of indirect and direct embryogenesis and grown under field conditions in a RCBD replicated three times. Significant (P between the test cultivars and regeneration method for the growth and yield variables. The highest tuber numbers and marketable yield was recorded with Zapallo. Conventional propagation method gave highest growth rates however the difference in yield between the conventional propagation and tissue culture regenerated plants did not vary significantly (P ELISA established that field plants had a higher virus titre compared to the tissue culture regenerated plants.