Initial Stages Of Culture Research Articles

Alterations in Soil Bacterial Community and Its Assembly Process within Paddy Field Induced by Integrated Rice–Giant River Prawn (Macrobrachium rosenbergii) Farming

Integrated rice–aquatic animal farming has become a vital strategy for enhancing food security. To assess the effects of integrated rice–giant river prawn (Macrobrachium rosenbergii) farming (IRPF) on agricultural ecosystems, we used 16S rRNA gene sequencing to analyze soil bacterial communities in comparison with traditional rice monoculture (RM). Our study revealed that the IRPF did not significantly affect the diversity of the soil bacterial community. However, during the initial culture stage, IRPF markedly increased the relative abundance of the phylum candidate division NC10 and the genus Candidatus Methylomirabilis, enhancing nitrogen-cycling-related functions within the bacterial community. Additionally, IRPF reduced the complexity and stability of these communities in the early to middle culture stages. While stochastic processes usually dominate the assembly of these communities, IRPF restricted bacterial migration and reduced the influence of these stochastic processes. Furthermore, IRPF had a significant impact on environmental factors within paddy soils, strongly correlating with shifts in bacterial communities, particularly through variations in soil nitrite concentration. In conclusion, the influence of IRPF on the bacterial community in paddy soils was primarily observed during the early and middle culture stages, and the impacts of IRPF on the soil bacterial community were primarily driven by environmental changes, especially soil nitrite concentration. These findings provide theoretical insights and a reference for understanding the microbiological impacts of different integrated rice–fish farming systems on agricultural ecosystems.

Mineralization Characteristics of Soil Organic Carbon and Its Relationship with Organic Carbon Components in Artificial Robinia pseudoacacia Forest in Loess Hilly Region

In order to explore the characteristics of organic carbon mineralization and the variation law of organic carbon components of an artificial forest in a loess hilly area, an artificial Robinia pseudoacacia forest restored for 13 years and the adjacent slope farmland were selected as the research objects, and indoor culture experiments under three different temperature treatments (15, 25, and 35℃) were carried out. The results indicated that the mineralization rate of soil organic carbon decreased sharply at first and then stabilized. The cumulative release of organic carbon increased rapidly in the initial stage of culture and gradually slowed in the later stage. Soil organic carbon mineralization in sloping farmland was more sensitive to temperature change, and its temperature sensitivity coefficient Q10 was 1.52, whereas that in R. pseudoacacia forest land was only 1.38. According to the fitting of the single reservoir first-order dynamic equation, the soil mineralization potential Cp of R. pseudoacacia forest land and slope farmland was between 2.02-4.32 g·kg-1 and 1.25-3.17 g·kg-1, respectively, that is, the mineralization potential of the R. pseudoacacia forest was higher. During the cultivation period, the content of various active organic carbon components decreased with time, and that in the R. pseudoacacia forest land was greater than that in the slope land. The cumulative carbon release of soil was significantly positively correlated with the contents of MBC and DOC (P<0.05), and Q10 (15-25℃) was negatively correlated with the contents of SOC, EOC, and SWC (P<0.05). These results could provide some reference for the study of soil carbon sequestration in loess hilly regions under climate change.

Combination of simultaneous saccharification and fermentation of corn stover with consolidated bioprocessing of cassava starch enhances lipid production by the amylolytic oleaginous yeast Lipomyces starkeyi

Highly integrated processes are crucial for the commercial success of microbial lipid production from low-cost substrates. Here, combination of simultaneous saccharification and fermentation (SSF) of corn stover with consolidated bioprocessing (CBP) of cassava starch by Lipomyces starkeyi was firstly developed as a novel strategy for lipid production. Starch was quickly hydrolyzed within 24 h by the amylolytic enzymes secreted by L. starkeyi to provide adequate fermentable sugars at the initial stage of culture, which eliminated the pre-hydrolysis step. More interestingly, synergistic effect for achieving higher lipid production by combined utilization of corn stover and cassava starch at relatively low enzyme dosage was realized, in comparison with the separate utilization of these two substrates. The fatty acid profiles indicated that lipid prepared by the combination strategy was suitable precursor for biodiesel production. The combined SSF&CBP strategy offers a simplified, highly-efficient, and economical route for co-valorization of low-cost substrates into lipids.

Establishment and characterization of a liver cell line from black porgy, Acanthopagrus schlegelii

In this study, a liver cell line (ASL) derived from black porgy (Acanthopagrus schlegelii) was established and characterised. After the initial primary culture, the cell line has been cultured for over 40 passages at 28 °C in Dulbecco's modified Eagle's medium (DMEM) with 15 % foetal bovine serum (FBS). Immunostaining results indicated that ASL cells contained at least two cytotypes in the initial culture stage; however, epithelioid cells dominated and replaced the other type during passage 6–20. ASL cells grow in temperature range 20–28 °C in DMEM containing 15–20 % FBS. Cytogenetic analysis showed that the modal chromosome number in ASL cells was 48. Partial amplification of the cytochrome c oxidase subunit I gene indicates that ASL originates from black porgy. ASL cells showed a transfection efficiency of approximately 15 % after transfection with the pEGFP-N3 plasmid. Cytopathic effects were observed in ASL cells after red-spotted grouper nervous necrosis virus (RGNNV) infection, and viral replication in RGNNV-infected ASL cells was confirmed using quantitative reverse transcription polymerase chain reaction, virus titre assays, western blot, and transmission electron microscopy assays. Furthermore, several antiviral genes were induced to varying degrees in RGNNV-infected ASL cells, indicating that the immune response was activated in ASL cells post RGNNV infection. In conclusion, the newly established ASL cell line is an ideal in vitro tool for studying NNV-black porgy interactions.

Prolonged Post-Polymerization Biocompatibility of Polymethylmethacrylate-Tri-n-Butylborane (PMMA-TBB) Bone Cement.

Polymethylmethacrylate (PMMA)-based acrylic bone cement is commonly used to fix bone and metallic implants in orthopedic procedures. The polymerization initiator tri-n-butylborane (TBB) has been reported to significantly reduce the cytotoxicity of PMMA-based bone cement compared to benzoyl peroxide (BPO). However, it is unknown whether this benefit is temporary or long-lasting, which is important to establish given that bone cement is expected to remain in situ permanently. Here, we compared the biocompatibility of PMMA-TBB and PMMA-BPO bone cements over several days. Rat femur-derived osteoblasts were seeded onto two commercially-available PMMA-BPO bone cements and experimental PMMA-TBB polymerized for one day, three days, or seven days. Significantly more cells attached to PMMA-TBB bone cement during the initial stages of culture than on both PMMA-BPO cements, regardless of the age of the materials. Proliferative activity and differentiation markers including alkaline phosphatase production, calcium deposition, and osteogenic gene expression were consistently and considerably higher in cells grown on PMMA-TBB than on PMMA-BPO, regardless of cement age. Although osteoblastic phenotypes were more favorable on older specimens for all three cement types, biocompatibility increased between three-day-old and seven-day-old PMMA-BPO specimens, and between one-day-old and three-day-old PMMA-TBB specimens. PMMA-BPO materials produced more free radicals than PMMA-TBB regardless of the age of the material. These data suggest that PMMA-TBB maintains superior biocompatibility over PMMA-BPO bone cements over prolonged periods of at least seven days post-polymerization. This superior biocompatibility can be ascribed to both low baseline cytotoxicity and a further rapid reduction in cytotoxicity, representing a new biological advantage of PMMA-TBB as a novel bone cement material.

The study of selective primary culture and determination of a breast cancer cell line in vitro*

Abstract Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach. In this experiment, we used primary culture to select and detect breast cancer cells in vitro, which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank. Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital. Cells were isolated and used to establish successful cell culture. Cell activity and cell purity were measured before liquid nitrogen cryopreservation. Results (1) At the initial culture stage, cells grew with adherence. Cell multiplication could be seen after the cell medium was exchanged three times. Cell viability was above 86%, while the viability of the target cells was above 75%, as detected by hematoxylin and eosin (HE) staining. (2) The number of breast cancer cells decreased, while the number of fibroblasts increased after five rounds of passage. (3) The success rate was 73.08%, which did not include polluted cells and those that were not successfully cryopreserved. Conclusion (1) breast cancer cells could be selected from primary culture in vitro through an appropriate method. (2) Exchange of the cell medium and further cell passage improved cell multiplication. (3) The experimental results could be monitored using trypan blue and HE staining. (4) The success of breast cancer cell culture in vitro could be used as a reference for other cell culture, so as to establish a tumor tissue cell bank.

Early responses of in vitro cultured fish‐growing glochidia: The effects of taurine, lipids and sera onMargaritifera margaritifera

In vitro culture has great potential for the propagation of freshwater mussels in both commercial and conservation aquaculture. The use of in vitro techniques precludes the need for host fish, thus decreasing costs and increasing efficiency. However, protocols are still lacking for many species that grow substantially during the parasitic stage. In this study, we tested the effects of taurine addition, serum type and source of lipids on the survival rate and increase in length of Margaritifera margaritifera larvae during the initial stage of culture (first 11 days of exposure to media). Our results show that taurine has no significant effect on the early survival rate of glochidia; however, the possible importance of this amino acid in subsequent stages is discussed. The use of an emulsified lipid mixture instead of traditional fish oil showed significantly higher rates of survival. Finally, the addition of serum showed variable effects, with both horse serum and newborn calf serum having higher survival in trials using mussel populations from different sources. These findings can contribute to the invention or improvement of in vitro protocols for species growing during infection and at the same time show the potential that early survival assessments could have for the development of in vitro methods in species with long parasitic stages.

Variations in soil organic carbon decompositions of different land use patterns on the tableland of Loess Plateau.

Land use patterns are one of the critical factors affecting soil carbon sequestration or decomposition and greenhouse gas emissions. The accurate evaluation of its change is particularly crucial for the carbon cycle of the terrestrial ecosystem and global climate change. This paper examined the dynamic soil respiration by means of the indoor airtight culture method and static box-gas chromatography under the land uses of the farmland, natural grassland, shrub forestland, arbor and shrub mixed forestland, and arbor forestland on the tableland of Loess Plateau. The results showed that soil organic carbon mineralization and soil respiration rate were influenced by the land use type. (1) The content and rate of soil mineralizable carbon in grassland were significantly higher than that in forestland and farmland, and that in forestland significantly higher than that in the farmland (P < 0.05). The trend of organic carbon mineralization was rapid in the initial stage of culture (by the rate of 30.02~238.56mgkg-1h-1 in the first 0.5h) and slowed down in the later stage (by the rate of 1.07~1.95mgkg-1h-1 in 1575h). In the soil of grassland, the accumulation of mineralizable organic carbon in the 0-5-cm layer was 1.20~1.64 times that in the soil of forestland and 1.82 times that of farmland. Compared with the 0-5-cm soil layer, there were decreases in the 5-20-cm soil layer under all land uses, with a decline of up to 48% in farmland. (2) The soil carbon mineralization potential of different land use types ranged from 0.81 to 2.70mgkg-1, that of grassland was significantly higher than that of farmland (P < 0.05), and the soil organic carbon decomposition rate constant (k) under shrub forestland was significantly higher than that under other land use types (P < 0.05). The highest organic carbon mineralization capacity and the lowest sequestration were in the soil of farmland, while that in grassland had the lowest organic carbon mineralization capacity and the highest sequestration. (3) The cumulative amount of soil respiration was highest under the natural grassland, followed by the shrub forestland, and lowest under the farmland. The soil respiration rate was positively correlated with soil temperature, and its correlation with soil water content was positive in the wet season (May through October) and negative in the dry season (November through April in the following year). (4) The soil respiration rate showed obvious seasonal differences. After the conversion of farmland to forestland, it would be rising in the content of soil organic carbon and labile organic carbon, and be going down in the temperature sensitivity (Q10). In summary, by converting farmland to forestland or grassland, the sequestration of soil organic carbon had greatly been enhanced, and the possibility of soil greenhouse gas emissions had reduced on the tableland of Loess Plateau.

Nacre formation by epithelial cell cultures from mantle of the black-lip pearl oyster, Pinctada margaritifera.

Mantle tissue from the black-lip pearl oyster, Pinctada margaritifera, was cultured in vitro using sterilized seawater supplemented with 0.1% yeast extract as the culture medium. Granular and agranular epithelial cells, hyalinocytes, and fibroblast-like cells were observed in the initial stages of culture. Epithelial cells later formed pseudopodial cell networks containing clusters of granulated cells, which upon maturation released their colored granules. These granules induced formation of nacre crystal deposits on the bottom of the culture plate. Cultures comprised of only granulated epithelial cells were established through periodic sub-culturing of mantle cells and maintained for over 18mo in a viable condition. Reverse transcriptase PCR of cultured cells demonstrated gene expression of the shell matrix protein, nacrein. To further evaluate the functional ability of cultured granulated epithelial cells, nuclear shell beads were incubated in culture medium containing these cells to induce nacre formation on the beads. Observation of the bead surface under a stereomicroscope at periodic intervals showed the gradual formation of blackish yellow colored nacre deposits. Examination of the bead surface by scanning electron microscopy and energy dispersive X-ray analysis at periodic intervals revealed a distinct brick and mortar formation characteristic of nacre, comprised of aragonite platelets and matrix proteins. Calcium, carbon, and oxygen were the major elements in all stages examined. Our study shows that mantle epithelial cells in culture retain the ability to secrete nacre and can therefore form the basis for future studies on the biomineralization process and its application in development of sustainable pearl culture.

Radar chart to visualize connectivity of neuronal network on planner type MEA

Event Abstract Back to Event Radar chart to visualize connectivity of neuronal network on planner type MEA Gu-Haeng Lee1 and Yoonkey Nam1* 1 College of Engineering, KAIST, Department of Bio and Brain Engineering, Republic of Korea 1. Motivation Many researchers use connectivity analysis in neuronal network research. In particular, when studying plasticity, it is necessary to analyze the connectivity of consecutive data. In this case, many visualization methods are used. Since MEAs can simultaneously measure signals from multiple neurons, they are suitable for neuronal network studies as compared to studies using patch clamps. Methods for studying connectivity using MEA data are cross-correlation analysis of two specific channels, using cross-correlation matrix and dendrogram, and drawing connectivity map. However, existing connectivity maps are difficult to identify overall connectivity due to too many connection lines if all neurons in the network are strongly connected. [1] Therefore, in this study, we studied how to see the connection trend more easily. 2. Material and Methods For the measurement, a 60-channel MEA from MCS with a 30 um diameter titanium oxide electrodes and a 200 um space was used. E18 SD rat hippocampal neurons were randomly cultured on MEA. Spike data were measured from 8 DIV to 30 DIV for one hour daily. All analysis was done using Matlab. After cross-correlation analysis, the connection direction was identified through peak timing and the connection strength was defined using normalized peak amplitude. The connection score of one channel had a direction and size of 360 degrees. The size of the score was determined by the connection strength and distance between channels. The direction and range of the score were determined according to the direction and distance between the channels. To see the direction and distance of the connection at a glance, if the two channels were close to each other, the score had a smaller value in a wider range, and in a far distance, the score had a narrower range in a larger value. The obtained connection score was drawn as a radar chart at the position corresponding to each channel. In order to show the distance of connectivity, the distribution of scores was shown as uniform, triangle, and Gaussian distribution. 3. Result & Discussion Even in the case of data with a maximum of 47 active channels, the connectivity of all channels can be seen without overlapping. At the initial stage of culture, even though there was an active channel, the spike number was not enough to confirm the connectivity. After two weeks of culture, connectivity began to appear. As the time passes, the connection strength increases. After 3 weeks, the active channel was reduced, but connectivity was still present. Even if spike recording data has thousands of channels, it will be able to see the connectivity at a glance by normalizing the score. Through the shape of the radar chart, we can see that the direction and size of connectivity change in a short time. It can be considered that the visualization method using the radar chart can be used for the study of plasticity. 4. Conclusion Rader chart was used to visualize the connectivity of neuronal networks cultured in MEA. Even fully developed neuronal networks with very complex connectivity were able to see connectivity at a glance using this method. Acknowledgements This work was supported by National Research Foundation grant(NRF-2015K1A3A1A14021018) funded by Korean government.

Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stemborer, Chilo partellus (Swinhoe)

Sweet sorghum is a climate smart crop with multiple uses. The crop is susceptible to attack by the spotted stemborer, Chilo partellus (Swinhoe). This causes deadheart formation, leading to lodging of plants and consequent high economic losses. Lack of stable sources of resistance make any genetic enhancement through breeding difficult. We report a study to build up host plant resistance using transgenic technology by introducing two different classes of Bt genes (cry1Aa and cry1B) into two elite sweet sorghum genotypes of India (SSV84 and RSSV9). We devised tissue culture methods to suit the genotypes of our interest, SSV84 and RSSV9, and employed two methods of genetic transformation: the particle bombardment and in planta method of Agrobacterium. Modification of in vitro culture methods involved subculture every 3 days in the initial stages of culture and the use of precultured embryos as target tissues. For the in planta method, a floral dip for 1 h in Agrobacterium suspension supplemented with l-cysteine and Tween-20 was used. Sixteen transgenic events were generated; inheritance, integration and stable expression of the transgenes till the T4 generation were confirmed. The amount of Bt Cry1Aa protein at 25–30 days of growth ranged from 24.8 to 72.8 ng/g of fresh leaf tissue. We recorded 78.4 % larval mortality, reduced leaf damage (3.0 out of 9.0) and reduced feeding (41.0 %) over the controls in insect feed assays. Stable inheritance and expression in the in planta-derived transgenics are presented.

A comparative study of three different methods of shoot meristem excision for induction of embryogenic calli in coconut

&lt;p&gt;&lt;strong&gt;A protocol was standardized to maximize yields of embryogenic calli from shoot meristem culture of coconut. Three different shoot meristem excision methods were tested &lt;em&gt;viz&lt;/em&gt;., excision of shoot meristem aseptically from &lt;em&gt;in vitro&lt;/em&gt; germinated embryo after 10-12 days, excision of shoot meristem from &lt;em&gt;in vitro&lt;/em&gt; germinated embryo subjected to GA3 treatment for five days and excision of shoot meristem from fresh embryo. The primary calli induction after 30 days of culture incubation for the three treatments were 21%, 27% and 79% respectively. Further, the primary calli formed from the shoot meristem excised from fresh embryo gave rise to 56% of embryogenic calli. The calli obtained from the shoot meristem which were excised from &lt;em&gt;in vitro&lt;/em&gt; germinated embryo formed less percentage of embryogenic calli because of the presence of cotyledonary tissues which inhibited the multiplication of meristematic tissues. In the case of shoot meristem extracted from GA3-treated embryos, the percentage of non-embryogenic calli was more compared to the shoot meristem excised from fresh embryo. It was observed that the addition of GA3 in the initial stages of culture inhibited the formation of embryogenic calli and favored direct shoot development. Currently, the shoot meristem excised from fresh embryo is being employed for scaling up the planting material production from released varieties of coconut.&lt;/strong&gt;&lt;/p&gt;

The influence of particle size and static magnetic fields on the uptake of magnetic nanoparticles into three dimensional cell-seeded collagen gel cultures.

Over recent decades there has been and continues to be major advances in the imaging, diagnosis and potential treatment of medical conditions, by the use of magnetic nanoparticles. However, to date the majority of cell delivery studies employ a traditional 2D monolayer culture. This article aims to determine the ability of various sized magnetic nanoparticles to penetrate and travel through a cell seeded collagen gel model, in the presence or absence of a magnetic field. Three different sized (100, 200, and 500 nm) nanoparticles were employed in the study. The results showed cell viability was unaffected by the presence of nanoparticles over a 24-h test period. The initial uptake of the 100 nm nanoparticle into the collagen gel structure was superior compared to the larger sized nanoparticles under the influence of a magnetic field and incubated for 24 h. Interestingly, it was the 200 nm nanoparticles, which proved to penetrate the gel furthest, under the influence of a magnetic field, during the initial culture stage after 1-h incubation.

Detection, isolation, and preliminary characterization of bacteria contaminating plant tissue cultures

In order to limit the contamination problem in plant tissue cultures experiments on selection of media suitable for detection and isolation of bacteria contaminating plant tissue explants, and preliminary characterization of isolates were made. In the first experiment aiming at detection of bacteria in plant explants four strains representing genera most often occurring at our survey of plant tissue cultures, and earlier isolated and identified (&lt;em&gt;Bacillus, Methylobacterium&lt;/em&gt;, &lt;em&gt;Pseudomonas &lt;/em&gt;and &lt;em&gt;Xanthomonas&lt;/em&gt;) were streaked on five bacteriological media (NA, King B, K, R2A and 523) and on the medium used for plant culture initiation – ½ MS with milk albumin (IM). All strains grew on all media but on K and IM at the slowest rate and on 523 medium at the fastest. The IM medium proved to be useful for immediate bacteria detection at the initial stage of culture. In the second experiment, aiming at characterization of isolates on the basis of colony growth and morphology 14 strains (&lt;em&gt;Agrobacterium&lt;/em&gt;, &lt;em&gt;Bacillus&lt;/em&gt;, &lt;em&gt;Curtobacterium, Flavobacterium&lt;/em&gt;, &lt;em&gt;Lactobacillus&lt;/em&gt;, &lt;em&gt;Methylobacterium &lt;/em&gt;– 2 strains &lt;em&gt;Mycobacterium&lt;/em&gt;, &lt;em&gt;Paenibacillus&lt;/em&gt;, &lt;em&gt;Plantibacterium&lt;/em&gt;, &lt;em&gt;Pseudomonas, Stenotrophomonas&lt;/em&gt;, &lt;em&gt;Xanthomonas, &lt;/em&gt;and species &lt;em&gt;Serratia marcescens&lt;/em&gt;) were streaked on five microbiological media: KB, NBY, YDC, YNA and YPGA&lt;em&gt;. &lt;/em&gt;All strains grew on all those media but at different rates. The only exception was the strain of &lt;em&gt;Lactobacillus &lt;/em&gt;spp., which did not grow on King B medium. This medium allowed the detection of such characteristic traits as fluorescence (&lt;em&gt;Pseudomonas&lt;/em&gt;) and secretion of inclusions (&lt;em&gt;Stenotrophomonas&lt;/em&gt;). The third experiment was focussed on assessment of the sensitivity of detection of specific bacteria in pure cultures and in plant tis- sue cultures using standard PCR and BIO-PCR techniques with genus specific primers and 2 methods of DNA isolation. Results showed that the use of Genomic Mini kit enabled an increase of the sensitivity by 100 times as compared to extraction of DNA by boiling. Moreover, the application of BIO-PCR increased sensitivity of detection from 102 to 105 times over the standard PCR. If looking for unknown cultivable bacteria more effective detection seems to be use of microbiological method enabling detection on bacteriological media single cells in the fragments of explants or in wash liquids, in which fragmented explants were shaken.

HE-cadherin–Fc fusion protein coated surface enhances the adhesion and proliferation of human mesenchymal stem cells

A fusion protein consisting of human E-cadherin extracellular domain and the immunoglobulin G Fc region (hE-cadherin–Fc) was prepared and used as a cell–cell adhesion biomimicking matrix for the in vitro expansion of human mesenchymal stem cells (hMSCs) for use in regenerative medicine. The hE-cadherin–Fc was stably immobilized onto a polystyrene plate due to the hydrophobicity of the Fc domain, enhancing the surface wettability and topography of the plate. The hE-cadherin–Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs compared with the tissue culture-treated plate (TC-PS) and the gelatin-coated plate. Furthermore, the expanded hMSCs on the hE-cadherin–Fc were positive for CD105, similar to those from the gelatin. Additionally, the expression of E-cadherin and β-catenin in the hMSCs was improved on the hE-cadherin–Fc matrix, suggesting that the interactions of the hE-cadherin–Fc matrix with the hMSCs were substitutes for the cell–cell adhesion junctions during the initial culture stage in the absence of intercellular interactions. The hE-cadherin–Fc was shown to be a promising artificial ECM for the in vitro expansion of hMSCs.

Culture surface influence on T-cell phenotype and function

When dealing with T lymphocyte culture there is currently very less information available about the interaction between T-cells and the culture system. In this study we look at the influence of the culture chamber on T-cell proliferation in two main aspects of the culture system, namely: culture chamber material and geometry. The study was carried out using unique polymeric closed cell culture inserts, which were processed via injection moulding from polystyrene (PS), polycarbonate (PC), polyetherurethane (PEU), polystyrene-co-acrylonitrile (PSAN) and polyetherimide (PEI). Furthermore culture chamber geometry was studied using commercially available 24, 12 and 6-well plates prepared from tissue culture plastic (TCP). For T lymphocyte stimulation two methods were used involving either EBV peptide pools or MACS iBead particles depending on the experiment performed. Culture was done with 1645 RPMI medium supplemented with foetal calf serum, penicillin, streptomycin and rhIL-2. We found four materials out of five we tested (PS, PC, PSAN and PEI) exhibited similar fold expansions with minimal influence on proportions of CD4 and CD8, while PEU had a negative influence on T cell growth along with adversely affected CD4/CD8 proportions. Changes in the geometry of TCP had no effect on T cell growth or maturation rather the size of geometry seems to have more influence on proliferation. T-cells appear to prefer smaller geometries during initial stages of culture while towards the end of the culture size becomes less significant to cell proliferation. The parameters tested in this study have significant influences on T-cell growth and are necessary to consider when designing and constructing expansion systems for antigen specific T lymphocytes. This is important when culturing T-cells for immunotherapeutic applications where antigen specificity, T-cell maturation and function should remain unaffected during culture.

PHENOTYPIC CONVERSION OF RALSTONIA SOLANACEARUM IN WATER EXTRACT OF SOLANUM TOXICARIUM

Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt in numerous plant species worldwide. In soil and culture media, the pathogen can undergo spontaneous phenotypic conversion (PC) from a pathogenic to a non-pathogenic form. This PC occurs also in Solanum plants and their homogenates. Little is known about the PC mechanism so that, to elucidate it, the culture conditions that influence bacterial PC were investigated using water extract of Solanum toxicarium. The pathogen was incubated in static culture in the water extract at various initial bacterial concentrations and incubation temperatures. PC did not occur during the exponential phase, but the pathogen underwent PC at the 4-5th day after reaching the level of 108 CFU ml-1. However, PC occurred infrequently when the bacterium was grown under conditions that did not allow a high concentration. Thus, R. solanacearum undergoes PC when the bacteria are present in the water extract for a certain period (4-5 days) at a high concentration (108 CFU ml-1). It is thought that the bacteria consume the nutrients in the water extract at the initial stage of culture, and that a starvation state will be reached if the pathogen is present for a certain period at a high concentration. Thus, it is supposed that the pathogen undergoes PC in order to express a phenotype advantageous to growth under nutrient starvation stress. Bacterial PC was not influenced by low temperature stress in the water extract, though it was induced at high temperature.

The effect of VEGF-immobilized nickel-free high-nitrogen stainless steel on viability and proliferation of vascular endothelial cells

Using ester bonds, vascular endothelial growth factor-A (VEGF-A) was immobilized on the surface of a novel biometal, nickel-free high-nitrogen stainless steel (HNS). The biological activity of immobilized VEGF-A was investigated after the culture of human umbilical vein endothelial cells (HUVECs) on the substrate. Immobilization of VEGF-A onto the HNS surface was performed using trisuccinimidyl citrate (TSC) as a linker. Firstly, UV irradiation was employed to amplify hydroxyl groups on the HNS surface. Next, the HNS was dipped into TSC/dimethyl sulfoxide solution at room temperature. From the results of water contact angle measurement and X-ray photoelectron spectroscopy (XPS) analysis, TSC was found to be immobilized on the HNS surface via ester bonds. Quantitative analysis demonstrated that immobilized VEGF-A remained even after immersion in culture medium for 7 days; however, it was gradually deimmobilized by hydrolysis of the ester bonds at the TSC–metal interface. As a result, VEGF-A-immobilized HNS significantly contributed to the stimulation of HUVEC growth for the initial stage of culture, even though the gradual reduction in growth stimulation of HUVECs occurred by the sequential deimmobilization of VEGF-A, which was caused by the hydrolysis of the ester groups. Therefore, VEGF-A-immobilized HNS could be applied as a basic material for coronary stents.

Various fates of neuronal progenitor cells observed on several different chemical functional groups

Neuronal progenitor cells cultured on gold-coated glass surfaces modified by different chemical functional groups, including hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), bromo (-Br), mercapto (-SH), - Phenyl and methyl (-CH3), were studied here to investigate the influence of surface chemistry on the cells’ adhesion, morphology, proliferation and functional gene expression. Focal adhesion staining indicated in the initial culture stage cells exhibited morphological changes in response to different chemical functional groups. Cells cultured on -NH2 grafted surface displayed focal adhesion plaque and flattened morphology and had the largest contact area. However, their counter parts on -CH3 grafted surface displayed no focal adhesion and rounded morphology and had the smallest contact area. After 6 days culture, the proliferation trend was as follows: -NH2 > -SH> -COOH> - Phenyl > - Br > -OH> -CH3. To determine the neural functional properties of the cells affected by surface chemistry, the expression of glutamate decarboxylase (GAD67), nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF) were characterized. An increase of GAD67 expression was observed on -NH2, -COOH and -SH grafted surfaces, while no increase in NGF and BDNF expression was observed on any chemical surfaces. These results highlight the importance of surface chemistry in the fate determination of neuronal progenitor cells, and suggest that surface chemistry must be considered in the design of biomaterials for neural tissue engineering.

PHOTOAUTOTROPHIC MICROPROPAGATION SYSTEMS - APPLICATIONS TO CONSERVATION BIOTECHNOLOGY PROGRAMMES

The IUCN updates information on the conservation status of plants which are threatened in their natural habitats. Several species are facing serious threats to their existence due to over collection, alien species, ecological and reproductive constraints. The Global Strategy for Plant Conservation aims to halt the current loss of plant diversitv bv means of conservation measures, training and capacity building. Over 50 percent of the world's plant species are endemic to the 34 biodiversity hotspots. In vitro conservation tools have been an integral part of ex situ conservation initiatives. However conservation practitioners from biodiversity hotspot countries need cost effective, sustainable and simple in vitro plant growing methods to rescue plants of high conservation rating that are facing threat to their existence. Photoautotrophic Micropropagation Systems (PMS) can specifically be of use in the initial stage of culture, rooting and weaning of recalcitrant species. Ventilation, supporting systems, improved illumination and CO 2 enrichment are the significant factors in developing a successful PMS. About twelve threatened plant species were tested for developing PMS in the last four years at the Conservation Biotechnology Unit (CBU), Royal Botanic Garden, Kew. Except for two species, all other species performed better in PMS compared to conventional micropropagation. Plants developed in PMS had leaves with well developed cuticle, stomata and vasculature. Fresh and dry weights of root and shoot systems of PMS derived plants were significantly better than mixotrophic plants. On average less than 50% of the plants survived transplantation following conventional micropropagation, but more than 80% of photoautotrophically grown plants were successfully transplanted. Superior success rate in developing propagules of high quality is very important in species of high conservation rating due to the limited availability of explant material from the wild or ex situ. The techniques outlined here can provide robust propagules of threatened plants required for reintroduction back to the wild and restoration programmes.