Showing papers on "Tissue culture published in 2015"

The impact of adiposity on adipose tissue-resident lymphocyte activation in humans

Abstract: The presence of T lymphocytes in human adipose tissue has only recently been demonstrated and relatively little is known of their potential relevance in the development of obesity-related diseases. We aimed to further characterise these cells and in particular to investigate how they interact with modestly increased levels of adiposity typical of common overweight and obesity. Subcutaneous adipose tissue and fasting blood samples were obtained from healthy males aged 35–55 years with waist circumferences in lean ( 102 cm) categories. Adipose tissue-resident CD4+ and CD8+ T lymphocytes together with macrophages were identified by gene expression and flow cytometry. T lymphocytes were further characterised by their expression of activation markers CD25 and CD69. Adipose tissue inflammation was investigated using gene expression analysis and tissue culture. Participants reflected a range of adiposity from lean to class I obesity. Expression of CD4 (T-helper cells) and CD68 (macrophage), as well as FOXP3 RNA transcripts, was elevated in subcutaneous adipose tissue with increased levels of adiposity (P<0.001, P<0.001 and P=0.018, respectively). Flow cytometry revealed significant correlations between waist circumference and levels of CD25 and CD69 expression per cell on activated adipose tissue-resident CD4+ and CD8+ T lymphocytes (P-values ranging from 0.053 to <0.001). No such relationships were found with blood T lymphocytes. This increased T lymphocyte activation was related to increased expression and secretion of various pro- and anti-inflammatory cytokines from subcutaneous whole adipose tissue explants. This is the first study to demonstrate that even modest levels of overweight/obesity elicit modifications in adipose tissue immune function. Our results underscore the importance of T lymphocytes during adipose tissue expansion, and the presence of potential compensatory mechanisms that may work to counteract adipose tissue inflammation, possibly through an increased number of T-regulatory cells.

Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice

Abstract: Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

Effects of low crystalline carbonate apatite on proliferation and osteoblastic differentiation of human bone marrow cells

Abstract: Carbonated apatite (CO3Ap) is the inorganic component of bone. We have proposed a new method for the fabrication of CO3Ap blocks based on a dissolution-precipitation method using a synthetic precursor. The aim of this study is to examine the effects of low crystalline CO3Ap on initial cell attachment, proliferation and osteoblastic differentiation of human bone marrow cells (hBMCs) using sintered hydroxyapatite and tissue culture plates as controls. Initial cell attachment and proliferation were assessed with a MTT assay. Expression of osteoblastic markers was examined by reverse transcription-polymerase chain reaction. XRD and FT-IR results showed formation of B-type carbonate apatite with lower crystallinity. No difference was observed for initial cell attachment between HAp and CO3Ap discs. hBMSC attached more significantly on tissue culture plate than on HAp and CO3Ap discs. The number of cells on HAp was higher than that on CO3Ap until day 7, after which the number of cells was similar. hBMSC proliferated more significantly on tissue culture plate than on HAp and CO3Ap discs. In contrast, hBMCs incubated on CO3Ap demonstrated much higher expression of osteoblastic markers of differentiation, such as type I collagen, alkaline phosphatase, osteopontin and osteocalcin, than hBMCs on HAp. On the tissue culture plate, they were not any change throughout the culture period. These results demonstrated that low crystalline CO3Ap exhibit higher osteoinductivity than HAp.

Slow-freezing versus vitrification for human ovarian tissue cryopreservation.

Abstract: Ovarian tissue can be cryopreserved prior to chemotherapy using either the slow-freezing or the vitrification method; however, the data on the equality of the procedures are still conflicting. In this study, a comparison of the cryo-damage of human ovarian tissue induced by either vitrification or slow-freezing was performed. Ovarian tissue from 23 pre-menopausal patients was cryopreserved with either slow-freezing or vitrification. After thawing/warming, the tissue was histologically and immunohistochemically analyzed and cultured in vitro. During tissue culture the estradiol release was assessed. No significant difference was found in the proportion of high-quality follicles after thawing/warming in the slow-freezing and vitrification group, respectively (72.7 versus 66.7 %, p = 0.733). Estradiol secretion by the ovarian tissue was similar between groups during 18 days in vitro culture (area-under-the-curve 5,411 versus 13,102, p = 0.11). Addition of Sphingosine-1-Phosphate or Activin A to the culture medium did not alter estradiol release in both groups. The proportion of Activated Caspase-3 or ‘Proliferating-Cell-Nuclear-Antigen’ positive follicles at the end of the culture period was similar between slow-freezing and vitrification. Slow-freezing and vitrification result in similar morphological integrity after cryopreservation, a similar estradiol release in culture, and similar rates of follicular proliferation and apoptosis after culture.

Printing cancer cells into intact microvascular networks: a model for investigating cancer cell dynamics during angiogenesis

Abstract: While cancer cell invasion and metastasis are dependent on cancer cell–stroma, cancer cell–blood vessel, and cancer cell–lymphatic vessel interactions, our understanding of these interactions remain largely unknown. A need exists for physiologically-relevant models that more closely mimic the complexity of cancer cell dynamics in a real tissue environment. The objective of this study was to combine laser-based cell printing and tissue culture methods to create a novel ex vivo model in which cancer cell dynamics can be tracked during angiogenesis in an intact microvascular network. Laser direct-write (LDW) was utilized to reproducibly deposit breast cancer cells (MDA-MB-231 and MCF-7) and fibroblasts into spatially-defined patterns on cultured rat mesenteric tissues. In addition, heterogeneous patterns containing co-printed MDA-MB-231/fibroblasts or MDA-MB-231/MCF-7 cells were generated for fibroblast-directed and collective cell invasion models. Printed cells remained viable and the cells retained the ability to proliferate in serum-rich media conditions. Over a culture period of five days, time-lapse imaging confirmed fibroblast and MDA-MB-231 cell migration within the microvascular networks. Confocal microscopy indicated that printed MDA-MB-231 cells infiltrated the tissue thickness and were capable of interacting with endothelial cells. Angiogenic network growth in tissue areas containing printed cancer cells was characterized by significantly increased capillary sprouting compared to control tissue areas containing no printed cells. Our results establish an innovative ex vivo experimental platform that enables time-lapse evaluation of cancer cell dynamics during angiogenesis within a real microvascular network scenario.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

Abstract: Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

Abstract: To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/− 0.0154 and 0.0171 +/− 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/− 0.0079 virions/image) than glans tissue (0.0167 +/− 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/− 0.0188 vs. 0.0151 +/− 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/− 3.908 vs. 12.466 +/− 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants

Abstract: Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review

Abstract: Main conclusion The present review discusses not only advances in coconut tissue culture and associated biotechnological interventions but also future research directions toward the resilience of this important palm crop. Coconut (Cocos nucifera L.) is commonly known as the ‘tree of life’. Every component of the palm can be used to produce items of value and many can be converted into industrial products. Coconut cultivation faces a number of acute problems that reduce its productivity and competitiveness. These problems include various biotic and abiotic challenges as well as an unstable market for its traditional oil-based products. Around 10 million small-holder farmers cultivate coconut palms worldwide on c. 12 million hectares of land, and many more people own a few coconut palms that contribute to their livelihoods. Inefficiency in the production of seedlings for replanting remains an issue; however, tissue culture and other biotechnological interventions are expected to provide pragmatic solutions. Over the past 60 years, much research has been directed towards developing and improving protocols for (i) embryo culture; (ii) clonal propagation via somatic embryogenesis; (iii) homozygote production via anther culture; (iv) germplasm conservation via cryopreservation; and (v) genetic transformation. Recently other advances have revealed possible new ways to improve these protocols. Although effective embryo culture and cryopreservation are now possible, the limited frequency of conversion of somatic embryos to ex vitro seedlings still prevents the large-scale clonal propagation of coconut. This review illustrates how our knowledge of tissue culture and associated biotechnological interventions in coconut has so far developed. Further improvement of protocols and their application to a wider range of germplasm will continue to open up new horizons for the collection, conservation, breeding and productivity of coconut.

Alternative induction of de novo shoot organogenesis or somatic embryogenesis from in vitro cultures of mature zygotic embryos of passion fruit ( Passiflora edulis Sims) is modulated by the ratio between auxin and cytokinin in the medium

Abstract: Several reports on tissue culture-based techniques have been published for passion fruit (Passiflora edulis), an important tropical fruit crop. However, a system in which de novo shoot organogenesis (DNSO) or somatic embryogenesis (SE) can be induced from the same type of explant was not available so far. The present study describes an in vitro system that enables alternative induction of both morphogenetic pathways from P. edulis mature zygotic embryo. DNSO was observed when explants were cultured on Murashige and Skoog medium supplemented with high 6-benzyladenine (BA) to 2,4-dichloro-phenoxyacetic acid (2,4-D) ratios, such as 72.4 μM BA and 4.5 μM 2,4-D. Embryogenic calli were observed when low BA/2,4-D ratios, such as 4.5 μM BA and 72.4 μM 2,4-D were used. Morpho-anatomical characterization revealed that epidermal and sub-epidermal cells were involved with the regeneration process via both DNSO and SE pathways. These cells became meristematic and divided extensively to form protuberances after 12–15 days of culture in both systems. These protuberances led to the formation of either meristemoids or pro-embryogenic cell clusters, which differentiated into shoot buds or somatic embryos, respectively. The use of this system coupled with techniques that allow gene expression studies might greatly contribute to further studies on in vitro morphogenesis in Passiflora.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

Abstract: In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.

Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.

Abstract: Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

Stem cell tissue culture seedling raising technique

Abstract: The invention discloses a stem cell tissue culture seedling raising technique. The technique comprises the steps of 1, taking a fresh plant, conducting sterilization on the surface and cutting the plant; 2, conducting tissue differentiation and reserving a cambium; 3, culturing the cambium on an isolation medium. According to the technique, existing methods are improved based on a traditional tissue culture technique, plant stem cells are taken as a target, induction, separation and explant culture are conducted, and a corresponding stem cell culture system is established. By the adoption of the plant stem cell culture technique, the plant culture technique is enriched, and a novel research direction and opportunity is provided for commercialized production of natural products and development of the plant biotechnology.

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Abstract: Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

Thidiazuron induced efficient in vitro multiplication and ex vitro conservation of Rauvolfia serpentina – a potent antihypertensive drug producing plant

Abstract: An efficient system for in vitro propagation of the endangered medicinal plant Rauvolfia serpentina has been developed. Proliferation of shoots was achieved from nodal segment explants, excised fro...

The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis.

Abstract: This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2•−) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2•− was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2•− is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth.

Isolation, culture, characterization, and adipogenic differentiation of heifer endometrial mesenchymal stem cells

Abstract: Mesenchymal stem cells (MSCs) have been isolated from various tissues of different species. The mammalian endometrium has morphological and functional modifications throughout the estrous cycle undergoing periodic proliferation and degeneration. The aim of this study was to isolate, culture, and characterize and to determine adipogenic differentiation of endometrial mesenchymal stem cells (En-MSCs) in heifers. Uteri of healthy heifers were collected from Shiraz Slaughterhouse, Iran and transferred to Stem Cell Laboratory of Stem Cell and Transgenic Technology Research Center to isolate En-MSCs from endometrial tissue samples. The tissue samples were exposed to collagenase type IA to reach the primary culture of En-MSCs. Isolated En-MSCs were sub-cultured up to passage 4. A specified number of En-MSCs were seeded into 12- and 24-well culture plates, and the number of cells was counted to evaluate the growth behavior of isolated cells and the population doubling time (PDT). RT-PCR for CD45 marker (hematopoietic stem cells) and CD73 marker (MSCs) and differentiation to adipocytes were performed for MSCs confirmation of En-MSCs of heifer. After cell culture, spindle-shape En-MSCs were visible adherent to the culture flasks. The cell count and the growth curves using 12- and 24-well culture plates showed that the PDT of En-MSCs was 37, 159.5, 52.9, and 136.3 h after seeding 2.2 × 104 and 20 × 104 (12-well) and 5 × 104 and 10 × 104 (24-well) En-MSCs, respectively. Using RT-PCR, heifer En-MSCs were negative for CD45 marker and positive for CD73 marker. Moreover, after culture of En-MSCs in differentiation medium, the cells differentiated toward adipocytes as verified by positive staining with Oil Red O staining. The morphology, growth kinetic, and differentiation of bovine En-MSCs demonstrated that these cells may be a good choice in bovine cell therapy and if necessary, they can be easily and safely administered in bovine tissue regeneration.

Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture.

Abstract: We describe a steroid-inducible BABY BOOM system that improves plant regeneration in Arabidopsis leaf cultures and yields fertile plants. Regeneration of Arabidopsis thaliana plants for extended periods of time in tissue culture may result in sterile plants. We report here a novel approach for A. thaliana regeneration using a regulated system to induce embryogenic cultures from leaf tissue. The system is based on BABY BOOM (BBM), a transcription factor that turns on genes involved in embryogenesis. We transformed the nucleus of A. thaliana plants with BBM:GR, a gene in which the BBM coding region is fused with the glucocorticoid receptor (GR) steroid-binding domain. In the absence of the synthetic steroid dexamethasone (DEX), the BBM:GR fusion protein is localized in the cytoplasm. Only when DEX is included in the culture medium does the BBM transcription factor enter the nucleus and turn on genes involved in embryogenesis. BBM:GR plant lines show prolific shoot regeneration from leaf pieces on media containing DEX. Removal of DEX from the culture media allowed for flowering and seed formation. Therefore, use of BBM:GR leaf tissue for regeneration of plants for extended periods of time in tissue culture will facilitate the recovery of fertile plants.

Polyacrylamide gel substrates that simulate the mechanical stiffness of normal and malignant neuronal tissues increase protoporphyin IX synthesis in glioma cells

Abstract: Protoporphyrin IX (PPIX) produced following the administration of exogenous 5d-aminolevulinic acid is clinically approved for photodynamic therapy and fluorescence-guided resection in various jurisdictions around the world. For both applications, quantification of PPIX forms the basis for accurate therapeutic dose calculation and identification of malignant tissues for resection. While it is well established that the PPIX synthesis and accumulation rates are subject to the cell’s biochemical microenvironment, the effect of the physical microenvironment, such as matrix stiffness, has received little attention to date. Here we studied the proliferation rate and PPIX synthesis and accumulation in two glioma cell lines U373 and U118 cultured under five different substrate conditions, including the conventional tissue culture plastic and polyacrylamide gels that simulated tissue stiffness of normal brain (1 kPa) and glioblastoma tumors (12 kPa). We found that the proliferation rate increased with substrate stiffness for both cell lines, but not in a linear fashion. PPIX concentration was significantly higher in cells cultured on tissue-simulating gels than on the much stiffer tissue culture plastic for both cell lines. These findings, albeit preliminary, suggest that the physical microenvironment might be an important determinant of tumor aggressiveness and PPIX synthesis in glioma cells.

Injury Response of Resected Human Brain Tissue In Vitro

Abstract: Brain injury affects a significant number of people each year. Organotypic cultures from resected normal neocortical tissue provide unique opportunities to study the cellular and neuropathological consequences of severe injury of adult human brain tissue in vitro. The in vitro injuries caused by resection (interruption of the circulation) and aggravated by the preparation of slices (severed neuronal and glial processes and blood vessels) reflect the reaction of human brain tissue to severe injury. We investigated this process using immunocytochemical markers, reverse transcriptase quantitative polymerase chain reaction and Western blot analysis. Essential features were rapid shrinkage of neurons, loss of neuronal marker expression and proliferation of reactive cells that expressed Nestin and Vimentin. Also, microglia generally responded strongly, whereas the response of glial fibrillary acidic protein-positive astrocytes appeared to be more variable. Importantly, some reactive cells also expressed both microglia and astrocytic markers, thus confounding their origin. Comparison with post-mortem human brain tissue obtained at rapid autopsies suggested that the reactive process is not a consequence of epilepsy.

Immunomodulatory potential of shatavarins produced from Asparagus racemosus tissue cultures.

Abstract: Medicinal properties of Asparagus racemosus (vernacular name: Shatavari) are attributed to its steroidal saponins called shatavarins. This plant is facing the threat of being endangered due to several developmental, seasonal constrains and malpractices involved in its collection and storage. To support its conservation, a tissue culture protocol is standardized which produces 20 fold higher levels of shatavarin. Here we evaluate the bioactivity and immunomodulatory potential of in vitro produced shatavarins from cell cultures of AR using human peripheral blood lymphocytes. In vitro produced shatavarin stimulated immune cell proliferation and IgG secretion in a dose dependent manner. It stimulated interleukin (IL)-12 production and inhibited production of IL-6. It also had strong modulatory effects on Th1/Th2 cytokine profile, indicating its potential application for immunotherapies where Th1/Th2 balance is envisaged. Our study demonstrating the bioactivity of tissue cultured AR extracts supports further in vivo evaluation of its immunomodulatory efficacy.

In vitro spermatogenesis using bovine testis tissue culture techniques

Abstract: Spermatogenesis is a complex process initiated by spermatogonial stem cells (SSCs) that have the ability to differentiate into mature spermatozoa or to self-renew to maintain the SSC population and long-term fertility. However, a technique for complete spermatogenesis in vitro using cell culture has not yet been developed. In the present study, we developed in vitro spermatogenesis techniques using bovine testis tissue culture. The effects of specific temperatures and different media on maintaining tubule and germ cell competency were investigated. We found that the optimal temperature and media were 37°C and mouse serum-free medium (mSFM), respectively. In addition, the efficacy of various hormones and growth factors on spermatogenesis in bovine testis tissues maintained in vitro was evaluated. We found that the addition of triiodothyronine (T3) and stem cell factor (SCF) induced spermatogenesis of bovine SSCs in vitro. Therefore, tissue fragments were cultured in the presence of T3 and SCF for three months to induce spermatogenesis in vitro. Overall, in vitro spermatogenesis was enhanced 2.4- to 2.7-fold. Our tissue culture technique may serve as a model system that leads to a more comprehensive understanding of the biology of SSCs as well as the factors that regulate male fertility. Furthermore, the results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.

In Vitro Micropropagation of a Valuable Medicinal Plant, Plectranthus amboinicus

Abstract: The effect of the plant growth regulators benzyl amino purine (BAP), 1-naphthaleneacetic acid (NAA) and kinetin (KIN) on in vitro shoot induction and proliferation of Plectranthus amboinicus was examined. Explants obtained from lateral shoots and apical shoots of P. amboinicus were inoculated on Murashige and Skoog (MS) culture medium supplemented with different concentrations of BAP, NAA and KIN. When the effect of each growth regulator was considered singly, the highest rate of shoot induction (80% of explants producing shoots) and highest number of shoots produced (2.4 shoots per explant) were obtained from lateral shoot explants cultured on MS media supplemented with 3.0 mg/L BAP within 6 - 7 weeks. Better results were obtained using MS medium supplemented with 1 mg/L BAP + 5 mg/L NAA. Shoot proliferation rose to 85%, while 5.7 shoots per explants were recorded. Among the different media tested for rooting, MS medium supplemented with 1.0 mg/L IBA was the most effective for root induction. The quality of the roots obtained was better than that obtained using MS media supplemented with NAA or IAA.

Conifer somatic embryogenesis - an efficient plant regeneration system for theoretical studies and mass propagation.

Abstract: Since the first report of somatic embryogenesis in Norway spruce in 1985, the in vitro process has been initiated for a number of conifer species belonging to different genera. The process of somatic embryogenesis involves initiation, proliferation, maturation and plantlets (emblings) regeneration. The initiation of somatic embryogenesis is restricted mostly to juvenile explants, although recently explants taken from adult trees produced embryogenic tissues. The successful initiation, maturation and emblings regeneration are affected by factors as developmental stage of primary explants, genotype, plant growth regulators content in the culture medium, light conditions. Optimisation of mentioned factors resulted in regeneration of emblings capable of growing after transfer to soil. Additional key words: in vitro, plant biotechnology, plant growth regulators (PGRs) Address: T. Salaj, R. Matusova, J. Salaj, Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.Box 39A, 950 07 Nitra, Slovak Republic, e-mail: terezia.salaj@savba.sk

Intestinal Crypt Organoids as Experimental Models

Abstract: When it comes to studying the effect of food bioactives on gut health, one of the essential steps that needs to be assessed is characterizing specific effects of the bioactives on the physical barrier of the lumen, the gastrointestinal tissue. In addition to studying the effects on transport function (e.g. by using Ussing chambers or cell culture systems), it is of great interest to evaluate the effects on morphology, cell biology, gene expression, and relevant functions of different cell types that are resident in the gastrointestinal (GI) tract. An ideal near-physiological model should contain a mixture of different GI epithelial cells (e.g. Paneth cells, goblet cells, absorptive and hormone secretive epithelial cells), which can be cultured indefinitely. Recently, the culture and applications of long-term primary multi-cellular cluster structures gastrointestinal organoids (or enteroids) have been demonstrated, and within the last 5 years the number of researchers that commonly use this tissue culture model has increased rapidly. This multi-cellular system may be a promising addition for existing ex vivo and alternative for animal models for testing effects of food bioactives on the intestinal tissue, and could provide a model for pre-screening of compounds prior to moving to the large scale testing systems. Moreover, intestinal organoids can be cultured from different species (e.g. human, pig and mouse). In this chapter we will focus on organoids cultured from mouse and pig crypt cells. We will give a short overview on how to isolate, culture, incubate, and apply them in different research fields.

Tissue culture technique of aquilaria sinensis

Abstract: The invention discloses a tissue culture technique of aquilaria sinensis and relates to a seedling growing method for obtaining good-quality seedlings from the aquilaria sinensis by use of an in-vitro rapid propagation technique. According to the tissue culture technique, the tissue culture rapid propagation system of the aquilaria sinensis is established by taking the stem with buds of the aquilaria sinensis as the explant and by virtue of the processes of explant sterilization, multiple shoot induction, rooting culture, acclimatization and transplanting and the like; the tissue culture technique has important practical significance on acceleration of the popularization of the good-quality aquilaria sinensis variety and the development and utilization of resources.