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Sample records for epithelial tissue culture

  1. The autologus graft of epithelial tissue culture

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    Minaee B

    1999-08-01

    Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

  2. Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

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    Hahn, Soojung; Yoo, Jongman

    2017-08-17

    An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

  3. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

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    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  4. Organoid culture systems for prostate epithelial and cancer tissue

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    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans

    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material

  5. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

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    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  6. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

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    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  7. Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model.

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    Speroni, Lucia; Whitt, Gregory S; Xylas, Joanna; Quinn, Kyle P; Jondeau-Cabaton, Adeline; Barnes, Clifford; Georgakoudi, Irene; Sonnenschein, Carlos; Soto, Ana M

    2014-01-01

    The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone-sensitive 3D culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose-dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

  8. Treatment of chronic desquamative gingivitis using tissue-engineered human cultured gingival epithelial sheets: a case report.

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    Okuda, Kazuhiro; Momose, Manabu; Murata, Masashi; Saito, Yoshinori; lnoie, Masukazu; Shinohara, Chikara; Wolff, Larry F; Yoshie, Hiromasa

    2004-04-01

    Human cultured gingival epithelial sheets were used as an autologous grafting material for regenerating gingival tissue in the maxillary left and mandibular right quadrants of a patient with chronic desquamative gingivitis. Six months post-surgery in both treated areas, there were gains in keratinized gingiva and no signs of gingival inflammation compared to presurgery. In the maxillary left quadrant, preoperative histopathologic findings revealed the epithelium was separated from the connective tissue and inflammatory cells were extensive. After grafting with the gingival epithelial sheets, inflammatory cells were decreased and separation between epithelium and connective tissue was not observed. The human cultured gingival epithelial sheets fabricated using tissue engineering technology showed significant promise for gingival augmentation in periodontal therapy.

  9. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  10. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

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    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

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    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

  12. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts

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    Iskandar, Anita R.; Xiang, Yang; Frentzel, Stefan; Talikka, Marja; Leroy, Patrice; Kuehn, Diana; Guedj, Emmanuel; Martin, Florian; Mathis, Carole; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

    2015-01-01

    Organotypic 3D cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model. PMID:26085348

  13. Epithelial Cell Cultures

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    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  14. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

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    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  15. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

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    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  16. Force transmission in epithelial tissues.

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    Vasquez, Claudia G; Martin, Adam C

    2016-03-01

    In epithelial tissues, cells constantly generate and transmit forces between each other. Forces generated by the actomyosin cytoskeleton regulate tissue shape and structure and also provide signals that influence cells' decisions to divide, die, or differentiate. Forces are transmitted across epithelia because cells are mechanically linked through junctional complexes, and forces can propagate through the cell cytoplasm. Here, we review some of the molecular mechanisms responsible for force generation, with a specific focus on the actomyosin cortex and adherens junctions. We then discuss evidence for how these mechanisms promote cell shape changes and force transmission in tissues. © 2016 Wiley Periodicals, Inc.

  17. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

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    Schlage, Walter K; Iskandar, Anita R; Kostadinova, Radina; Xiang, Yang; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Talikka, Marja; Geertz, Marcel; Mathis, Carole; Ivanov, Nikolai; Hoeng, Julia; Peitsch, Manuel C

    2014-10-01

    Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

  18. ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

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    Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

    2012-01-01

    The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

  19. Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model.

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    Tanaka, Yohei; Nakayama, Jun

    2016-01-01

    Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000-1,800 nm wavelengths and excluded 1,400-1,500 nm wavelengths. A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm(2) irradiation (Psolar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage.

  20. Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

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    Tanaka Y

    2016-08-01

    Full Text Available Yohei Tanaka,1,2 Jun Nakayama2 1Department of Plastic Surgery, Clinica Tanaka Plastic, Reconstructive Surgery and Anti-aging Center, 2Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan Background and objective: Humans are increasingly exposed to near-infrared (NIR radiation from both natural (eg, solar and artificial (eg, electrical appliances sources. Although the biological effects of sun and ultraviolet (UV exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues.Materials and methods: DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C. The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths.Results: A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05.Conclusion: We found that NIR irradiation induced the

  1. Segmentation and Quantitative Analysis of Epithelial Tissues.

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    Aigouy, Benoit; Umetsu, Daiki; Eaton, Suzanne

    2016-01-01

    Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

  2. Studying cytokinesis in Drosophila epithelial tissues.

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    Pinheiro, D; Bellaïche, Y

    2017-01-01

    Epithelial tissue cohesiveness is ensured through cell-cell junctions that maintain both adhesion and mechanical coupling between neighboring cells. During development, epithelial tissues undergo intensive cell proliferation. Cell division, and particularly cytokinesis, is coupled to the formation of new adhesive contacts, thereby preserving tissue integrity and propagating cell polarity. Remarkably, the geometry of the new interfaces is determined by the combined action of the dividing cell and its neighbors. To further understand the interplay between the dividing cell and its neighbors, as well as the role of cell division for tissue morphogenesis, it is important to analyze cytokinesis in vivo. Here we present methods to perform live imaging of cell division in Drosophila epithelial tissues and discuss some aspects of image processing and analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Plant tissue culture techniques

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    Rolf Dieter Illg

    1991-01-01

    Full Text Available Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus or organized tissues or organs put in culture, under controlled sterile conditions.

  4. Plant Tissue Culture

    Indian Academy of Sciences (India)

    Admin

    Plant tissue culture is a technique of culturing plant cells, tissues and organs on ... working methods (Box 2) and discovery of the need for B vita- mins and auxins for ... Kotte (Germany) reported some success with growing isolated root tips.

  5. Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

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    Ye, P; Yu, H; Simonian, M; Hunter, N

    2014-04-01

    Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin

  6. Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

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    Le-Bel, Gaëtan; Ghio, Sergio Cortez; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

    2018-05-27

    Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

  7. Lingual Epithelial Stem Cells and Organoid Culture of Them

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    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  8. Emergent material properties of developing epithelial tissues.

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    Machado, Pedro F; Duque, Julia; Étienne, Jocelyn; Martinez-Arias, Alfonso; Blanchard, Guy B; Gorfinkiel, Nicole

    2015-11-23

    Force generation and the material properties of cells and tissues are central to morphogenesis but remain difficult to measure in vivo. Insight is often limited to the ratios of mechanical properties obtained through disruptive manipulation, and the appropriate models relating stress and strain are unknown. The Drosophila amnioserosa epithelium progressively contracts over 3 hours of dorsal closure, during which cell apices exhibit area fluctuations driven by medial myosin pulses with periods of 1.5-6 min. Linking these two timescales and understanding how pulsatile contractions drive morphogenetic movements is an urgent challenge. We present a novel framework to measure in a continuous manner the mechanical properties of epithelial cells in the natural context of a tissue undergoing morphogenesis. We show that the relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is consistent with a linear behaviour, although with a lag. We thus used myosin fluorescence intensity as a proxy for active force generation and treated cells as natural experiments of mechanical response under cyclic loading, revealing unambiguous mechanical properties from the hysteresis loop relating stress to strain. Amnioserosa cells can be described as a contractile viscoelastic fluid. We show that their emergent mechanical behaviour can be described by a linear viscoelastic rheology at timescales relevant for tissue morphogenesis. For the first time, we establish relative changes in separate effective mechanical properties in vivo. Over the course of dorsal closure, the tissue solidifies and effective stiffness doubles as net contraction of the tissue commences. Combining our findings with those from previous laser ablation experiments, we show that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures. Our results show that in an epithelial

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

    Directory of Open Access Journals (Sweden)

    Han Hu

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

  10. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

  11. Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

    Science.gov (United States)

    Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

  12. High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.B.; Harney, J.; Hennessy, T.P.

    1994-01-01

    When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in open-quotes edgeclose quotes cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed. 33 refs., 2 figs., 3 tabs

  13. The plant tissue culture

    International Nuclear Information System (INIS)

    Crocomo, O.J.; Sharp, W.R.

    1973-01-01

    Progress in the field of plant tissue culture at the Plant Biochemistry Sector, Centro de Energia na Agricultura (CENA), Piracicaba, S.P., Brazil, pertains to the simplification of development in 'Phaseolus vulgaris' by dividing the organism into its component organs, tissues, and cells and the maintenance of these components on defined culture media 'in vitro'. This achievement has set the stage for probing the basis for the stability of the differentiated states and/or the reentry of mature differentiated cells into the mitotic cell cycle and their subsequent redifferentiation. Data from such studies at the cytological and biochemical level have been invaluable in the elucidation of the control mechanisms responsible for expression of the cellular phenotype. Unlimited possibilities exist for the application of tissue culture in the vegetative propagation of 'Phaseolus' and other important cultivars in providing genocopies or a large scale and/or readily obtaining plantlets from haploid cell lines or from protoplast (wall-less cells) hybridization products following genetic manipulation. These tools are being applied in this laboratory for the development and selection of high protein synthesizing 'Phaseolus' cultivars

  14. Crossroads of Wnt and Hippo in epithelial tissues.

    Science.gov (United States)

    Bernascone, Ilenia; Martin-Belmonte, Fernando

    2013-08-01

    Epithelial tissues undergo constant growth and differentiation during embryonic development and to replace damaged tissue in adult organs. These processes are governed by different signaling pathways that ultimately control the expression of genes associated with cell proliferation, patterning, and death. One essential pathway is Wnt, which controls tubulogenesis in several epithelial organs. Recently, Wnt has been closely linked to other signaling pathways, such as Hippo, that orchestrate proliferation and apoptosis to control organ size. There is evidence that epithelial cell junctions may sequester the transcription factors that act downstream of these signaling pathways, which would represent an important aspect of their functional regulation and their influence on cell behavior. Here, we review the transcriptional control exerted by the Wnt and Hippo signaling pathways during epithelial growth, patterning, and differentiation and recent advances in understanding of the regulation and crosstalk of these pathways in epithelial tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

    Science.gov (United States)

    Flood, P; Alvarez, L; Reynaud, E G

    2016-10-11

    Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

  16. A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

    Science.gov (United States)

    Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

    2014-01-01

    The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

  17. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    OpenAIRE

    Liu, Z.; Zhang, P.; Zhou, Y.; Qin, H.; Shen, T.

    2010-01-01

    Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithel...

  18. From cells to tissue: A continuum model of epithelial mechanics

    Science.gov (United States)

    Ishihara, Shuji; Marcq, Philippe; Sugimura, Kaoru

    2017-08-01

    A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

  19. Epithelial-mesenchymal transition: An emerging target in tissue fibrosis

    Science.gov (United States)

    Li, Meirong; Luan, Fuxin; Zhao, Yali; Hao, Haojie; Zhou, Yong; Han, Weidong

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is involved in a variety of tissue fibroses. Fibroblasts/myofibroblasts derived from epithelial cells contribute to the excessive accumulation of fibrous connective tissue in damaged tissue, which can lead to permanent scarring or organ malfunction. Therefore, EMT-related fibrosis cannot be neglected. This review highlights the findings that demonstrate the EMT to be a direct contributor to the fibroblast/myofibroblast population in the development of tissue fibrosis and helps to elucidate EMT-related anti-fibrotic strategies, which may enable the development of therapeutic interventions to suppress EMT and potentially reverse organ fibrosis. PMID:26361988

  20. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  1. Turbulent Dynamics of Epithelial Cell Cultures

    Science.gov (United States)

    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  2. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

  3. A multicellular view of cytokinesis in epithelial tissue.

    Science.gov (United States)

    Herszterg, Sophie; Pinheiro, Diana; Bellaïche, Yohanns

    2014-05-01

    The study of cytokinesis in single-cell systems provided a wealth of knowledge on the molecular and biophysical mechanisms controlling daughter cell separation. In this review, we outline recent advances in the understanding of cytokinesis in epithelial tissues. These findings provide evidence for how the cytokinetic machinery adapts to a multicellular context and how the cytokinetic machinery is itself exploited by the tissue for the preservation of tissue function and architecture during proliferation. We propose that cytokinesis in epithelia should be viewed as a multicellular process, whereby the biochemical and mechanical interactions between the dividing cell and its neighbors are essential for successful daughter cell separation while defining epithelial tissue organization and preserving tissue integrity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Three-dimensional epithelial tissues generated from human embryonic stem cells.

    Science.gov (United States)

    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  5. Two-layer tissue engineered urethra using oral epithelial and muscle derived cells.

    Science.gov (United States)

    Mikami, Hiroshi; Kuwahara, Go; Nakamura, Nobuyuki; Yamato, Masayuki; Tanaka, Masatoshi; Kodama, Shohta

    2012-05-01

    We fabricated novel tissue engineered urethral grafts using autologously harvested oral cells. We report their viability in a canine model. Oral tissues were harvested by punch biopsy and divided into mucosal and muscle sections. Epithelial cells from mucosal sections were cultured as epithelial cell sheets. Simultaneously muscle derived cells were seeded on collagen mesh matrices to form muscle cell sheets. At 2 weeks the sheets were joined and tubularized to form 2-layer tissue engineered urethras, which were autologously grafted to surgically induced urethral defects in 10 dogs in the experimental group. Tissue engineered grafts were not applied to the induced urethral defect in control dogs. The dogs were followed 12 weeks postoperatively. Urethrogram and histological examination were done to evaluate the grafting outcome. We successfully fabricated 2-layer tissue engineered urethras in vitro and transplanted them in dogs in the experimental group. The 12-week complication-free rate was significantly higher in the experimental group than in controls. Urethrogram confirmed urethral patency without stricture in the complication-free group at 12 weeks. Histologically urethras in the transplant group showed a stratified epithelial layer overlying well differentiated submucosa. In contrast, urethras in controls showed severe fibrosis without epithelial layer formation. Two-layer tissue engineered urethras were engineered using cells harvested by minimally invasive oral punch biopsy. Results suggest that this technique can encourage regeneration of a functional urethra. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  6. Is tissue CA125 expression in epithelial ovarian adenocarcinoma heterogenic?

    DEFF Research Database (Denmark)

    Sparholt, Morten H; Høgdall, Claus K; Nedergaard, Lotte

    2013-01-01

    To evaluate if heterogeneity of tissue cancer antigen 125 (CA125) expression is present in epithelial serous adenocarcinomas. Furthermore, to investigate whether there is a correlation between levels of CA125 tissue expression, serum level of CA125, stage, and grade. A total of 10 patients...... diagnosed with serous ovarian adenocarcinomas were included. Preoperative blood samples were collected to determine serum CA125 levels. Tumor tissue from primary surgery was collected and processed for immunohistochemical analyses. CA125 was expressed in varying degrees in tumor tissues from all patients....... Mean tissue CA125 expression for each patient ranged from 36% to 98%. Intrapatient variations in tissue expression ranged from 10% to 90% point. No significant correlations between levels of CA125 tissue expression, serum level of CA125, stage, and grade were found. We found that the tissue expression...

  7. Engineered human broncho-epithelial tissue-like assemblies

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    2012-01-01

    Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

  8. Epithelial-Mesenchymal Transition in Tissue Repair and Fibrosis

    Science.gov (United States)

    Stone, Rivka C.; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I.; Tomic-Canic, Marjana

    2016-01-01

    Epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics which confer migratory capacity. EMT and its converse, MET (mesenchymal-to-epithelial transition), are integral stages of many physiologic processes, and as such are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes - the resident skin epithelial cells - migrate across the wound bed to restore the epidermal barrier. Moreover, EMT also plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblast arises from cells of epithelial lineage in response to injury but is pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the impaired repair of fibrotic wounds may identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. PMID:27461257

  9. Epithelial-mesenchymal transition in tissue repair and fibrosis.

    Science.gov (United States)

    Stone, Rivka C; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I; Tomic-Canic, Marjana

    2016-09-01

    The epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including the loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics that confer migratory capacity. EMT and its converse, MET (mesenchymal-epithelial transition), are integral stages of many physiologic processes and, as such, are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes (the resident skin epithelial cells) migrate across the wound bed to restore the epidermal barrier. Moreover, EMT plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblasts arise from cells of the epithelial lineage in response to injury but are pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the faulty repair of fibrotic wounds might identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. Graphical Abstract Model for injury-triggered EMT activation in physiologic wound repair (left) and fibrotic wound healing (right).

  10. A dynamic cellular vertex model of growing epithelial tissues

    Science.gov (United States)

    Lin, Shao-Zhen; Li, Bo; Feng, Xi-Qiao

    2017-04-01

    Intercellular interactions play a significant role in a wide range of biological functions and processes at both the cellular and tissue scales, for example, embryogenesis, organogenesis, and cancer invasion. In this paper, a dynamic cellular vertex model is presented to study the morphomechanics of a growing epithelial monolayer. The regulating role of stresses in soft tissue growth is revealed. It is found that the cells originating from the same parent cell in the monolayer can orchestrate into clustering patterns as the tissue grows. Collective cell migration exhibits a feature of spatial correlation across multiple cells. Dynamic intercellular interactions can engender a variety of distinct tissue behaviors in a social context. Uniform cell proliferation may render high and heterogeneous residual compressive stresses, while stress-regulated proliferation can effectively release the stresses, reducing the stress heterogeneity in the tissue. The results highlight the critical role of mechanical factors in the growth and morphogenesis of epithelial tissues and help understand the development and invasion of epithelial tumors.

  11. Culture of insect tissues

    International Nuclear Information System (INIS)

    Cestari, A.N.; Simoes, L.C.G.

    1978-01-01

    Several aspects are discussed related to the behavior of politenic chromosomes from Rhyncosciara salivary glands kept in culture during different periods of time, without interference of insect hormones. Nucleic acid-and protein synthesis in isolated nuclei and chromosomes are also investigated. Autoradiographic techniques and radioactive precursors for nucleic acids and proteins are used in the research. (M.A.) [pt

  12. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  13. Biotransformations with plant tissue cultures.

    Science.gov (United States)

    Carew, D P; Bainbridge, T

    1976-01-01

    Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium maculatum were examined for their capacity to transform aniline, anisole, acetanilide, benzoic acid and coumarin. None of the cultures transformed acetanilide but each produced acetanilide when fed aniline. All three cultures converted benzoic acid to its para-hydroxy derivative. Coumarin was selectively hydroxylated at the 7-position by Catharanthus and Conium and anisole was O-demethylated only by older Catharanthus tissue.

  14. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

    NARCIS (Netherlands)

    Huang, L.; van Loveren, C.; Ling, J.; Wei, X.; Crielaard, W.; Deng, D.M.

    2016-01-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated

  15. Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique.

    Science.gov (United States)

    Noruddin, Nur Adelina Ahmad; Saim, Aminuddin B; Chua, Kien Hui; Idrus, Ruszymah

    2007-12-01

    To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application. Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells. Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques. Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.

  16. Calcium as a signal integrator in developing epithelial tissues.

    Science.gov (United States)

    Brodskiy, Pavel A; Zartman, Jeremiah J

    2018-05-16

    Decoding how tissue properties emerge across multiple spatial and temporal scales from the integration of local signals is a grand challenge in quantitative biology. For example, the collective behavior of epithelial cells is critical for shaping developing embryos. Understanding how epithelial cells interpret a diverse range of local signals to coordinate tissue-level processes requires a systems-level understanding of development. Integration of multiple signaling pathways that specify cell signaling information requires second messengers such as calcium ions. Increasingly, specific roles have been uncovered for calcium signaling throughout development. Calcium signaling regulates many processes including division, migration, death, and differentiation. However, the pleiotropic and ubiquitous nature of calcium signaling implies that many additional functions remain to be discovered. Here we review a selection of recent studies to highlight important insights into how multiple signals are transduced by calcium transients in developing epithelial tissues. Quantitative imaging and computational modeling have provided important insights into how calcium signaling integration occurs. Reverse-engineering the conserved features of signal integration mediated by calcium signaling will enable novel approaches in regenerative medicine and synthetic control of morphogenesis.

  17. Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.

    Science.gov (United States)

    Larouche, Danielle; Cuffley, Kristine; Paquet, Claudie; Germain, Lucie

    2011-03-01

    The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.

  18. Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

    Science.gov (United States)

    Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

    2017-04-01

    The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

  19. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  20. Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

    Science.gov (United States)

    Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

    2016-07-10

    The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

  1. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    Directory of Open Access Journals (Sweden)

    Aaron H Fronk

    2016-07-01

    Full Text Available The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

  2. Epigenetics in plant tissue culture

    NARCIS (Netherlands)

    Smulders, M.J.M.; Klerk, de G.J.M.

    2011-01-01

    Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at

  3. Gastric tissue biopsy and culture

    Science.gov (United States)

    ... symptoms may include: Loss of appetite or weight loss Nausea and vomiting Pain in the upper part of the belly Black stools Vomiting blood or coffee ground-like material A gastric tissue biopsy and culture can help detect: Cancer Infections, most commonly Helicobacter ...

  4. Volumetric imaging of oral epithelial neoplasia by MPM-SHGM: epithelial connective tissue interface (Conference Presentation)

    Science.gov (United States)

    Pal, Rahul; Yang, Jinping; Qiu, Suimin; Resto, Vicente; McCammon, Susan; Vargas, Gracie

    2016-03-01

    The majority of oral cancers are comprised of oral squamous cell carcinoma in which neoplastic epithelial cells invade across the epithelial connective tissue interface (ECTI). Invasion is preceded by a multi-component process including epithelial hyperproliferation, loss of cell polarity, and remodeling of the extracellular matrix. Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia. In particular, volumetric imaging by these methods can reveal aspects of the 3D microstructure that are not possible by other methods and which could both further our understanding of neoplastic transformation and be explored for development of diagnostic approaches in this disease having only 55% 5-year survival rate. MPAM-SHG were applied to reveal the 3D structure of the critical ECTI interface that plays an integral part toward invasion. Epithelial dysplasia was induced in an established hamster model. MPAM-SHGM was applied to lesion sites, using 780 nm excitation (450-600nm emission) for autofluroescence of cellular and extracellular components; 840 nm using 420 nm bandpass filter for SHG. The ECTI surface was identified as the interface at which SHG signal began following the epithelium and was modeled as a 3D surface using Matlab. ECTI surface area and cell features at sites of epithelial expansion where ECTI was altered were measured; Imaged sites were biopsied and processed for histology. ROC analysis using ECTI image metrics indicated the ability to delineate normal from neoplasia with high sensitivity and specificity and it is noteworthy that inflammation did not significantly alter diagnostic potential of MPAM-SHGM .

  5. Response of cultured normal human mammary epithelial cells to X rays

    International Nuclear Information System (INIS)

    Yang, T.C.; Stampfer, M.R.; Smith, H.S.

    1983-01-01

    The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D 0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique

  6. Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

    Science.gov (United States)

    Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

    2018-05-03

    Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

  7. Connexin Communication Compartments and Wound Repair in Epithelial Tissue

    Directory of Open Access Journals (Sweden)

    Marc Chanson

    2018-05-01

    Full Text Available Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

  8. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Science.gov (United States)

    Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

    2017-11-01

    Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  9. γδ T cells in homeostasis and host defence of epithelial barrier tissues

    DEFF Research Database (Denmark)

    Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

    2017-01-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue...... homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body — namely, the epidermis and the intestine — and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity...

  10. Transforming growth factor-beta 1, 2, and 3 can inhibit epithelial tissue outgrowth on smooth and microgrooved substrates.

    NARCIS (Netherlands)

    Walboomers, X.F.; Dalton, B.A.; Evans, M.D.; Steele, J.G.; Jansen, J.A.

    2002-01-01

    In this study, we describe the influence of parallel surface microgrooves, and of TGF-beta, on the outgrowth of corneal epithelial tissue. Microgrooves (depth 1 microm, width 1-10 microm) were made in polystyrene culturing surfaces. These surfaces were left untreated, or loaded with TGF-beta 1, 2,

  11. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  12. Polarized Airway Epithelial Models for Immunological Co-Culture Studies

    DEFF Research Database (Denmark)

    Papazian, Dick; Würtzen, Peter A; Hansen, Søren Werner Karlskov

    2016-01-01

    Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation...... of the epithelial response towards environmental antigens and genetic susceptibility, resulting in inflammation and T cell-derived immune responses. In vivo animal models have long been used to study immune homeostasis of the airways but are limited by species restriction and lack of exposure to a natural...

  13. ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

    Science.gov (United States)

    Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

    2017-03-01

    The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (Pepithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

  14. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

    Directory of Open Access Journals (Sweden)

    Miessen Katrin

    2012-03-01

    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  15. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri.

    Science.gov (United States)

    Miessen, Katrin; Einspanier, Ralf; Schoen, Jennifer

    2012-03-19

    Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  16. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    International Nuclear Information System (INIS)

    Norgaard, J.O.

    1987-01-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

  17. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  18. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  19. Feedback amplification loop drives malignant growth in epithelial tissues.

    Science.gov (United States)

    Muzzopappa, Mariana; Murcia, Lada; Milán, Marco

    2017-08-29

    Interactions between cells bearing oncogenic mutations and the surrounding microenvironment, and cooperation between clonally distinct cell populations, can contribute to the growth and malignancy of epithelial tumors. The genetic techniques available in Drosophila have contributed to identify important roles of the TNF-α ligand Eiger and mitogenic molecules in mediating these interactions during the early steps of tumor formation. Here we unravel the existence of a tumor-intrinsic-and microenvironment-independent-self-reinforcement mechanism that drives tumor initiation and growth in an Eiger-independent manner. This mechanism relies on cell interactions between two functionally distinct cell populations, and we present evidence that these cell populations are not necessarily genetically different. Tumor-specific and cell-autonomous activation of the tumorigenic JNK stress-activated pathway drives the expression of secreted signaling molecules and growth factors to delaminating cells, which nonautonomously promote proliferative growth of the partially transformed epithelial tissue. We present evidence that cross-feeding interactions between delaminating and nondelaminating cells increase each other's sizes and that these interactions can explain the unlimited growth potential of these tumors. Our results will open avenues toward our molecular understanding of those social cell interactions with a relevant function in tumor initiation in humans.

  20. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  1. Tissue culture of ornamental cacti

    Directory of Open Access Journals (Sweden)

    Eugenio Pérez-Molphe-Balch

    2015-12-01

    Full Text Available Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

  2. Establishment of primary keratinocyte culture from horse tissue biopsates

    Directory of Open Access Journals (Sweden)

    Jernej OGOREVC

    2015-12-01

    Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

  3. Cell-size distribution in epithelial tissue formation and homeostasis.

    Science.gov (United States)

    Puliafito, Alberto; Primo, Luca; Celani, Antonio

    2017-03-01

    How cell growth and proliferation are orchestrated in living tissues to achieve a given biological function is a central problem in biology. During development, tissue regeneration and homeostasis, cell proliferation must be coordinated by spatial cues in order for cells to attain the correct size and shape. Biological tissues also feature a notable homogeneity of cell size, which, in specific cases, represents a physiological need. Here, we study the temporal evolution of the cell-size distribution by applying the theory of kinetic fragmentation to tissue development and homeostasis. Our theory predicts self-similar probability density function (PDF) of cell size and explains how division times and redistribution ensure cell size homogeneity across the tissue. Theoretical predictions and numerical simulations of confluent non-homeostatic tissue cultures show that cell size distribution is self-similar. Our experimental data confirm predictions and reveal that, as assumed in the theory, cell division times scale like a power-law of the cell size. We find that in homeostatic conditions there is a stationary distribution with lognormal tails, consistently with our experimental data. Our theoretical predictions and numerical simulations show that the shape of the PDF depends on how the space inherited by apoptotic cells is redistributed and that apoptotic cell rates might also depend on size. © 2017 The Author(s).

  4. Tissue architecture: the ultimate regulator of breast epithelial function

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, Mina J; Rizki, Aylin; Mian, Saira

    2003-10-20

    A problem in developmental biology that continues to take center stage is how higher organisms generate diverse tissues and organs given the same cellular genotype. In cell and tumor biology, the key question is not the production of form, but its preservation: how do tissues and organs maintain homeostasis, and how do cells within tissues lose or overcome these controls in cancer? Undoubtedly, mechanisms that maintain tissue specificity should share features with those employed to drive formation of the tissues. However, they are unlikely to be identical. At a simplistic level, developmental pathways may be thought of as a series of extremely rapid short-term events. Each new step depends on what came before, and the outcome is the organism itself at birth. All organs, with a few notable exceptions, such as the mammary gland and the brain, 'arrive' together and are complete when the organism is born. In mice and humans, these events occur in a mere 21 days and 9 months respectively. The stability of the differentiated state and the homeostasis of the organism, on the other hand, will last 40-110 times longer. How does the organism achieve this feat? How are tissues maintained? These questions also relate fundamentally to how tissues become malignant and, although not discussed here, to aging. While there is much literature on differentiation - loosely defined as the gain of a single or a series of functions - we know much less about the forces and the pathways that maintain organ morphology and function as a unit. This may be partly because it is difficult to study a tissue as a unit in vivo and there are few techniques that allow maintenance of organs in vitro long enough and in such a way as to make cell and molecular biology experiments possible. Techniques for culturing cells in three-dimensional gels (3D) as a surrogate for tissues, however, have been steadily improving and the method is now used by several laboratories. In this commentary we

  5. Nedd4L expression is decreased in ovarian epithelial cancer tissues compared to ovarian non-cancer tissue.

    Science.gov (United States)

    Yang, Qiuyun; Zhao, Jinghe; Cui, Manhua; Gi, Shuting; Wang, Wei; Han, Xiaole

    2015-12-01

    Recent studies have demonstrated that the neural precursor cell expressed, developmentally downregulated 4-like (Nedd4L) gene plays a role in the progression of various cancers. However, reports describing Nedd4L expression in ovarian cancer tissues are limited. A cohort (n = 117) of archival formalin-fixed, paraffin embedded resected normal ovarian epithelial tissues (n = 10), benign ovarian epithelial tumor tissues (n = 10), serous borderline ovarian epithelial tumor tissues (n = 14), mucous borderline ovarian epithelial tumor tissues (n = 11), and invasive ovarian epithelial cancer tissues (n = 72) were assessed for Nedd4L protein expression using immunohistochemistry. Nedd4L protein expression was significantly decreased in invasive ovarian epithelial cancer tissues compared to non-cancer tissues (P < 0.05). Decreased Nedd4L protein expression correlated with clinical stage, pathological grade, lymph node metastasis and survival (P < 0.05). Nedd4L protein expression may be an independent prognostic marker of ovarian cancer development. © 2015 Japan Society of Obstetrics and Gynecology.

  6. Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

    Science.gov (United States)

    2013-01-01

    Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

  7. Replication and interaction of herpes simplex virus and human papillomavirus in differentiating host epithelial tissue

    International Nuclear Information System (INIS)

    Meyers, Craig; Andreansky, Samita S.; Courtney, Richard J.

    2003-01-01

    We have investigated the interactions and consequences of superinfecting and coreplication of human papillomavirus (HPV) and herpes simplex virus (HSV) in human epithelial organotypic (raft) culture tissues. In HPV-positive tissues, HSV infection and replication induced significant cytopathic effects (CPE), but the tissues were able to recover and maintain a certain degree of tissue integrity and architecture. HPV31b not only maintained the episomal state of its genomic DNA but also maintained its genomic copy number even during times of extensive HSV-induced CPE. E2 transcripts encoded by HPV31b were undetectable even though HPV31b replication was maintained in HSV- infected raft tissues. Expression of HPV31b oncogenes (E6 and E7) was also repressed but to a lesser degree than was E2 expression. The extent of CPE induced by HSV is dependent on the magnitude of HPV replication and gene expression at the time of HSV infection. During active HSV infection, HPV maintains its genomic copy number even though genes required for its replication were repressed. These studies provide new insight into the complex interaction between two common human sexually transmitted viruses in an in vitro system, modeling their natural host tissue in vivo

  8. Advanced three-dimensional culture of equine intestinal epithelial stem cells.

    Science.gov (United States)

    Stewart, A Stieler; Freund, J M; Gonzalez, L M

    2018-03-01

    Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease

  9. Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs?

    Science.gov (United States)

    Nigam, Sanjay K

    2013-12-01

    Branching morphogenesis is critical to the development of organs such as kidney, lung, mammary gland, prostate, pancreas, and salivary gland. Essentially, an epithelial bud becomes an iterative tip-stalk generator (ITSG) able to form a tree of branching ducts and/or tubules. In different organs, branching morphogenesis is governed by similar sets of genes. Epithelial branching has been recapitulated in vitro (or ex vivo) using three-dimensional cell culture and partial organ culture systems, and several such systems relevant to kidney tissue engineering are discussed here. By adapting systems like these it may be possible to harness the power inherent in the ITSG program to propagate and engineer epithelial tissues and organs. It is also possible to conceive of a universal ITSG capable of propagation that may, by recombination with organ-specific mesenchymal cells, be used for engineering many organ-like tissues similar to the organ from which the mesenchyme cells were derived, or toward which they are differentiated (from stem cells). The three-dimensional (3D) branched epithelial structure could act as a dynamic branching cellular scaffold to establish the architecture for the rest of the tissue. Another strategy-that of recombining propagated organ-specific ITSGs in 3D culture with undifferentiated mesenchymal stem cells-is also worth exploring. If feasible, such engineered tissues may be useful for the ex vivo study of drug toxicity, developmental biology, and physiology in the laboratory. Over the long term, they have potential clinical applications in the general fields of transplantation, regenerative medicine, and bioartificial medical devices to aid in the treatment of chronic kidney disease, diabetes, and other diseases.

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

    Science.gov (United States)

    Jayasankar, Vidya; Vasudevan, Srinivasa Raghavan; Poulose, Suja C; Divipala, Indira

    2018-06-12

    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 18 mo 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.

  11. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling

    Science.gov (United States)

    ITO, TAKUJI; BAI, TAO; TANAKA, TETSUJI; YOSHIDA, KENJI; UEYAMA, TAKASHI; MIYAJIMA, MASAYASU; NEGISHI, TAKAYUKI; KAWASAKI, TAKAHIKO; TAKAMATSU, HYOTA; KIKUTANI, HITOSHI; KUMANOGOH, ATSUSHI; YUKAWA, KAZUNORI

    2015-01-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of

  12. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    Science.gov (United States)

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The

  13. γδ T cells in homeostasis and host defence of epithelial barrier tissues.

    Science.gov (United States)

    Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

    2017-12-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

  14. Walnut tissue culture: research and field applications

    Science.gov (United States)

    2004-01-01

    Vitrotech Biotecnologia Vegetal began researching propagating Juglans regia (English walnut) and various Juglans hybrids by tissue culture in 1993 and has operated on a commercial scale since 1996. Since this time, more than one and a half million walnuts of different species have been propagated and field planted. Tissue cultured...

  15. Enamel tissue engineering using subcultured enamel organ epithelial cells in combination with dental pulp cells.

    Science.gov (United States)

    Honda, Masaki J; Shinmura, Yuka; Shinohara, Yoshinori

    2009-01-01

    We describe a strategy for the in vitro engineering of enamel tissue using a novel technique for culturing enamel organ epithelial (EOE) cells isolated from the enamel organ using 3T3-J2 cells as a feeder layer. These subcultured EOE cells retain the capacity to produce enamel structures over a period of extended culture. In brief, enamel organs from 6-month-old porcine third molars were dissociated into single cells and subcultured on 3T3-J2 feeder cell layers. These subcultured EOE cells were then seeded onto a collagen sponge in combination with primary dental pulp cells isolated at an early stage of crown formation, and these constructs were transplanted into athymic rats. After 4 weeks, complex enamel-dentin structures were detected in the implants. These results show that our culture technique maintained ameloblast lineage cells that were able to produce enamel in vivo. This novel subculture technique provides an important tool for tooth tissue engineering. Copyright 2008 S. Karger AG, Basel.

  16. Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

    Science.gov (United States)

    Pistone Creydt, Virginia; Fletcher, Sabrina Johanna; Giudice, Jimena; Bruzzone, Ariana; Chasseing, Norma Alejandra; Gonzalez, Eduardo Gustavo; Sacca, Paula Alejandra; Calvo, Juan Carlos

    2013-02-01

    Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

  17. Prevalence of human papillomavirus in epithelial ovarian cancer tissue. A meta-analysis of observational studies

    DEFF Research Database (Denmark)

    Svahn, Malene F; Faber, Mette Tuxen; Christensen, Jane

    2014-01-01

    The role of human papillomavirus (HPV) in the pathogenesis of ovarian cancer is controversial, and conflicting results have been published. We conducted a systematic review and meta-analysis to estimate the prevalence of HPV in epithelial ovarian cancer tissue....

  18. Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

    Science.gov (United States)

    Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

    2018-06-26

    Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

  19. Development of an Autonomous, Dual Chamber Bioreactor for the Growth of 3-Dimensional Epithelial-Stromal Tissues in Microgravity

    Science.gov (United States)

    Patel, Zarana S.; Wettergreen, Matthew A.; Huff, Janice L.

    2014-01-01

    We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3-D organotypic epithelial-stromal cultures that require an air-liquid interface. These complex 3-D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air-liquid interface, these 3-D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue-like human epithelial-stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air-liquid, liquid-liquid, and liquid-air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add-on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.

  20. Screening of the residual normal ovarian tissue adjacent to orthotopic epithelial ovarian carcinomas in nude mice.

    Science.gov (United States)

    Zhu, G H; Wang, S T; Yao, M Z; Cai, J H; Chen, C Y; Yang, Z X; Hong, L; Yang, S Y

    2014-04-16

    The objective of this study was to explore the feasibility and methods of screening the residual normal ovarian tissue adjacent to orthotopic ovarian carcinomas in nude mice. Human epithelial ovarian cancer cells (OVCAR3) were subcutaneously implanted for a tumor source and ovarian orthotopic transplantation. The cancer tissue, proximal paraneoplastic tissue, middle paraneoplastic tissue, remote paraneoplastic tissue, and normal ovarian tissue were removed. CK-7, CA125, p53, survivin, MMP-2, and TIMP-2 expression was detected by reverse transcription polymerase chain reaction. We obtained 35 paraneoplastic residual ovarian tissues with normal biopsies from 40 cases of an orthotopic epithelial ovarian carcinoma model (87.5%). CK-7, CA125, p53, survivin, MMP-2, and TIMP-2 expression was lower in proximal paraneoplastic tissue than in cancer tissue (P tissue (P tissue as well as among residual normal ovarian tissues with different severity (P > 0.05). In ovarian tissues of 20 normal nude mice, the expression of CK- 7, CA125, p53, survivin, MMP-2, and TIMP-2 was negative. Overall, the expression levels of CK-7, CA125, p53, survivin, MMP-2, TIMP-2, and other molecular markers showed a decreasing trend in the non-cancer tissue direction. The expression levels can be used as standards to screen residual normal ovarian tissue. We can obtain relatively safe normal ovarian tissues adjacent to epithelial ovarian cancer.

  1. The expression of Egfl7 in human normal tissues and epithelial tumors.

    Science.gov (United States)

    Fan, Chun; Yang, Lian-Yue; Wu, Fan; Tao, Yi-Ming; Liu, Lin-Sen; Zhang, Jin-Fan; He, Ya-Ning; Tang, Li-Li; Chen, Guo-Dong; Guo, Lei

    2013-04-23

    To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors.
 RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results.
 Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. 
 Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.

  2. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    International Nuclear Information System (INIS)

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-01-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

  3. Progress in planta transformation without tissue culture

    International Nuclear Information System (INIS)

    Gu Yunhong; Chinese Academy of Sciences, Hefei; Qin Guangyong; Huo Yuping; Yu Zengliang

    2004-01-01

    With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly. (authors)

  4. Contraction and elongation: Mechanics underlying cell boundary deformations in epithelial tissue.

    Science.gov (United States)

    Hara, Yusuke

    2017-06-01

    The cell-cell boundaries of epithelial cells form cellular frameworks at the apical side of tissues. Deformations in these boundaries, for example, boundary contraction and elongation, and the associated forces form the mechanical basis of epithelial tissue morphogenesis. In this review, using data from recent Drosophila studies on cell boundary contraction and elongation, I provide an overview of the mechanism underlying the bi-directional deformations in the epithelial cell boundary, that are sustained by biased accumulations of junctional and apico-medial non-muscle myosin II. Moreover, how the junctional tensions exist on cell boundaries in different boundary dynamics and morphologies are discussed. Finally, some future perspectives on how recent knowledge about single cell boundary-level mechanics will contribute to our understanding of epithelial tissue morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

  5. Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

    Science.gov (United States)

    Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

    2018-02-01

    Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

  6. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...

  7. The development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold.

    Science.gov (United States)

    O'Leary, Cian; Cavanagh, Brenton; Unger, Ronald E; Kirkpatrick, C James; O'Dea, Shirley; O'Brien, Fergal J; Cryan, Sally-Ann

    2016-04-01

    Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 μm and 80 μm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Lead, selenium and nickel concentrations in epithelial ovarian cancer, borderline ovarian tumor and healthy ovarian tissues.

    Science.gov (United States)

    Canaz, Emel; Kilinc, Metin; Sayar, Hamide; Kiran, Gurkan; Ozyurek, Eser

    2017-09-01

    Wide variation exists in ovarian cancer incidence rates suggesting the importance of environmental factors. Due to increasing environmental pollution, trace elements and heavy metals have drawn attention in studies defining the etiology of cancer, but scant data is available for ovarian cancer. Our aim was to compare the tissue concentrations of lead, selenium and nickel in epithelial ovarian cancer, borderline tumor and healthy ovarian tissues. The levels of lead, selenium and nickel were estimated using atomic absorption spectrophotometry in formalin-fixed paraffin-embedded tissue samples. Tests were carried out in 20 malignant epithelial ovarian cancer, 15 epithelial borderline tumor and 20 non-neoplastic healthy ovaries. Two samples were collected for borderline tumors, one from papillary projection and one from the smooth surface of cyst wall. Pb and Ni concentrations were found to be higher both in malignant and borderline tissues than those in healthy ovaries. Concentrations of Pb and Ni in malignant tissues, borderline papillary projections and capsular tissue samples were not different. Comparison of Se concentrations of malignant, borderline and healthy ovarian tissues did not reveal statistical difference. Studied metal levels were not found to be different in either papillary projection or in cyst wall of the borderline tumors. This study revealed the accumulation of lead and nickel in ovarian tissue is associated with borderline and malignant proliferation of the surface epithelium. Accumulation of these metals in epithelial ovarian cancer and borderline ovarian tumor has not been demonstrated before. Copyright © 2017 Elsevier GmbH. All rights reserved.

  9. [Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

    Science.gov (United States)

    Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

    2016-11-20

    To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (Ptissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (Ptissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

  10. A hybrid computational model to explore the topological characteristics of epithelial tissues.

    Science.gov (United States)

    González-Valverde, Ismael; García-Aznar, José Manuel

    2017-11-01

    Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Polarity in Mammalian Epithelial Morphogenesis

    OpenAIRE

    Roignot, Julie; Peng, Xiao; Mostov, Keith

    2013-01-01

    Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture model...

  12. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    Science.gov (United States)

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

  13. A functional model for adult stem cells in epithelial tissues.

    NARCIS (Netherlands)

    Verstappen, J.; Katsaros, C.; Torensma, R.; Hoff, J.W. Von den

    2009-01-01

    Tissue turnover, regeneration, and repair take place throughout life. Stem cells are key players in these processes. The characteristics and niches of the stem cell populations in different tissues, and even in related tissues, vary extensively. In this review, stem cell differentiation and stem

  14. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-01-01

    Human thyroid epithelial tissues from 23 individuals were obtained from surgical tissue, and cultured in vitro. Dose response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X-rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (D q ) values and extrapolation number (n) values greater than 1) at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X-rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  15. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-09-01

    Human thyroid epithelial tissue from 23 individuals was obtained from surgical tissue, and cultured in vitro. Dose-response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (Dsub(q)) values and extrapolation number (n) values greater than 1)* at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  16. Study on tissue culture for Gelidium seedling

    Science.gov (United States)

    Pei, Lu-Qing; Luo, Qi-Jun; Fei, Zhi-Qing; Ma, Bin

    1996-06-01

    As seedling culture is a crucial factor for successful cultivation of Gelidium, the authors researched tissue culture technology for producing seedlings. The morphogeny and experimental ecology were observed and studied fully in 2 5 mm isolated tissue fragments. Regeneration, appearance of branching creepers and attaching structure and new erect seedlings production and development were studied. Fragments were sown on bamboo slice and vinylon rope. The seedlings were cultured 20 30 days indoor, then cultured in the sea, where the density of erect seedlings was 3 19 seedlings/cm2, growth rate was 3.84% day. The frond arising from seedlings directly was up to 10 cm per year. The ecological conditions for regenerated seedlings are similar to the natural ones. The regenerated seedlings are suitable for raft culture in various sea areas.

  17. Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Kopecky, K.J.; Nakamura, Nori; Jones, M.P.; Ito, Toshio; Clifton, K.H.

    1986-09-01

    Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and X-irradiated to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5 %, the average mean lethal dose D 0 was 97.6 cGy for HY cells, and 96.7 cGy and 94.3 cGy, respectively, for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average D 0 of 85.0 cGy and PC cells a significantly (p = .001) lower average D 0 of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (p = .82) or sex (p = .10). These results suggest that malignant thyroid cells may be especially radiosensitive. (author)

  18. A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

    International Nuclear Information System (INIS)

    Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

    2005-01-01

    Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

  19. Active Vertex Model for cell-resolution description of epithelial tissue mechanics.

    Science.gov (United States)

    Barton, Daniel L; Henkes, Silke; Weijer, Cornelis J; Sknepnek, Rastko

    2017-06-01

    We introduce an Active Vertex Model (AVM) for cell-resolution studies of the mechanics of confluent epithelial tissues consisting of tens of thousands of cells, with a level of detail inaccessible to similar methods. The AVM combines the Vertex Model for confluent epithelial tissues with active matter dynamics. This introduces a natural description of the cell motion and accounts for motion patterns observed on multiple scales. Furthermore, cell contacts are generated dynamically from positions of cell centres. This not only enables efficient numerical implementation, but provides a natural description of the T1 transition events responsible for local tissue rearrangements. The AVM also includes cell alignment, cell-specific mechanical properties, cell growth, division and apoptosis. In addition, the AVM introduces a flexible, dynamically changing boundary of the epithelial sheet allowing for studies of phenomena such as the fingering instability or wound healing. We illustrate these capabilities with a number of case studies.

  20. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  1. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  2. Apoptosis related genes expressed in cultured Fallopian tube epithelial cells infected in vitro with Neisseria gonorrhoeae

    Directory of Open Access Journals (Sweden)

    PAZ A REYES

    2007-01-01

    Full Text Available Background: Infection of the Fallopian tubes (FT by Neisseria gonorrhoeae (Ngo can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. Aim: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. Materials and Methods: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Results: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1 without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I and TNFRSF1B (TNFR-II. Conclusion: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. Results strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed

  3. Fibronectin distribution in epithelial and associated tissues of the rat

    DEFF Research Database (Denmark)

    Couchman, J R; Gibson, W T; Thom, D

    1979-01-01

    Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated...... parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring...... in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues...

  4. Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

    Science.gov (United States)

    Deatly, Anne M.; Lin, Yen-Huei; McCarthy, Maureen; Chen, Wei; Miller, Lynn Z.; Quiroz, Jorge; Nowak, Becky M.; Lerch, Robert A.; Udem, Stephen A.; Goodwin, Thomas J.

    2012-01-01

    Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse

  5. Robust cell tracking in epithelial tissues through identification of maximum common subgraphs.

    Science.gov (United States)

    Kursawe, Jochen; Bardenet, Rémi; Zartman, Jeremiah J; Baker, Ruth E; Fletcher, Alexander G

    2016-11-01

    Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterizing cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a 'maximum common subgraph' to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as phototoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell-cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues. © 2016 The Authors.

  6. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

    Science.gov (United States)

    Lai, Jui-Yang; Ma, David Hui-Kang

    2013-01-01

    Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

  7. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

    Science.gov (United States)

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  8. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    Directory of Open Access Journals (Sweden)

    Babak Qasemi-Panahi

    2013-02-01

    Full Text Available Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1 on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 μL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  9. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues

    International Nuclear Information System (INIS)

    O'Grady, S.M.; Palfrey, H.C.; Field, M.

    1987-01-01

    This review summarizes our present understanding of Na-K-Cl cotransport and its physiological role in absorption and secretion of electrolytes and water in epithelial tissues. In the past several years an extensive literature about this cotransporter has developed due to its widespread distribution in a variety of cell types and its essential role in fluid and electrolyte transport in several epithelial tissues. We summarize this literature and speculate on the future characterization of this transport system. Although this review focuses on cotransport as it relates to absorptive and secretory processes in epithelia, important information concerning the pharmacology, stoichiometry, and regulation of Na-K-Cl cotransport in nonepithelial systems (i.e., erythrocytes, fibroblasts, squid axon, etc.) has been included to supplement areas that are less well established in the epithelial literature. 114 references

  10. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    Energy Technology Data Exchange (ETDEWEB)

    Aslanova, Afag [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Takagi, Ryo; Yamato, Masayuki; Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yamamoto, Masakazu, E-mail: yamamoto.ige@twmu.ac.jp [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  11. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    International Nuclear Information System (INIS)

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-01-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  12. Substituted Indoleacetic Acids Tested in Tissue Cultures

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1978-01-01

    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable...

  13. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Raquel Rodrigues-Diez

    2014-01-01

    Full Text Available Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β. Epithelial mesenchymal transition (EMT is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2 with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription. The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β.

  14. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

  15. Functional imaging and assessment of the glucose diffusion rate in epithelial tissues in optical coherence tomography

    International Nuclear Information System (INIS)

    Larin, K V; Tuchin, V V

    2008-01-01

    Functional imaging, monitoring and quantitative description of glucose diffusion in epithelial and underlying stromal tissues in vivo and controlling of the optical properties of tissues are extremely important for many biomedical applications including the development of noninvasive or minimally invasive glucose sensors as well as for therapy and diagnostics of various diseases, such as cancer, diabetic retinopathy, and glaucoma. Recent progress in the development of a noninvasive molecular diffusion biosensor based on optical coherence tomography (OCT) is described. The diffusion of glucose was studied in several epithelial tissues both in vitro and in vivo. Because OCT provides depth-resolved imaging of tissues with high in-depth resolution, the glucose diffusion is described not only as a function of time but also as a function of depth. (special issue devoted to application of laser technologies in biophotonics and biomedical studies)

  16. SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

    Directory of Open Access Journals (Sweden)

    Chengpeng Zhu

    Full Text Available We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM. The cultured pterygial epithelial cells (PECs were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4, which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(PH dehydrogenase (quinone 1 (NQO1, NF-E2-related factor-2 (NRF2 and superoxide dismutases (SOD2. Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: β-catenin, nonphospho-β-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6. We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

  17. Modulation of epithelial tissue and cell migration by microgrooves.

    NARCIS (Netherlands)

    Dalton, B.A.; Walboomers, X.F.; Dziegielewski, M.; Evans, M.D.; Taylor, S.; Jansen, J.A.; Steele, J.G.

    2001-01-01

    We used a polystyrene substratum to study the response of migrating epithelium to 1- or 5-microm depth microgrooves with groove/ridge widths of 1, 2, 5, or 10 microm. The migration of a tissue sheet was enhanced along the microgrooves, while migration across the microgrooves was inhibited. Changing

  18. NOD-Like Receptors in Intestinal Homeostasis and Epithelial Tissue Repair

    Science.gov (United States)

    Parlato, Marianna; Yeretssian, Garabet

    2014-01-01

    The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease. PMID:24886810

  19. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  20. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  1. Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

    Science.gov (United States)

    Rottgen, Trey S; Nickerson, Andrew J; Rajendran, Vazhaikkurichi M

    2018-05-10

    Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca 2+ -activated Cl − secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca 2+ -activated Cl − channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca 2+ -sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

  2. Esophageal 3D Culture Systems as Modeling Tools in Esophageal Epithelial Pathobiology and Personalized MedicineSummary

    Directory of Open Access Journals (Sweden)

    Kelly A. Whelan

    Full Text Available The stratified squamous epithelium of the esophagus shows a proliferative basal layer of keratinocytes that undergo terminal differentiation in overlying suprabasal layers. Esophageal pathologies, including eosinophilic esophagitis, gastroesophageal reflux disease, Barrett's esophagus, squamous cell carcinoma, and adenocarcinoma, cause perturbations in the esophageal epithelial proliferation-differentiation gradient. Three-dimensional (3D culture platforms mimicking in vivo esophageal epithelial tissue architecture ex vivo have emerged as powerful experimental tools for the investigation of esophageal biology in the context of homeostasis and pathology. Herein, we describe types of 3D culture that are used to model the esophagus, including organotypic, organoid, and spheroid culture systems. We discuss the development and optimization of various esophageal 3D culture models; highlight the applications, strengths, and limitations of each method; and summarize how these models have been used to evaluate the esophagus under homeostatic conditions as well as under the duress of inflammation and precancerous/cancerous conditions. Finally, we present future perspectives regarding the use of esophageal 3D models in basic science research as well as translational studies with the potential for personalized medicine. Keywords: Organotypic Culture, Organoid, Spheroid Culture, Esophageal Disease

  3. Tissue culture as a plant production technique for horticultural crops ...

    African Journals Online (AJOL)

    Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture. Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments ...

  4. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    Science.gov (United States)

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

  5. Tissue culture of surgically prepared temporalis fascia.

    Science.gov (United States)

    Walby, A P; Kerr, A G; Nevin, N C; Woods, G

    1982-10-01

    Temporalis fascia which is used to graft the tympanic membrane has been shown to be viable in tissue culture by a previous pilot study. This present study reports the effect on the viability of the fascia by scraping loose connective tissue from it and allowing it to dry. Pieces of fascia from 30 patients were each divided in 4 and prepared to give explants, fresh, fresh and scraped, dried, and dried and scraped. The fascia grew from 17 patients when cultured fresh, 5 when fresh and scraped, 1 when dried, and none when dried and scraped. These results are significantly different and show that the fascia is devitilized when prepared by the normal method for use in tympanoplasty.

  6. Looking beyond the genes: the interplay between signaling pathways and mechanics in the shaping and diversification of epithelial tissues

    OpenAIRE

    Urdy, Severine; Goudemand, Nicolas; Pantalacci, Sophie

    2016-01-01

    The core of Evo-Devo lies in the intuition that the way tissues grow during embryonic development, the way they sustain their structure and function throughout lifetime, and the way they evolve are closely linked. Epithelial tissues are ubiquitous in metazoans, covering the gut and internal branched organs, as well as the skin and its derivatives (ie, teeth). Here, we discuss in vitro, in vivo, and in silico studies on epithelial tissues to illustrate the conserved, dynamical, and complex asp...

  7. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  8. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

    Directory of Open Access Journals (Sweden)

    Lai JY

    2013-10-01

    Full Text Available Jui-Yang Lai,1–3 David Hui-Kang Ma4,5 1Institute of Biochemical and Biomedical Engineering, 2Biomedical Engineering Research Center, 3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 4Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 5Department of Chinese Medicine, Chang Gung University, Taoyuan, Taiwan Abstract: Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA cross-linked amniotic membrane (AM on limbal epithelial cell (LEC cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous

  9. Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens.

    Science.gov (United States)

    Chen, Jing; Toghi Eshghi, Shadi; Bova, George Steven; Li, Qing Kay; Li, Xingde; Zhang, Hui

    2013-12-01

    The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content. It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens. Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist's reading. We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results

  10. Canine Uterine Leiomyoma with Epithelial Tissue Foci, Adenomyosis, and Cystic Endometrial Hyperplasia

    OpenAIRE

    Karagiannis, George S.; Pelekanis, Mihalis; Loukopoulos, Panayiotis; Ververidis, Haris N.; Kaldrymidou, Eleni

    2011-01-01

    An 11-year-old Labrador Retriever bitch with a history of intermittent, sanguineous vaginal discharge of a six-month duration was presented. During exploratory laparotomy, two well-delineated, intramural masses were identified bilaterally in the uterine horns. Histopathologic examination of the mass on the left horn showed that it was a typical leiomyoma. However, the second mass appeared with an unusual coexistence of histological lesions, involving epithelial tissue foci, mild focal adenomy...

  11. The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues.

    Science.gov (United States)

    Santosh, Arvind Babu Rajendra; Jones, Thaon Jon

    2014-03-17

    In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs) are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.

  12. The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues

    Directory of Open Access Journals (Sweden)

    Arvind Babu Rajendra Santosh

    2014-03-01

    Full Text Available In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.

  13. Tumor suppressor roles of CENP-E and Nsl1 in Drosophila epithelial tissues.

    Science.gov (United States)

    Clemente-Ruiz, Marta; Muzzopappa, Mariana; Milán, Marco

    2014-01-01

    Depletion of spindle assembly checkpoint (SAC) genes in Drosophila epithelial tissues leads to JNK-dependent programmed cell death and additional blockade of the apoptotic program drives tumorigenesis. A recent report proposes that chromosomal instability (CIN) is not the driving force in the tumorigenic response of the SAC-deficient tissue, and that checkpoint proteins exert a SAC-independent tumor suppressor role. This notion is based on observations that the depletion of CENP-E levels or prevention of Bub3 from binding to the kinetochore in Drosophila tissues unable to activate the apoptotic program induces CIN but does not cause hyperproliferation. Here we re-examined this proposal. In contrast to the previous report, we observed that depletion of CENP-E or Nsl1-the latter mediating kinetochore targeting of Bub3-in epithelial tissues unable to activate the apoptotic program induces significant levels of aneuploidy and drives tumor-like growth. The induction of the JNK transcriptional targets Wingless, a mitogenic molecule, and MMP1, a matrix metaloproteinase 1 involved in basement membrane degradation was also observed in these tumors. An identical response of the tissue was previously detected upon depletion of several SAC genes or genes involved in spindle assembly, chromatin condensation, and cytokinesis, all of which have been described to cause CIN. All together, these results reinforce the role of CIN in driving tumorigenesis in Drosophila epithelial tissues and question the proposed SAC-independent roles of checkpoint proteins in suppressing tumorigenesis. Differences in aneuploidy rates might explain the discrepancy between the previous report and our results.

  14. Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

    Science.gov (United States)

    Krieg, Thomas; Abraham, David; Lafyatis, Robert

    2007-01-01

    Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

  15. Homeostatic pressure, tumor growth and fingering of epithelial tissues: Some generic physics arguments

    Science.gov (United States)

    Risler, Thomas

    2011-03-01

    We propose that one aspect of homeostasis is the regulation of tissues to preferred pressures, which can lead to a competition for space of purely mechanical origin and be an underlying mechanism for tumor growth. Surface and bulk contributions to pressure lead to the existence of a critical size that must be overcome by metastases to reach macroscopic sizes. This property qualitatively explains the observed size distributions of metastases, while size-independent growth rates cannot account for clinical and experimental data. It also potentially explains the observed preferential growth of metastases on tissue surfaces and membranes, suggests a mechanism underlying the seed and soil hypothesis introduced by Stephen Paget in 1889, and yields realistic values for metastatic inefficiency. Treating epithelial tissues as viscous fluids with effective cell division, we find a novel hydrodynamic instability that leads to the formation of fingering protrusions of the epithelium into the connective tissue. Arising from a combination of viscous friction effects and proliferation of the epithelial cells, this instability provides physical insight into a potential mechanism by which interfaces between epithelia and stroma undulate, and potentially by which tissue dysplasia leads to cancerous invasion. In collaboration with M. Basan, J.-F. Joanny, X. Sastre-Garau and J. Prost.

  16. Split-dose recovery in epithelial and vascular-connective tissue of pig skin

    International Nuclear Information System (INIS)

    Peel, D.M.; Hopewell, J.W.; Simmonds, R.H.; Dodd, P.; Meistrich, M.L.

    1984-01-01

    In the first 16 weeks after irradiation, two distinct waves of reaction can be observed in pig skin; the first wave (3-9 weeks) represents the expression of damage to the epithelium while the second is indicative of primary damage to the dermis, mediated through vascular injury. Following β-irradiation with a strontium-90 applicator, a severe epithelial reaction was seen with little subsequent dermal effects. X-rays (250 kV) on the other hand, produced a minimal epithelial response at doses which led to the development of dermal necrosis after 10-16 weeks. Comparison of single doses with two equal doses separated by 24 h produced a D 2 -D 1 value of 7.0 Gy at the doses which produced moist desquamation in 50% of fields (ED 50 ) after strontium-90 irradiation. After X-irradiation comparison of ED 50 doses for the later dermal reaction suggested a D 2 -D 1 value of 4.5 Gy. Over this same dose range of X-rays the D 2 -D 1 value for the first wave epithelial reaction was 3.5 Gy. These values of D 2 -D 1 for epithelial and dermal reactions in pig skin were compared with published data and were examined in relation to the theoretical predictions of a linear quadratic model for tissue target cell survival. The results were broadly in keeping with the productions of such a model. (Auth.)

  17. High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells. A relation between localization and function?

    NARCIS (Netherlands)

    Kooij, A.; Bosch, K. S.; Frederiks, W. M.; van Noorden, C. J.

    1992-01-01

    The localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells

  18. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Science.gov (United States)

    Cousins, Fiona L; Murray, Alison; Esnal, Arantza; Gibson, Douglas A; Critchley, Hilary O D; Saunders, Philippa T K

    2014-01-01

    In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These

  19. PRDM1 expression on the epithelial component but not on ectopic lymphoid tissues of Warthin tumour.

    Science.gov (United States)

    Wang, Y; Zhou, J; Zhang, Y; Wang, L; Liu, Y; Fan, L; Zhu, J; Xu, X; Huang, G; Li, X; Xun, W

    2015-05-01

    To determine the role of PRDM1, a key molecule for modulating the immune cells, in Warthin tumour (WT) pathogenesis. Forty paraffin-embedded parotid tissues of patients (mean age: 62.08 ± 11.90) with WT were retrieved from the pathology archives of Qindu Hospital from January 2012 to December 2012. The PRDM1 expression was investigated in a cohort of WT by immunohistochemistry. PRDM1 was expressed only on the epithelial component but not on ectopic lymphoid tissue of the tumour. Statistically, PRDM1 expression rates between WT glandular epithelial cells (40/40 cases) and the tumour-adjacent tissues (0/9 cases), and WT germinal centres (0/34 cases) and tonsil tissues (10/10 cases) were significantly different (P < 0.001), respectively. The PRDM1 expression appeared to play an essential role in WT pathogenesis. A better understanding of it might give options for revealing possible novel management strategies. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

    NARCIS (Netherlands)

    Kabgani, N.; Grigoleit, T.; Schulte, K.; Sechi, A.; Sauer-Lehnen, S.; Tag, C.; Boor, P.; Kuppe, C.; Warsow, G.; Schordan, S.; Mostertz, J.; Chilukoti, R.K.; Homuth, G.; Endlich, N.; Tacke, F.; Weiskirchen, R.; Fuellen, G.; Endlich, K.; Floege, J.; Smeets, B.; Moeller, M.J.

    2012-01-01

    Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or

  1. Interactions of virulent and avirulent leptospires with primary cultures of renal epithelial cells

    DEFF Research Database (Denmark)

    Ballard, S A; Williamson, M; Adler, B

    1986-01-01

    A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar cop...

  2. Dose response of tracheal epithelial cells to ionizing radiation in air-liquid interface cultures

    International Nuclear Information System (INIS)

    Fukutsu, K.; Yamada, Y.; Shimo, M.

    2002-01-01

    The dose-response relationships of tracheal epithelial cells to ionizing radiation was examined in air-liquid interface cultures, which were developed for the purpose of simulating in vivo conditions. The cultures investigated in this study were expected to be advantageous for the performance of irradiation experiments using short-range α rays. The level of dose response of air-liquid interface cultures to ionizing radiation proved to be the same as that for in vivo conditions. This result indicates that air-liquid interface cultures will prove most useful, to facilitate future studies for the investigation of the biological effects induced in tracheal epithelial cells by ionizing radiation, especially by α-rays. (orig.)

  3. Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Alice L Yu

    Full Text Available The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE induces cell loss, cellular senescence, and extracellular matrix (ECM synthesis in primary human retinal pigment epithelial (RPE cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J, connective tissue growth factor (CTGF, fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

  4. Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio

    Directory of Open Access Journals (Sweden)

    Wang YB

    2013-10-01

    Full Text Available Yanbo Wang, Xuxia Yan, Linglin Fu Marine Resources and Nutrition Biology Research Center, Food Quality and Safety Department, Zhejiang Gongshang University, Hangzhou, People's Republic of China Abstract: Nano-selenium (Se, with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp. Keywords: selenium nanoparticle, intestinal epithelial cell, crucian carp, primary culture

  5. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    This study was conducted with an objective of determining the correlates of adoption of tissue culture banana technology and its impacts on household incomes in Kenya. The results show that while some households have opted not to adopt tissue culture banana biotechnology, almost all the adopters are growing tissue ...

  6. Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

    Science.gov (United States)

    Song, Jiho; Shawky, Joseph H; Kim, YongTae; Hazar, Melis; LeDuc, Philip R; Sitti, Metin; Davidson, Lance A

    2015-07-01

    Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

    International Nuclear Information System (INIS)

    Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

    2005-01-01

    Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

  8. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  9. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

    Directory of Open Access Journals (Sweden)

    Ricardo Chaves Vilela

    2013-02-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  10. Bubbly vertex dynamics: A dynamical and geometrical model for epithelial tissues with curved cell shapes

    Science.gov (United States)

    Ishimoto, Yukitaka; Morishita, Yoshihiro

    2014-11-01

    In order to describe two-dimensionally packed cells in epithelial tissues both mathematically and physically, there have been developed several sorts of geometrical models, such as the vertex model, the finite element model, the cell-centered model, and the cellular Potts model. So far, in any case, pressures have not neatly been dealt with and the curvatures of the cell boundaries have been even omitted through their approximations. We focus on these quantities and formulate them in the vertex model. Thus, a model with the curvatures is constructed, and its algorithm for simulation is provided. The possible extensions and applications of this model are also discussed.

  11. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

    Science.gov (United States)

    Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

    2011-01-01

    We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

  12. Citrus tissue culture employing vegetative explants.

    Science.gov (United States)

    Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

    2001-11-01

    Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

  13. Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

    Science.gov (United States)

    Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

    2018-04-24

    Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. [Chromosome variability in the tissue culture of rare Gentiana species].

    Science.gov (United States)

    Tvardovs'ka, M O; Strashniuk, N M; Mel'nyk, V M; Adonin, V I; Kunakh, V A

    2008-01-01

    Cytogenetic analysis of plants and tissue culture of Gentiana lutea, G. punctata, G. acaulis has been carried out. Culturing in vitro was found to result in the changes of chromosome number in the calluses of the species involved. Species specificity for variation of the cultured cell genomes was shown. Contribution of the original plant genotypes to the cytogenetic structure of the tissue culture was established. Gentiana callus tissues (except for in vitro culture of G. punctata, derived from plant of Breskul'ska population) were found to exhibit modal class with the cells of diploid and nearly diploid chromosome sets.

  15. A minimal model of epithelial tissue dynamics and its application to the corneal epithelium

    Science.gov (United States)

    Henkes, Silke; Matoz-Fernandez, Daniel; Kostanjevec, Kaja; Coburn, Luke; Sknepnek, Rastko; Collinson, J. Martin; Martens, Kirsten

    Epithelial cell sheets are characterized by a complex interplay of active drivers, including cell motility, cell division and extrusion. Here we construct a particle-based minimal model tissue with only division/death dynamics and show that it always corresponds to a liquid state with a single dynamic time scale set by the division rate, and that no glassy phase is possible. Building on this, we construct an in-silico model of the mammalian corneal epithelium as such a tissue confined to a hemisphere bordered by the limbal stem cell zone. With added cell motility dynamics we are able to explain the steady-state spiral migration on the cornea, including the central vortex defect, and quantitatively compare it to eyes obtained from mice that are X-inactivation mosaic for LacZ.

  16. Expression of Tissue Factor in Epithelial Ovarian Carcinoma Is Involved in the Development of Venous Thromboembolism.

    Science.gov (United States)

    Sakurai, Manabu; Matsumoto, Koji; Gosho, Masahiko; Sakata, Akiko; Hosokawa, Yoshihiko; Tenjimbayashi, Yuri; Katoh, Takashi; Shikama, Ayumi; Komiya, Haruna; Michikami, Hiroo; Tasaka, Nobutaka; Akiyama-Abe, Azusa; Nakao, Sari; Ochi, Hiroyuki; Onuki, Mamiko; Minaguchi, Takeo; Yoshikawa, Hiroyuki; Satoh, Toyomi

    2017-01-01

    Our 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor (TF) in the development of venous thromboembolism (VTE) before treatment, especially in clear cell carcinoma (CCC). This follow-up study further investigated this possibility in a larger cohort. We investigated the intensity of TF expression (ITFE) and other variables for associations with VTE using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between November 2004 and December 2010, none of whom had received neoadjuvant chemotherapy. Before starting treatment, all patients were ultrasonographically screened for VTE. The ITFE was graded based on immunostaining of surgical specimens. Histological types were serous carcinoma (n = 42), CCC (n = 12), endometrioid carcinoma (n = 15), mucinous carcinoma (n = 53), and undifferentiated carcinoma (n = 6). The prevalence of VTE was significantly higher in CCC (34%) than in non-CCC (17%, P = 0.03). As ITFE increased, the frequencies of CCC and VTE increased significantly (P epithelial ovarian cancer may involve TF expression in cancer tissues.

  17. Active tension network model suggests an exotic mechanical state realized in epithelial tissues

    Science.gov (United States)

    Noll, Nicholas; Mani, Madhav; Heemskerk, Idse; Streichan, Sebastian J.; Shraiman, Boris I.

    2017-12-01

    Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behaviour remains an open problem. Here we formulate and analyse the active tension network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodelling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal--`isogonal'--deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit fly embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena.

  18. Mapping of Mechanical Strains and Stresses around Quiescent Engineered Three-Dimensional Epithelial Tissues

    Science.gov (United States)

    Gjorevski, Nikolce; Nelson, Celeste M.

    2012-01-01

    Understanding how physical signals guide biological processes requires qualitative and quantitative knowledge of the mechanical forces generated and sensed by cells in a physiologically realistic three-dimensional (3D) context. Here, we used computational modeling and engineered epithelial tissues of precise geometry to define the experimental parameters that are required to measure directly the mechanical stress profile of 3D tissues embedded within native type I collagen. We found that to calculate the stresses accurately in these settings, we had to account for mechanical heterogeneities within the matrix, which we visualized and quantified using confocal reflectance and atomic force microscopy. Using this technique, we were able to obtain traction forces at the epithelium-matrix interface, and to resolve and quantify patterns of mechanical stress throughout the surrounding matrix. We discovered that whereas single cells generate tension by contracting and pulling on the matrix, the contraction of multicellular tissues can also push against the matrix, causing emergent compression. Furthermore, tissue geometry defines the spatial distribution of mechanical stress across the epithelium, which communicates mechanically over distances spanning hundreds of micrometers. Spatially resolved mechanical maps can provide insight into the types and magnitudes of physical parameters that are sensed and interpreted by multicellular tissues during normal and pathological processes. PMID:22828342

  19. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Directory of Open Access Journals (Sweden)

    Fiona L Cousins

    Full Text Available BACKGROUND: In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. METHODOLOGY: A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4 withdrawal; mice received a single injection of bromodeoxyuridine (BrdU 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. PRINCIPAL FINDINGS: Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. CONCLUSIONS/SIGNIFICANCE: These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and

  20. 3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

    Science.gov (United States)

    Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

    2015-02-13

    Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

    Science.gov (United States)

    Gajewska, Malgorzata; McNally, Sara

    2017-01-01

    Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

  2. Cultured alveolar epithelial cells from septic rats mimic in vivo septic lung.

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    2010-06-01

    Full Text Available Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200-300 g were sacrificed 24 hours after cecal ligation and double puncture (2CLP or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A and MAPk (JNK, ERK, an p38 signaling activation, or barrier function was examined by measuring transepithelial resistance (TER or the flux of two molecular tracers (5 and 20 A. Inhibitors of JNK (SP600125, 20 microM and ERK (U0126, 10 microM were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm(2, respectively, however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique

  3. TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

    Science.gov (United States)

    Kannler, Martina; Lüling, Robin; Yildirim, Ali Önder; Gudermann, Thomas; Steinritz, Dirk; Dietrich, Alexander

    2018-05-12

    Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca 2+ ([Ca 2+ ] i ) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca 2+ ] i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca 2+ ] i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

  4. The effects of platelet gel on cultured human retinal pigment epithelial (hRPE cells

    Directory of Open Access Journals (Sweden)

    Sahar Balagholi

    2017-11-01

    Full Text Available The positive role of platelet gel (PG in tissue regeneration is well known, however, other characteristics of PG still remain to be determined. We investigated cellular and molecular changes in cultured human retinal pigment epithelial (hRPE cells when treated with different concentrations of PG named PG1, PG2, and PG3. hRPE cells were isolated from donor eyes of two newborn children, within 24 hours after their death. The cells were treated with three concentrations of PG for 7 days: 3 × 104/ml (PG1, 6 × 104/ml (PG2, and 9 × 104/ml (PG3. Fetal bovine serum was used as a control. Immunocytochemistry was performed with anti-RPE65 (H-85, anti-Cytokeratin 8/18 (NCL-5D3, and anti-PAX6 antibody. We used MTT assay to determine cell viability. Gene expressions of PAX6, MMP2, RPE65, ACTA2, MKI67, MMP9, and KDR were analyzed using real-time PCR. A significant increase in viability was observed for PG3-treated cells compared to control (p = 0.044 and compared to PG1 group (p = 0.027, on day 7. Cellular elongation together with dendritiform extensions were observed in PG-treated cells on days 1 and 3, while epithelioid morphology was observed on day 7. All cells were immunoreactive for RPE65, cytokeratin 8/18, and PAX6. No significant change was observed in the expression of MKI67 and PAX6, but the expressions of MMP2, MMP9, ACTA2, and KDR were significantly higher in PG2-treated cells compared to controls (p < 0.05. Our results indicate that increased concentration of PG and extended exposure time have positive effects on viability of hRPE cells. PG may be useful for hRPE cell encapsulation in retinal cell replacement therapy.

  5. The effect of mechanical extension stimulation combined with epithelial cell sorting on outcomes of implanted tissue-engineered muscular urethras.

    Science.gov (United States)

    Fu, Qiang; Deng, Chen-Liang; Zhao, Ren-Yan; Wang, Ying; Cao, Yilin

    2014-01-01

    Urethral defects are common and frequent disorders and are difficult to treat. Simple natural or synthetic materials do not provide a satisfactory curative solution for long urethral defects, and urethroplasty with large areas of autologous tissues is limited and might interfere with wound healing. In this study, adipose-derived stem cells were used. These cells can be derived from a wide range of sources, have extensive expansion capability, and were combined with oral mucosal epithelial cells to solve the problem of finding seeding cell sources for producing the tissue-engineered urethras. We also used the synthetic biodegradable polymer poly-glycolic acid (PGA) as a scaffold material to overcome issues such as potential pathogen infections derived from natural materials (such as de-vascular stents or animal-derived collagen) and differing diameters. Furthermore, we used a bioreactor to construct a tissue-engineered epithelial-muscular lumen with a double-layer structure (the epithelial lining and the muscle layer). Through these steps, we used an epithelial-muscular lumen built in vitro to repair defects in a canine urethral defect model (1 cm). Canine urethral reconstruction was successfully achieved based on image analysis and histological techniques at different time points. This study provides a basis for the clinical application of tissue engineering of an epithelial-muscular lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Versatile electrochemial sensor for tissue culturing and sample handling

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Kwasny, Dorota; Al Atraktchi, Fatima Al-Zahraa

    2014-01-01

    Culturing of organtypic brain tissues is a routine procedure in neural research. The visual inspection of the medium is the only way of determining the state of the tissue. At the end of culturing, post-processing techniques such as HPLC can be used to measure the concentration of the secreted...

  7. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    International Nuclear Information System (INIS)

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G.

    2007-01-01

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells

  8. Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

    Science.gov (United States)

    Murase, Hiromi; Tsuruma, Kazuhiro; Kuse, Yoshiki; Shimazawa, Masamitsu; Hara, Hideaki

    2017-12-01

    Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Effect of lunar materials on plant tissue culture.

    Science.gov (United States)

    Walkinshaw, C. H.; Venketeswaran, S.; Baur, P. S.; Croley, T. E.; Scholes, V. E.; Weete, J. D.; Halliwell, R. S.; Hall, R. H.

    1973-01-01

    Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials.

  10. A novel porous scaffold fabrication technique for epithelial and endothelial tissue engineering.

    Science.gov (United States)

    McHugh, Kevin J; Tao, Sarah L; Saint-Geniez, Magali

    2013-07-01

    Porous scaffolds have the ability to minimize transport barriers for both two- (2D) and three-dimensional tissue engineering. However, current porous scaffolds may be non-ideal for 2D tissues such as epithelium due to inherent fabrication-based characteristics. While 2D tissues require porosity to support molecular transport, pores must be small enough to prevent cell migration into the scaffold in order to avoid non-epithelial tissue architecture and compromised function. Though electrospun meshes are the most popular porous scaffolds used today, their heterogeneous pore size and intense topography may be poorly-suited for epithelium. Porous scaffolds produced using other methods have similar unavoidable limitations, frequently involving insufficient pore resolution and control, which make them incompatible with 2D tissues. In addition, many of these techniques require an entirely new round of process development in order to change material or pore size. Herein we describe "pore casting," a fabrication method that produces flat scaffolds with deterministic pore shape, size, and location that can be easily altered to accommodate new materials or pore dimensions. As proof-of-concept, pore-cast poly(ε-caprolactone) (PCL) scaffolds were fabricated and compared to electrospun PCL in vitro using canine kidney epithelium, human colon epithelium, and human umbilical vein endothelium. All cell types demonstrated improved morphology and function on pore-cast scaffolds, likely due to reduced topography and universally small pore size. These results suggest that pore casting is an attractive option for creating 2D tissue engineering scaffolds, especially when the application may benefit from well-controlled pore size or architecture.

  11. In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

    Science.gov (United States)

    Schmid, Richard; Tarau, Ioana-Sandra; Rossi, Angela; Leonhardt, Stefan; Schwarz, Thomas; Schuerlein, Sebastian; Lotz, Christian; Hansmann, Jan

    2018-01-01

    The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  12. Active Tension Network model reveals an exotic mechanical state realized in epithelial tissues

    Science.gov (United States)

    Noll, Nicholas; Mani, Madhav; Heemskerk, Idse; Streicha, Sebastian; Shraiman, Boris

    Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that mechanical balance of cells is dominated by cortical tension and introduces tension dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties: i) ATN behaves as a fluid at short times, but at long times it supports external tension, like a solid; ii) its mechanical equilibrium state has extensive degeneracy associated with a discrete conformal - ''isogonal'' - deformation of cells. ATN model predicts a constraint on equilibrium cell geometry, which we demonstrate to hold in certain epithelial tissues. We further show that isogonal modes are observed in a fruit fly embryo, accounting for the striking variability of apical area of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, understanding which helps understand biological phenomena.

  13. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  14. ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

    Science.gov (United States)

    Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

    2016-05-01

    ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells. © 2016 Federation of European Biochemical Societies.

  15. Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues.

    Science.gov (United States)

    Xiong, Jimin; Gronthos, Stan; Bartold, P Mark

    2013-10-01

    Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth-supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal-regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem-cell populations that demonstrate similar properties to mesenchymal stromal

  16. Canine Uterine Leiomyoma with Epithelial Tissue Foci, Adenomyosis, and Cystic Endometrial Hyperplasia

    Directory of Open Access Journals (Sweden)

    George S. Karagiannis

    2011-01-01

    Full Text Available An 11-year-old Labrador Retriever bitch with a history of intermittent, sanguineous vaginal discharge of a six-month duration was presented. During exploratory laparotomy, two well-delineated, intramural masses were identified bilaterally in the uterine horns. Histopathologic examination of the mass on the left horn showed that it was a typical leiomyoma. However, the second mass appeared with an unusual coexistence of histological lesions, involving epithelial tissue foci, mild focal adenomyosis, and cystic endometrial hyperplasia. Interestingly, such combination was never encountered before in dogs. Although uterine leiomyoma is quite usual in the reproductive system of female dogs, this case resembled relevant cases of human uterine adenomyomas in morphology, and thus it was offered a similar tentative diagnosis.

  17. Application of Hanging Drop Technique for Kidney Tissue Culture.

    Science.gov (United States)

    Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

    2017-01-01

    The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

  18. Application of Hanging Drop Technique for Kidney Tissue Culture

    Directory of Open Access Journals (Sweden)

    Shaohui Wang

    2017-05-01

    Full Text Available Background/Aims: The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. Methods: In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. Results: The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. Conclusions: We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli.

  19. Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants

    Science.gov (United States)

    Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

    2000-01-01

    Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. PMID:11085750

  20. Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

    Science.gov (United States)

    Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2013-01-01

    We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

  1. Leptin induction following irradiation is a conserved feature in mammalian epithelial cells and tissues.

    Science.gov (United States)

    Licursi, Valerio; Cestelli Guidi, Mariangela; Del Vecchio, Giorgia; Mannironi, Cecilia; Presutti, Carlo; Amendola, Roberto; Negri, Rodolfo

    2017-09-01

    Leptin (LEP) is a peptide hormone with multiple physiological functions. Besides its systemic actions, it has important peripheral roles such as a mitogen action on keratinocytes following skin lesions. We previously showed that LEP mRNA is significantly induced in response to neutron irradiation in mouse skin and that the protein increases in the irradiated epidermis and in the related subcutaneous adipose tissue. In this work, we investigated the post-transcriptional regulation of LEP by miRNAs and the conservation of LEP's role in radiation response in human cells. We used microarray analysis and real-time polymerase chain reaction (RT-PCR) to analyze modulation of miRNAs potentially targeting LEP in mouse skin following irradiation and bioinformatic analysis of transcriptome of irradiated human cell lines and cancer tissues from radiotherapy-treated patients to evaluate LEP expression. We show that a network of miRNAs potentially targeting LEP mRNA is modulated in irradiated mouse skin and that LEP itself is significantly modulated by irradiation in human epithelial cell lines and in breast cancer tissues from radiotherapy-treated patients. These results confirm and extend the previous evidence that LEP has a general and important role in the response of mammalian cells to irradiation.

  2. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  3. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  4. In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration.

    Science.gov (United States)

    Geng, Hua; Bu, Heng-Fu; Liu, Fangyi; Wu, Longtao; Pfeifer, Karl; Chou, Pauline M; Wang, Xiao; Sun, Jiaren; Lu, Lu; Pandey, Ashutosh; Bartolomei, Marisa S; De Plaen, Isabelle G; Wang, Peng; Yu, Jindan; Qian, Jiaming; Tan, Xiao-Di

    2018-04-03

    Inflammation affects regeneration of the intestinal epithelia; long non-coding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation. We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19 ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs. In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found levels of H19 only changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, mice with LPS-induced sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA

  5. Air exposure induced characteristics of dry eye in conjunctival tissue culture.

    Directory of Open Access Journals (Sweden)

    Hui Lin

    Full Text Available There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

  6. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera Hernández (Mónica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  7. Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

    NARCIS (Netherlands)

    Pyrc, Krzysztof; Sims, Amy C.; Dijkman, Ronald; Jebbink, Maarten; Long, Casey; Deming, Damon; Donaldson, Eric; Vabret, Astrid; Baric, Ralph; van der Hoek, Lia; Pickles, Raymond

    2010-01-01

    Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is

  8. Effects of ionizing radiation on plant tissue cultures

    International Nuclear Information System (INIS)

    Hell, K.G.

    1978-01-01

    A short review is done of the biological effects of ionizing radiations on plant tissues kept in culture, from the work of Gladys King, in 1949, with X-ray irradiated tobacco. The role of plant hormones is discussed in the processes of growth inhibition and growth restoration of irradiated tissues, as well as morphogenesis. Radioresistance of cells kept in culture and the use of ionizing radiations as mutagens are also commented. Some aspects of the biological effects of ionizing radiations that need to be investigated are discussed, and the problem of genome instability of plant tissues kept in culture is pointed out. (M.A.) [pt

  9. Effect of Zebularine loaded MePEG-PCL nanoparticles on viability, attachment of in vitro cultured lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Si-Wei Liu

    2015-01-01

    Full Text Available AIM: To investigate the effect of zebularine(Zebloaded Poly(ethylene glycol-block-poly(ε-caprolactonemethyl ether(MePEG-PCLnanoparticles(NPson the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells(LECs. METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L(ZebF1 group, 100μmol/L(ZebF2 group, Zeb -loaded MePEG-PCL NPs 50μmol/L(ZebNP1 group, Zeb -loaded MePEG-PCL NPs 100μmol/L(ZebNP2 group, MePEG-PCL empty NPs(NPs groupor blank medium(group Crespectively. A tetrazolium dye assay(MTTtest and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis. RESULTS: Determined by MTT colorimetric method: Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner(PPP ZebNP1>ZebF2(PCONCLUSION: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups, as well as enhancing the apoptosis.

  10. Gefarnate stimulates mucin-like glycoprotein secretion in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models

    Directory of Open Access Journals (Sweden)

    Dota A

    2013-01-01

    Full Text Available Atsuyoshi Dota, Yuko Takaoka-Shichijo, Masatsugu NakamuraOphthalmic Research and Development Center, Santen Pharmaceutical Co, Ltd, Ikoma-shi, Nara, JapanPurpose: The aim of this study was to evaluate the effect of gefarnate on mucin-like glycoprotein secretion in isolated rabbit conjunctival tissue, and on corneal epithelial damage in rabbit and cat dry-eye models.Methods: Conjunctival tissue isolated from rabbits was treated with gefarnate. Mucin-like glycoprotein was detected in the culture supernatant by an enzyme-linked lectin assay. Gefarnate ointment was topically applied to eyes once daily for 7 days in the rabbit dry-eye model, in which the lacrimal glands, Harderian gland, and nictitating membrane were removed, or for 4 weeks in the cat dry-eye model, in which the lacrimal gland and nictitating membrane were removed. Corneal epithelial damage was evaluated by measurement of corneal permeability by rose bengal in the rabbit model or by fluorescein staining in the cat model.Results: Gefarnate stimulated mucin-like glycoprotein secretion in conjunctival tissue in a dose-dependent manner. In the rabbit dry-eye model, application of gefarnate ointment to the eyes resulted in a dose-dependent decrease in rose bengal permeability in the cornea, with the effect being significant at concentrations of ≥0.3%. In the cat dry-eye model, application of gefarnate ointment resulted in a significant decrease in the corneal fluorescein staining score.Conclusion: These results suggest that gefarnate stimulates in vitro secretion of mucin-like glycoprotein in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models. Gefarnate may therefore be effective for treating dry eye.Keywords: gefarnate, fluorescein staining, rose bengal permeability, rabbit, cat, dry eye

  11. Epithelial tissue hyperplasia induced by the RAF inhibitor PF-04880594 is attenuated by a clinically well-tolerated dose of the MEK inhibitor PD-0325901.

    Science.gov (United States)

    Torti, Vince R; Wojciechowicz, Donald; Hu, Wenyue; John-Baptiste, Annette; Evering, Winston; Troche, Gabriel; Marroquin, Lisa D; Smeal, Tod; Yamazaki, Shinji; Palmer, Cynthia L; Burns-Naas, Leigh Ann; Bagrodia, Shubha

    2012-10-01

    Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers. ©2012 AACR.

  12. Banana Musa tissue culture plants enhanced by endophytic fungi

    African Journals Online (AJOL)

    Mo

    Merging biotechnology with biological control: Banana Musa tissue culture plants enhanced by endophytic .... While working in the laminar flow cabinet, sterile filter papers were placed in ..... University of Bonn, Bonn, Germany. Niere, B., 2001.

  13. Low technology tissue culture materials for initiation and ...

    African Journals Online (AJOL)

    Low technology tissue culture materials for initiation and multiplication of banana plants. ... African Crop Science Journal ... locally available macronutrients, micronutrients, sugar, equipment and facility reduced the cost of consumable material

  14. Response of cultured human airway epithelial cells to X-rays and energetic α-particles

    International Nuclear Information System (INIS)

    Yang, T.C.; Holley, W.R.; Curtis, S.B.; Gruenert, D.C.; California Univ., San Francisco, CA

    1990-01-01

    Radon and its progeny, which emit α-particles during decay, may play an important role in inducing human lung cancer. To gain a better understanding of the biological effects of α-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions. Experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus. The RBE for cell inactivation determined by the ratio of D 0 for X-rays to that for 8 MeV helium ions was 1.8-2.2. The cross-section for helium ions, calculated from the D 0 value, was about 24 μm 2 for cells of the primary culture. This cross-section is significantly smaller than the average geometric nuclear area (∼ 180 μm 2 ), suggesting that an average of 7.5 α-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion. (author)

  15. Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

    OpenAIRE

    Klinkspoor, J H; Yoshida, T; Lee, S P

    1998-01-01

    1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoprot...

  16. Lysophosphatidic acid signaling through its receptor initiates profibrotic epithelial cell fibroblast communication mediated by epithelial cell derived connective tissue growth factor.

    Science.gov (United States)

    Sakai, Norihiko; Chun, Jerold; Duffield, Jeremy S; Lagares, David; Wada, Takashi; Luster, Andrew D; Tager, Andrew M

    2017-03-01

    The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA 1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA 1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA 1 -deficient (LPA 1 -/- ) or -sufficient (LPA 1 +/+ ) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA 1 -/- Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA 1 -/- Col-GFP mice. LPA-LPA 1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA 1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues.

    Science.gov (United States)

    Greenwalt, D E; Mather, I H

    1985-02-01

    A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.

  18. Co-culture in cartilage tissue engineering.

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Riesle, J.U.; van Blitterswijk, Clemens

    2007-01-01

    For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to

  19. Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

    Science.gov (United States)

    Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

    2015-01-01

    PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

  20. Human meniscal proteoglycan metabolism in long-term tissue culture

    NARCIS (Netherlands)

    Verbruggen, G.; Verdonk, R.; Veys, E. M.; van Daele, P.; de Smet, P.; van den Abbeele, K.; Claus, B.; Baeten, D.

    1996-01-01

    For the purpose of human meniscal allografting, menisci have been maintained viable in in vitro culture. The influence of long-term tissue culture on the extracellular matrix metabolism of the meniscus has been studied. Fetal calf serum (FCS) was used as a supplement for the growth factors necessary

  1. Vascular endothelial growth factor polymorphisms and a synchronized examination of plasma and tissue expression in epithelial ovarian cancers.

    Science.gov (United States)

    Bhaskari, J; Premalata, C S; Shilpa, V; Rahul, B; Pallavi, V R; Ramesh, G; Krishnamoorthy, Lakshmi

    2016-01-01

    In this study, we have analyzed six genetic polymorphisms of the VEGF-A gene and correlated the genetic data with plasma and tissue expression of VEGF-A in epithelial ovarian carcinomas. A total of 130 cases including 95 malignant carcinomas, 17 low malignant potential and 18 benign tumours were studied. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were studied by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma levels of VEGF-A were estimated by enzyme-linked immunosorbent assay (ELISA) and tissue expression of VEGF-A by immunohistochemistry (IHC). Four polymorphisms of the above excluding rs699947 and rs3025039 showed significant association with malignancy, and we observed the presence of positive correlation between haplotype CCGGCC and increased expression of VEGF-A in both plasma and tissues which also correlated with poor prognosis and recurrence suggesting a probable increase in resistance to treatment in such carriers. Highly upregulated tissue expression of VEGF-A was seen in all epithelial ovarian carcinomas with intensity of expression increasing from benign to malignant cases. ELISA data from our study showed an increase in circulating levels of VEGF-A in malignancies. VEGF-A plasma levels can be employed as a biomarker for high-grade malignancy in epithelial ovarian cancers alongside tissue expression and CA-125 levels. This study is unique due to the fact that a simultaneous analysis of plasma and tissue expression has been demonstrated and is a first such study in epithelial ovarian cancers and representing the Indian population (South-east Asian) synchronized with genetic polymorphism data as well.

  2. Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

    Science.gov (United States)

    Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

    2014-12-01

    Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

  3. Epithelial-Mesenchymal Interactions in Urinary Bladder and Small Intestine and How to Apply Them in Tissue Engineering.

    Science.gov (United States)

    Jerman, Urška Dragin; Kreft, Mateja Erdani; Veranič, Peter

    2015-12-01

    Reciprocal interactions between the epithelium and mesenchyme are essential for the establishment of proper tissue morphology during organogenesis and tissue regeneration as well as for the maintenance of cell differentiation. With this review, we highlight the importance of epithelial-mesenchymal cross talk in healthy tissue and further discuss its significance in engineering functional tissues in vitro. We focus on the urinary bladder and small intestine, organs that are often compromised by disease and are as such in need of research that would advance effective treatment or tissue replacement. To date, the understanding of epithelial-mesenchymal reciprocal interactions has enabled the development of in vitro biomimetic tissue equivalents that have provided many possibilities in treating defective, damaged, or even cancerous tissues. Although research of the past several years has advanced the field of bladder and small intestine tissue engineering, one must be aware of its current limitations in successfully and above all safely introducing tissue-engineered constructs into clinical practice. Special attention is in particular needed when treating cancerous tissues, as initially successful tumor excision and tissue reconstruction may later on result in cancer recurrence due to oncogenic signals originating from an altered stroma. Recent rather poor outcomes in pioneering clinical trials of bladder reconstructions should serve as a reminder that recreating a functional organ to replace a dysfunctional one is an objective far more difficult to reach than initially foreseen. When considering effective tissue engineering approaches for diseased tissues in humans, it is imperative to introduce animal models with dysfunctional or, even more importantly, cancerous organs, which would greatly contribute to predicting possible complications and, hence, reducing risks when translating to the clinic.

  4. Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Sedarski, John A; Dylla, Brenda L; Shannon, Samantha K; Amirahmadi, Fazlollaah; Hughes, John G; Cheng, Allen C; Patel, Robin

    2017-09-01

    Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods. Copyright © 2017 American Society for Microbiology.

  5. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    Directory of Open Access Journals (Sweden)

    Shilpi Verghese

    2016-09-01

    Full Text Available Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1 and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.

  6. Chemo-mechanical modeling of tumor growth in elastic epithelial tissue

    Energy Technology Data Exchange (ETDEWEB)

    Bratsun, Dmitry A., E-mail: bratsun@pspu.ru [Department of Applied Physics, Perm National Research Polytechnical University, Perm, 614990 (Russian Federation); Zakharov, Andrey P. [Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa, 32000 Israel (Israel); Theoretical Physics Department, Perm State Humanitarian Pedagogical University, Perm, 614990 (Russian Federation); Pismen, Len [Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa, 32000 Israel (Israel)

    2016-08-02

    We propose a multiscale chemo-mechanical model of the cancer tumor development in the epithelial tissue. The epithelium is represented by an elastic 2D array of polygonal cells with its own gene regulation dynamics. The model allows the simulation of the evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm includes the division and intercalation of cells as well as the transformation of normal cells into a cancerous state triggered by a local failure of the spatial synchronization of the cellular rhythms driven by transcription/translation processes. Both deterministic and stochastic descriptions of the system are given for chemical signaling. The transformation of cells means the modification of their respective parameters responsible for chemo-mechanical interactions. The simulations reproduce a distinct behavior of invasive and localized carcinoma. Generally, the model is designed in such a way that it can be readily modified to take account of any newly understood gene regulation processes and feedback mechanisms affecting chemo-mechanical properties of cells.

  7. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  8. Chemo-mechanical modeling of tumor growth in elastic epithelial tissue

    Science.gov (United States)

    Bratsun, Dmitry A.; Zakharov, Andrey P.; Pismen, Len

    2016-08-01

    We propose a multiscale chemo-mechanical model of the cancer tumor development in the epithelial tissue. The epithelium is represented by an elastic 2D array of polygonal cells with its own gene regulation dynamics. The model allows the simulation of the evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm includes the division and intercalation of cells as well as the transformation of normal cells into a cancerous state triggered by a local failure of the spatial synchronization of the cellular rhythms driven by transcription/translation processes. Both deterministic and stochastic descriptions of the system are given for chemical signaling. The transformation of cells means the modification of their respective parameters responsible for chemo-mechanical interactions. The simulations reproduce a distinct behavior of invasive and localized carcinoma. Generally, the model is designed in such a way that it can be readily modified to take account of any newly understood gene regulation processes and feedback mechanisms affecting chemo-mechanical properties of cells.

  9. Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

    Science.gov (United States)

    Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

    2016-01-01

    The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

  10. Clonal propagation of eucalyptus by tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Mehra-Palta, A.

    1982-07-01

    Multiple adventitious buds were induced on cotyledons, shoot tips and nodal stem segments of Eucalyptus species cultured on a defined nutrient medium supplemented with the cytokinin zeatin and the auxin indole-3-butyric acid (IBA). The adventitious buds could be recycled on cytokinin medium to produce more buds thus providing the possibility of producing large clones from selected genotypes. The adventitious shoots were rooted in auxin medium and some of the resulting propagules were outplanted in the field. These techniques have the potential for use in the genetic improvement of Eucalyptus. (Refs. 15).

  11. Looking Beyond the Genes: The Interplay Between Signaling Pathways and Mechanics in the Shaping and Diversification of Epithelial Tissues.

    Science.gov (United States)

    Urdy, S; Goudemand, N; Pantalacci, S

    2016-01-01

    The core of Evo-Devo lies in the intuition that the way tissues grow during embryonic development, the way they sustain their structure and function throughout lifetime, and the way they evolve are closely linked. Epithelial tissues are ubiquitous in metazoans, covering the gut and internal branched organs, as well as the skin and its derivatives (ie, teeth). Here, we discuss in vitro, in vivo, and in silico studies on epithelial tissues to illustrate the conserved, dynamical, and complex aspects of their development. We then explore the implications of the dynamical and nonlinear nature of development on the evolution of their size and shape at the phenotypic and genetic levels. In rare cases, when the interplay between signaling and mechanics is well understood at the cell level, it is becoming clear that the structure of development leads to covariation of characters, an integration which in turn provides some predictable structure to evolutionary changes. We suggest that such nonlinear systems are prone to genetic drift, cryptic genetic variation, and context-dependent mutational effects. We argue that experimental and theoretical studies at the cell level are critical to our understanding of the phenotypic and genetic evolution of epithelial tissues, including carcinomas. © 2016 Elsevier Inc. All rights reserved.

  12. KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts.

    Science.gov (United States)

    Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C; Warwar, Ronald; Brown, Thomas L; Lauf, Peter K

    2006-11-01

    We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

  13. A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

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    Yu Takahashi

    2018-01-01

    Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

  14. Development of a vinasse culture medium for plant tissue culture

    International Nuclear Information System (INIS)

    Silva, A.L.L.D.; Gollo, L.

    2014-01-01

    Vinasse is the main pollutant (effluent) obtained from the distillation of sugarcane in the production of fuel alcohol. However, this residue is rich in nutrients that are required by plants. We developed a new culture medium using vinasse for the In vitro propagation of an orchid. The vinasse was treated (decanted and filtered), and the nutrients were determined and quantified. Different formulations using vinasse were tested for an In vitro culture. The vinasse dilutions demonstrated a good buffering effect. The ideal vinasse dilution for media formulation was 2.5%. The best KC formulations with vinasse were KCV1 and KCV5. Compared to KC medium, these formulations demonstrated similar results for In vitro multiplication, with the exception of protocorm-like body number, which was inferior in the vinasse formulations. Conversely, for In vitro elongation and rooting, these vinasse media were superior to KC medium. KC medium promotes a low rooting rate (8%) compared to 68 and 100% obtained by KCV1 and KCV5, respectively. Moreover, plantlets cultured on KC medium become protocorm-like body clusters, which impeded the acclimatization of these explants. Plantlets elongated and rooted on KCV1 and KCV5 were successfully acclimatized with a 91% survival rate for both KC vinasse formulations. This study shows the great potential of this technology as a rational alternative to vinasse disposal and adds value to what is currently considered a waste product. (author)

  15. Culture of Iris Pigment Epithelial Cells on Expanded-Polytetrafluroethylene (ePTFE Substrates for the Treatment of Age-Related Macular Degeneration

    Directory of Open Access Journals (Sweden)

    S Nian

    2011-05-01

    Full Text Available Introduction: Transplantation of an intact differentiated retinal pigment epithelial (RPE cell layer may provide a means to treat Age-Related Macular Degeneration (AMD. However, harvesting RPE cells can be a technically complicated procedure. Our current work aimed to prepare intact differentiated iris pigment epithelial (IPE cell layers, which are easy to obtain and have the same embryonic origin and similar properties as RPE cells, on ePTFE substrates for transplantation purposes to rescue deteriorated photoreceptors in AMD. Methods: IPE cells isolated from rat eyes were seeded on different substrates, including fibronectin n-heptylamine (HA ePTFE substrates, HA ePTFE substrates, ePTFE substrates and fibronectin tissue culture polystyrene (TCPS as control. Cell number and morphology were assessed at each time interval. The formation of tight junction was examined by immunostaining of junction proteins. Results: An obvious increasing trend of cell number was observed in IPE cells on fibronectin n-heptylamine (HA ePTFE substrate, exhibiting heavy pigmentation and epithelial morphology. At Day 28, tight junction formation was indicated by cell-cell junctional proteins along cell borders. Conclusion: Harvested IPE cells cultured on fibronectin HA-ePTFE substrates can differentiate and form a cell monolayer that may be suitable for transplantation.

  16. Epithelial-mesenchymal transition in keloid tissues and TGF-β1-induced hair follicle outer root sheath keratinocytes.

    Science.gov (United States)

    Yan, Li; Cao, Rui; Wang, Lianzhao; Liu, Yuanbo; Pan, Bo; Yin, Yanhua; Lv, Xiaoyan; Zhuang, Qiang; Sun, Xuejian; Xiao, Ran

    2015-01-01

    Keloid is a skin fibrotic disease with the characteristics of recurrence and invasion, its pathogenesis still remains unrevealed. The epithelial-mesenchymal transition (EMT) is critical for wound healing, fibrosis, recurrence, and invasion of cancer. We sought to investigate the EMT in keloid and the mechanism through which the EMT regulates keloid formation. In keloid tissues, the expressions of EMT-associated markers and transforming growth factor (TGF)-β1/Smad3 signaling were examined by immunohistochemistry. In the keloid epidermis and dermal tissue, the expressions of genes related to the regulation of skin homeostasis, fibroblast growth factor receptor 2 (FGFR2) and p63, were analyzed using quantitative real-time polymerase chain reaction. The results showed that accompanying the loss of the epithelial marker E-cadherin and the gain of the mesenchymal markers fibroblast-specific protein 1 (FSP1) and vimentin in epithelial cells from epidermis and skin appendages, and in endothelial cells from dermal microvessels, enhanced TGF-β1 expression and Smad3 phosphorylation were noted in keloid tissues. Moreover, alternative splicing of the FGFR2 gene switched the predominantly expressed isoform from FGFR2-IIIb to -IIIc, concomitant with the decreased expression of ΔNp63 and TAp63, which changes might partially account for abnormal epidermis and appendages in keloids. In addition, we found that TGF-β1-induced hair follicle outer root sheath keratinocytes (ORSKs) and normal skin epithelial cells underwent EMT in vitro with ORSKs exhibiting more obvious EMT changes and more similar expression profiles for EMT-associated and skin homeostasis-related genes as in keloid tissues, suggesting that ORSKs might play crucial roles in the EMT in keloids. Our study provided insights into the molecular mechanisms mediating the EMT pathogenesis of keloids. © 2015 by the Wound Healing Society.

  17. Oxygen and tissue culture affect placental gene expression.

    Science.gov (United States)

    Brew, O; Sullivan, M H F

    2017-07-01

    Placental explant culture is an important model for studying placental development and functions. We investigated the differences in placental gene expression in response to tissue culture, atmospheric and physiologic oxygen concentrations. Placental explants were collected from normal term (38-39 weeks of gestation) placentae with no previous uterine contractile activity. Placental transcriptomic expressions were evaluated with GeneChip ® Human Genome U133 Plus 2.0 arrays (Affymetrix). We uncovered sub-sets of genes that regulate response to stress, induction of apoptosis programmed cell death, mis-regulation of cell growth, proliferation, cell morphogenesis, tissue viability, and protection from apoptosis in cultured placental explants. We also identified a sub-set of genes with highly unstable pattern of expression after exposure to tissue culture. Tissue culture irrespective of oxygen concentration induced dichotomous increase in significant gene expression and increased enrichment of significant pathways and transcription factor targets (TFTs) including HIF1A. The effect was exacerbated by culture at atmospheric oxygen concentration, where further up-regulation of TFTs including PPARA, CEBPD, HOXA9 and down-regulated TFTs such as JUND/FOS suggest intrinsic heightened key biological and metabolic mechanisms such as glucose use, lipid biosynthesis, protein metabolism; apoptosis, inflammatory responses; and diminished trophoblast proliferation, differentiation, invasion, regeneration, and viability. These findings demonstrate that gene expression patterns differ between pre-culture and cultured explants, and the gene expression of explants cultured at atmospheric oxygen concentration favours stressed, pro-inflammatory and increased apoptotic transcriptomic response. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Culture promotes transfer of thyroid epithelial cell hyperplasia and proliferation by reducing regulatory T cell numbers.

    Science.gov (United States)

    Kayes, Timothy D; Braley-Mullen, Helen

    2013-01-01

    IFN-γ(-/-) NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ(-/-) SCID mice by splenocytes from mice with severe (4-5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ(-/-) NOD.H-2h4 mice have elevated numbers of FoxP3(+) T reg cells, up to 2-fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Sildenafil Effect on Nitric Oxide Secretion by Normal Human Endometrial Epithelial Cells Cultured In vitro

    Directory of Open Access Journals (Sweden)

    Farzaneh Chobsaz

    2011-01-01

    Full Text Available Background: Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specificphosphodiesterase type 5. It increases intracellular nitric oxide (NO production in some cells.There are reports on its positive effect on uterine circulation, endometrial thickness, and infertilityimprovement. Endometrial epithelial cells (EEC play an important role in embryo attachment andimplantation. The present work investigates the effect of sildenafil on human EEC and their NOsecretion in vitro.Materials and Methods: In this experimental in vitro study, endometrial biopsies (n=10 werewashed in a phosphate buffered solution (PBS and digested with collagenase I (2 mg/ml in DMEM/F12 medium at 37°C for 90 minutes. Epithelial glands were collected by sequential filtrationthrough nylon meshes (70 and 40 μm pores, respectively. Epithelial glands were then treated withtrypsin to obtain individual cells. The cells were counted and divided into four groups: control and1, 10, and 20 μM sildenafil concentrations. Cells were cultured for 15 days at 37ºC and 5% CO2; themedia were changed every 3 days, and their supernatants were collected for the NO assay. NO wasmeasured by standard Greiss methods. Data were analyzed by one way ANOVA.Results: There was no significant difference between groups in cell count and NO secretion, but thelevel of NO increased slightly in the experimental groups. The 10 μM dose showed the highest cellcount. EEC morphology changed into long spindle cells in the case groups.Conclusion: Sildenafil (1, 10, and 20 μM showed a mild proliferative effect on human EECnumbers, but no significant change was seen in NO production.

  20. Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

    Science.gov (United States)

    AbouZid, S

    2014-01-01

    Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture.

  1. Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

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    Nadine Wittkopf

    Full Text Available Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

  2. Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells.

    Directory of Open Access Journals (Sweden)

    Vincent M Bruno

    2009-08-01

    Full Text Available Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP kinase and NF-kappaB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.

  3. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Directory of Open Access Journals (Sweden)

    Susanne C. Hammer

    2016-09-01

    Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

  4. Identification of Stevioside Using Tissue Culture-Derived Stevia () Leaves

    OpenAIRE

    Ziaul Karim Md.; Daisuke Uesugi; Noriyuki Nakayama; M. Monzur Hossain; Kohji Ishihara; Hiroki Hamada

    2015-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for iden...

  5. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  6. Examination of epithelial tissue cytokine response to natural peste des petits ruminants virus (PPRV) infection in sheep and goats by immunohistochemistry.

    Science.gov (United States)

    Atmaca, H T; Kul, O

    2012-01-01

    In this study, we aimed to evaluate expression of IL-4, IL-10, TNF-α, IFN-γ and iNOS in lingual, buccal mucosa and lung epithelial tissue using immunoperoxidase technique and to compare with the tissues of control animals. The tissues used in the study were collected from 17 PPRV-affected and 5 healthy sheep and goats. In PPRV positive animals, the lungs, lingual and buccal mucosa had significantly higher iNOS, IFN-γ and TNF-α expressions compared to control group animals. There was no significant difference between PPRV positive and control groups for IL-4 and IL-10 expressions of epithelial tissues. In conclusion, the epithelial tissues infected by PPRV showed significant iNOS, IFN-γ and TNF-α expressions and they might play an important role in the initiation and regulation of cytokine response, as they take place in the first host barrier to be in contact with PPRV. It is suggested that the more epithelial damage produced by PPRV the more cytokine response may result in the infected epithelial cells. The first demonstration of iNOS expression and epithelial cytokine response to PPRV in natural cases is important because it may contribute to an early initiation of systemic immunity against PPRV infection, in addition to direct elimination of the virus during the initial epithelial phase of the infection.

  7. Expression of transcription factors Slug in the lens epithelial cells undergoing epithelial-mesenchymal transition induced by connective tissue growth factor

    Directory of Open Access Journals (Sweden)

    Ying-Na Wang

    2015-10-01

    Full Text Available AIM:To investigate the expression of transcription factors Slug in human lens epithelial cells (HLECs undergoing epithelial-mesenchymal transition (EMT induced by connective tissue growth factor (CTGF.METHODS: HLECs were treated with CTGF of different concentrations (20, 50 and 100 ng/mL or without CTGF (control for 24h. The morphological changes of HLECs were analysed by microscopy. The expression and cellular localization of Slug was evaluated by immumo-fluorescence. Expressions of Slug, E-cadherin and alpha smooth muscle actin (α-SMA were further determined by Western blot analysis. RESULTS: HLECs showed spidle fibrolasts-like characteristics and loosely connected each other after CTGF treatment. The immuno-fluorescence staining indicated that Slug was localized in the nuclei and its expression was induced by CTGF. The relative expressions of Slug protein were 1.64±0.11, 1.96 ±0.03, 3.12 ±0.10, and 4.08±0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, PCONCLUSION: Transcription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro.

  8. The use of animal tissues alongside human tissue: Cultural and ethical considerations.

    Science.gov (United States)

    Kaw, Anu; Jones, D Gareth; Zhang, Ming

    2016-01-01

    Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

  9. Alpha-particles induce preneoplastic transformation of rat tracheal epithelial cells in culture

    International Nuclear Information System (INIS)

    Thomassen, D.G.; Seiler, F.A.; Shyr, L.-J.; Griffith, W.C.

    1990-01-01

    To characterize the potential role of high-l.e.t. radiation in respiratory carcinogenesis, the cytotoxic and transforming potency of 5.5 MeV α-particles from electroplated sources of 238 Pu were determined using primary cultures of rat tracheal epithelial cells. RBE for cell killing by α-particles versus X-rays varied with dose, and ranged between 4 and 1.5 for α doses in the range 0.2-4 Gy. At equally toxic doses (relative survival 0.18-0.2), all three agents induced similar frequencies of preneoplastic transformation. For preneoplastic transformation induced by doses of α- and X-radiations giving 80 per cent toxicity, an α RBE of 2.4 was derived. The similar RBEs for cell killing and for preneoplastic transformation suggest an association between the type or degree of radiation-induced damage responsible for both cell killing and cell transformation. (author)

  10. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues

    NARCIS (Netherlands)

    Parsons, Linda M.; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

    2017-01-01

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein

  11. Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

    Science.gov (United States)

    Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

    2017-10-01

    In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

  12. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE

    Science.gov (United States)

    Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in CultureE.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.Previous studies have ...

  13. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19...

  14. Effects of estradiol and medroxyprogesterone acetate on morphology, proliferation and apoptosis of human breast tissue in organ cultures

    International Nuclear Information System (INIS)

    Eigėlienė, Natalija; Härkönen, Pirkko; Erkkola, Risto

    2006-01-01

    Human breast tissue undergoes phases of proliferation, differentiation and regression regulated by changes of the levels of circulating sex hormones during the menstrual cycle or aging. Ovarian hormones also likely play a key role in the etiology and biology of breast cancer. Reports concerning the proliferative effects of steroid hormones on the normal epithelium of human breast have been conflicting. Some studies have shown that steroid hormones may predispose breast epithelial cells to malignant changes by stimulating their proliferation, which is known to be regulated tightly by stromal cells. The aim of this study was to investigate the effects of 17β-estradiol and medroxyprogesterone acetate on proliferation, apoptosis, expression of differentiation markers and steroid hormone receptors in breast epithelium using an in vitro model of freshly isolated human breast tissue, in which a proper interaction of breast epithelium and stroma has been maintained. Human breast tissues were obtained from women undergoing surgery for breast tumours. Peritumoral tissues were excised and explants were cultured for 3 weeks in medium supplemented with E 2 or MPA or with E 2 +MPA. Endpoints included histopathological, histomorphometric and immunohistochemical assessment of the breast explants. Culture of breast explants for 14 or 21 days with steroid hormones increased proliferative activity and the thickness of acinar and ductal epithelium. E 2 -treatment led to hyperplastic epithelial morphology, MPA to hypersecretory single-layered epithelium and E 2 +MPA to multilayered but organised epithelium. The proliferative response to E 2 in comparison to control (p < 0.001) was more pronounced than to MPA (p < 0.05) or E 2 +MPA (p < 0.05) at 7 and 14 days for Ki-67 and PCNA. E 2 treatment also decreased the proportion of apoptotic cells after 7 (p < 0.01) and 14 (p < 0.01) days. In addition, the relative number of ERα, ERβ and PR positive epithelial cells was decreased by all

  15. Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

    Science.gov (United States)

    Trujillo, Kristina A.; Heaphy, Christopher M.; Mai, Minh; Vargas, Keith M.; Jones, Anna C.; Vo, Phung; Butler, Kimberly S.; Joste, Nancy E.; Bisoffi, Marco; Griffith, Jeffrey K

    2011-01-01

    Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors. PMID:21105047

  16. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

    Directory of Open Access Journals (Sweden)

    Mehta Bhavya C

    2005-10-01

    the in vitro culture of arachnoidal cells grown from human AG tissue. We demonstrated that these cells in vitro continue to express some of the cytoskeletal and junctional proteins characterized previously in human AG tissue, such as proteins involved in the formation of gap junctions, desmosomes, epithelial specific adherens junctions, as well as tight junctions. These junctional proteins in particular may be important in allowing these arachnoidal cells to regulate CSF outflow.

  17. A Method to Preclude Moisture Condensation in Plated Tissue Cultures

    Science.gov (United States)

    Alex M. Diner

    1992-01-01

    Excessive condensate normally accumulates in in vitro-illuminated petri dishes containing plant tissue cultures, causing avariety of problems. A dark-colored rubber net-mesh placed over the petri dishes prevented such condensation, even when charcoal-supplemented media are used under high light intensity in a growth chamber.

  18. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... ISSN 1684–5315 © 2009 Academic Journals. Full Length ... Key words: Biotechnology, adoption, tissue culture bananas, Kenya. INTRODUCTION ... Recent studies about the agronomic and economic impacts of biotech- ..... accused scientist for 'playing God', others have supported biotechnologies.

  19. YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.

    Science.gov (United States)

    Liu, Ming; Zhao, Shuangyun; Lin, Qingjie; Wang, Xiu-Ping

    2015-04-01

    Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Mathematical modelling of tissue formation in chondrocyte filter cultures.

    Science.gov (United States)

    Catt, C J; Schuurman, W; Sengers, B G; van Weeren, P R; Dhert, W J A; Please, C P; Malda, J

    2011-12-17

    In the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue. Experimental data was collected to allow comparison with the predictions of the simulation and refinement of the initial models. Healthy mature equine chondrocytes were expanded and subsequently seeded on collagen-coated filters and cultured for up to 7 weeks. Resulting samples were characterised biochemically, as well as histologically. The simulations showed a good representation of the experimentally obtained cell and matrix distribution within the cultures. The mathematical results indicate that the experimental GAG and cell distribution is critically dependent on the rate at which the cell differentiation process takes place, which has important implications for interpreting experimental results. This study demonstrates that large regions of the tissue are inactive in terms of proliferation and growth of the layer. In particular, this would imply that higher seeding densities will not significantly affect the growth rate. A simple mathematical model was developed to predict the observed experimental data and enable interpretation of the principal underlying mechanisms controlling growth-related changes in tissue composition.

  1. Propagation of Aquilaria malaccensis seedlings through tissue culture techniques

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Zaiton Ahmad; Mohd Rafaie Abdul Salam; Nurhayati Irwan; Affrida Abu Hassan; Rusli Ibrahim

    2010-01-01

    Aquilaria malaccensis or karas is the principal source of gaharu resin, which is used in many cultures for incense, perfumes and traditional medicines. The species is mainly propagated conventionally through seeds, cuttings and graftings. Propagation by seeds is usually a reliable method for other forest species, but for karas, this technique is inadequate to meet the current demand of seedling supplies. This is principally due to its low seed viability, low germination rate, delayed rooting of seedlings, long life-cycle and rare seed production. Tissue culture has several advantages over conventional propagation, especially for obtaining large number of uniform and high-yielding plantlets or clones. This paper presents the current progress on mass-propagation of Aquilaria malaccensis seedlings through tissue culture technique at Nuclear Malaysia. (author)

  2. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    Science.gov (United States)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  3. Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

    Science.gov (United States)

    Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

    2013-11-01

    Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

  4. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  5. Engineering stromal-epithelial interactions in vitro for ...

    Science.gov (United States)

    Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo tissue recombination, and in vitro co-cultures. Although these approaches have elucidated signaling mechanisms underlying morphogenetic processes and adult mammalian epithelial tissue function, they are limited by the availability of human tissue, low throughput, and human developmental or physiological relevance. Objectives: Bioengineering strategies to promote EMIs using human epithelial and mesenchymal cells have enabled the development of human in vitro models of adult epidermal and glandular tissues. In this review, we describe recent bioengineered models of human epithelial tissue and organs that can instruct the design of organotypic models of human developmental processes.Methods: We reviewed current bioengineering literature and here describe how bioengineered EMIs have enabled the development of human in vitro epithelial tissue models.Discussion: Engineered models to promote EMIs have recapitulated the architecture, phenotype, and function of adult human epithelial tissue, and similar engineering principles could be used to develop models of developmental morphogenesis. We describe how bioengineering strategies including bioprinting and spheroid culture could be implemented to

  6. Protein Profiling of Isolated Leukocytes, Myofibroblasts, Epithelial, Basal, and Endothelial Cells from Normal, Hyperplastic, Cancerous, and Inflammatory Human Prostate Tissues

    Directory of Open Access Journals (Sweden)

    Zahraa I. Khamis, Kenneth A. Iczkowski, Ziad J. Sahab, Qing-Xiang Amy Sang

    2010-01-01

    Full Text Available In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prsotate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.

  7. The role of L-type amino acid transporters in the uptake of glyphosate across mammalian epithelial tissues.

    Science.gov (United States)

    Xu, Jiaqiang; Li, Gao; Wang, Zhuoyi; Si, Luqin; He, Sijie; Cai, Jialing; Huang, Jiangeng; Donovan, Maureen D

    2016-02-01

    Glyphosate is one of the most commonly used herbicides worldwide due to its broad spectrum of activity and reported low toxicity to humans. Glyphosate has an amino acid-like structure that is highly polar and shows low bioavailability following oral ingestion and low systemic toxicity following intravenous exposures. Spray applications of glyphosate in agricultural or residential settings can result in topical or inhalation exposures to the herbicide. Limited systemic exposure to glyphosate occurs following skin contact, and pulmonary exposure has also been reported to be low. The results of nasal inhalation exposures, however, have not been evaluated. To investigate the mechanisms of glyphosate absorption across epithelial tissues, the permeation of glyphosate across Caco-2 cells, a gastrointestinal epithelium model, was compared with permeation across nasal respiratory and olfactory tissues excised from cows. Saturable glyphosate uptake was seen in all three tissues, indicating the activity of epithelial transporters. The uptake was shown to be ATP and Na(+) independent, and glyphosate permeability could be significantly reduced by the inclusion of competitive amino acids or specific LAT1/LAT2 transporter inhibitors. The pattern of inhibition of glyphosate permeability across Caco-2 and nasal mucosal tissues suggests that LAT1/2 play major roles in the transport of this amino-acid-like herbicide. Enhanced uptake into the epithelial cells at barrier mucosae, including the respiratory and gastrointestinal tracts, may result in more significant local and systemic effects than predicted from glyphosate's passive permeability, and enhanced uptake by the olfactory mucosa may result in further CNS disposition, potentially increasing the risk for brain-related toxicities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The role of activated charcoal in plant tissue culture.

    Science.gov (United States)

    Thomas, T Dennis

    2008-01-01

    Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

  9. Variations on metabolic activities of legume tissues through radiation in tissue culture

    International Nuclear Information System (INIS)

    Batra, Amla

    1977-01-01

    Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content. (author)

  10. Variations on metabolic activities of legume tissues through radiation in tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Batra, A [Rajasthan Univ., Jaipur (India). Dept. of Botany

    1977-12-01

    Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content.

  11. Cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung tumors

    International Nuclear Information System (INIS)

    Kolesnichenko, T.S.; Gor'kova, T.G.

    1985-01-01

    Local factors such as proliferative activity and the numerical ratio between epithelial and mesenchymal cells, and also the character of interaction between the tissue components in ontogeny may play an important role in the realization of sensitivity of mice of a particular line to the development of lung tumors. These characteristics of lung tissue in mice of lines A and C57BL are investigated under normal conditions and during induced carcinogenesis. Results are given of a comparative study of the relative numbers of epithelial and mesenchymal cells in organ cultures of embryonic lungs. 3 H-thymidine was added to the cultures on the 14th day of the experiment in a concentration of 1 microCi/m1 medium. An autoradiographic study of the cultures was performed

  12. Application of Synthetic Polymeric Scaffolds in Breast Cancer 3D Tissue Cultures and Animal Tumor Models

    Directory of Open Access Journals (Sweden)

    Girdhari Rijal

    2017-01-01

    Full Text Available Preparation of three-dimensional (3D porous scaffolds from synthetic polymers is a challenge to most laboratories conducting biomedical research. Here, we present a handy and cost-effective method to fabricate polymeric hydrogel and porous scaffolds using poly(lactic-co-glycolic acid (PLGA or polycaprolactone (PCL. Breast cancer cells grown on 3D polymeric scaffolds exhibited distinct survival, morphology, and proliferation compared to those on 2D polymeric surfaces. Mammary epithelial cells cultured on PLGA- or PCL-coated slides expressed extracellular matrix (ECM proteins and their receptors. Estrogen receptor- (ER- positive T47D breast cancer cells are less sensitive to 4-hydroxytamoxifen (4-HT treatment when cultured on the 3D porous scaffolds than in 2D cultures. Finally, cancer cell-laden polymeric scaffolds support consistent tumor formation in animals and biomarker expression as seen in human native tumors. Our data suggest that the porous synthetic polymer scaffolds satisfy the basic requirements for 3D tissue cultures both in vitro and in vivo. The scaffolding technology has appealing potentials to be applied in anticancer drug screening for a better control of the progression of human cancers.

  13. Effect of Twist, Snail and YB-1 gene expression in cervical cancer tissue on cell invasion and epithelial-mesenchymal transition

    Directory of Open Access Journals (Sweden)

    Xin-Qin Kang1

    2017-05-01

    Full Text Available Objective: To study the effect of Twist, Snail and YB-1 gene expression in cervical cancer tissue on cell invasion and epithelial-mesenchymal transition. Methods: Cervical cancer tissue samples and tissue samples adjacent to carcinoma were collected from 138 patients with radical operation for cervical cancer, fluorescence quantitative PCR method was used to detect the mRNA expression of Twist, Snail and YB-1 genes, cell invasion-related genes and epithelial-mesenchymal transition marker genes, the Pearson test was used to analyze the correlation of Twist, Snail and YB-1 gene mRNA expression in cervical cancer tissue with cell invasion and epithelial-mesenchymal transition. Results: Twist, Snail and YB-1 gene mRNA expression in cervical cancer tissue were higher than those in tissue adjacent to carcinoma, the invasion genes STAT3, YAP1, TUG1, FoxM1 and Rab11 mRNA expression were higher than those in tissue adjacent to carcinoma, and the epithelial-mesenchymal transition markers E-cadherin and β-catenin gene mRNA expression were lower than those in tissue adjacent to carcinoma while vimentin gene mRNA expression was higher than that in tissue adjacent to carcinoma. Pearson test showed that Twist, Snail and YB-1 gene mRNA expression in cervical cancer tissue were directly correlated with cell invasion and epithelial-mesenchymal transition. Conclusion: Twist, Snail and YB-1 genes are highly expressed in cervical cancer tissue, and their abnormal expression directly leads to the increased tumor cell invasion activity and the aggravated epithelial-mesenchymal transition.

  14. Blue-light filtering alters angiogenic signaling in human retinal pigmented epithelial cells culture model.

    Science.gov (United States)

    Vila, Natalia; Siblini, Aya; Esposito, Evangelina; Bravo-Filho, Vasco; Zoroquiain, Pablo; Aldrees, Sultan; Logan, Patrick; Arias, Lluis; Burnier, Miguel N

    2017-11-02

    Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein

  15. Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

    Science.gov (United States)

    Klinkspoor, J H; Yoshida, T; Lee, S P

    1998-05-15

    1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with beta-cyclodextrin (2.5-15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (>100 microM Triton X-100, >0.8 mM Tween-20), caused increased LDH release. Incubation with beta-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.

  16. Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

    Directory of Open Access Journals (Sweden)

    Beales Ian LP

    2002-04-01

    Full Text Available Abstract Background Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study. Methods AGS cells were cultured with IL-1β. DNA synthesis was assed by [3H]thymidine incorporation and total viable cell numbers by MTT assay. Results IL-1β dose dependently increased DNA synthesis and cell numbers. The enhanced proliferation was blocked by interleukin-1 receptor antagonist. Addition of neutralising antibody to GM-CSF reduced IL-1β-stimulated proliferation by 31 ± 4 %. GM-CSF alone significantly stimulated proliferation. Addition or neutralisation of IL-8 had no effect on basal or IL-1β-stimulated proliferation. The tyrosine kinase inhibitor genistein completely blocked IL-1β-stimulated proliferation and inhibition of the extracellular signal related kinase pathway with PD 98059 inhibited IL-1β stimulated proliferation by 58 ± 5 %. Conclusions IL-1β stimulates proliferation in gastric epithelial cells. Autocrine stimulation by GM-CSF contributes to this proliferative response. Signalling via tyrosine kinase activity is essential to the mitogenic response to IL-1β. The extracellular signal related kinase pathway is involved in, but not essential to downstream signalling. IL-1β may contribute to the hyperproliferation seen in H. pylori- infected gastric mucosa, and be involved in the carcinogenic process.

  17. Tissue types (image)

    Science.gov (United States)

    ... are 4 basic types of tissue: connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports ... binds them together (bone, blood, and lymph tissues). Epithelial tissue provides a covering (skin, the linings of the ...

  18. Study of the effects of low-dose radiation and rhEGF on growth of cultured human epithelial cells

    International Nuclear Information System (INIS)

    Yang Jicheng; Zhao Xiaoyu; Sheng Weihua; Tang Zhongyi

    1998-01-01

    In authors' study, the method of taking skin sample, mincing and trypsinizing the sample are presented. The cells were inoculated on adherent membrane or, for sublethally injured 3T3 cells, in culture dish fed with Eargles' medium supplemented with fetal calf serum and various growth-stimulating factors. The cultures were incubated at 37 degree C in an atmosphere containing 5% CO 2 . The medium was changed every three days. The cultured cells became confluent in about two weeks. At the same time, low-dose-radiation and rhEGF were used to influence the growth of the epithelial cells and to test the effects of dosage and concentration. The results showed that low-dose-radiation in the conditions like authors' study could enhance the growth of human epithelial cells just like rhEGF, and it has synergetic effects with rhEGF. The mechanism is discussed

  19. A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

    Science.gov (United States)

    Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

    2017-01-01

    Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

  20. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

    Directory of Open Access Journals (Sweden)

    Trisha N. Peel

    2016-01-01

    Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

  1. Cultivated vaginal microbiomes alter HIV-1 infection and antiretroviral efficacy in colonized epithelial multilayer cultures.

    Science.gov (United States)

    Pyles, Richard B; Vincent, Kathleen L; Baum, Marc M; Elsom, Barry; Miller, Aaron L; Maxwell, Carrie; Eaves-Pyles, Tonyia D; Li, Guangyu; Popov, Vsevolod L; Nusbaum, Rebecca J; Ferguson, Monique R

    2014-01-01

    There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

  2. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    Science.gov (United States)

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    NARCIS (Netherlands)

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; de Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing

  4. Connective tissue growth factor activates pluripotency genes and mesenchymal-epithelial transition in head and neck cancer cells.

    Science.gov (United States)

    Chang, Cheng-Chi; Hsu, Wen-Hao; Wang, Chen-Chien; Chou, Chun-Hung; Kuo, Mark Yen-Ping; Lin, Been-Ren; Chen, Szu-Ta; Tai, Shyh-Kuan; Kuo, Min-Liang; Yang, Muh-Hwa

    2013-07-01

    The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC. ©2013 AACR.

  5. Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface.

    Science.gov (United States)

    Chang-Lin, Joan-En; Kim, Kwang-Jin; Lee, Vincent H L

    2005-06-01

    ECL cultured from pigmented rabbits, while 85+/-0.7%, 36+/-1.6%, 38+/-1.8% and 15+/-3.5% decreases are observed for RCrECL from albino rabbits, respectively. Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.

  6. Identification of differences in gene expression in primary cell cultures of human endometrial epithelial cells and trophoblast cells following their interaction

    DEFF Research Database (Denmark)

    Høgh, Mette; Islin, Henrik; Møller, Charlotte

    2006-01-01

    The interaction between the cell types was simulated in vitro by growing primary cell cultures of human endometrial epithelial cells and trophoblast cells together (co-culture) and separately (control cultures). Gene expression in the cell cultures was compared using the Differential Display method and confirmed...

  7. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Liara M Gonzalez

    Full Text Available Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA, Minichromosome Maintenance Complex 2 (MCM2, Bromodeoxyuridine (BrdU and phosphorylated Histone H3 (pH3 distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2; enteroendocrine cells by Chromogranin A (CGA, Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII and sucrase isomaltase (SIM. Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

  8. A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

    Science.gov (United States)

    Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

    2017-06-01

    Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

    Science.gov (United States)

    Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

    2015-08-01

    Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

  10. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pearlman, Andrew Leonard [Univ. of California, Berkeley, CA (United States)

    1978-08-01

    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G2M by about 50%. When added to G1 cells, DE delayed recruitment of apparently quiescent (G0) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  11. Lemongrass-Incorporated Tissue Conditioner Against Candida albicans Culture

    Science.gov (United States)

    Amornvit, Pokpong; Srithavaj, Theerathavaj

    2014-01-01

    Background: Tissue conditioner is applied popularly with dental prosthesis during wound healing process but it becomes a reservoir of oral microbiota, especially Candida species after long-term usage. Several antifungal drugs have been mixed with this material to control fungal level. In this study, lemongrass essential oil was added into COE-COMFORT tissue conditioner before being determined for anti-Candida efficacy. Materials and Methods: Lemongrass (Cymbopogon citratus) essential oil was primarily determined for antifungal activity against C. albicans American type culture collection (ATCC) 10231 and MIC (minimum inhibitory concentration) value by agar disk diffusion and broth microdilution methods, respectively. COE-COMFORT tissue conditioner was prepared as recommended by the manufacturer after a fixed volume of the oil at its MIC or higher concentrations were mixed thoroughly in its liquid part. Antifungal efficacy of the tissue conditioner with/without herb was finally analyzed. Results: Lemongrass essential oil displayed potent antifungal activity against C. albicans ATCC 10231and its MIC value was 0.06% (v/v). Dissimilarly, the tissue conditioner containing the oil at MIC level did not cease the growth of the tested fungus. Both reference and clinical isolates of C. albicans were completely inhibited after exposed to the tissue conditioner containing at least 0.25% (v/v) of the oil (approximately 4-time MIC). The tissue conditioner without herb or with nystatin was employed as negative or positive control, respectively. Conclusion: COE-COMFORT tissue conditioner supplemented with lemongrass essential oil obviously demonstrated another desirable property as in vitro anti-Candida efficacy to minimize the risk of getting Candidal infection. PMID:25177638

  12. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  13. Human airway epithelial cell cultures for modeling respiratory syncytial virus infection.

    Science.gov (United States)

    Pickles, Raymond J

    2013-01-01

    Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.

  14. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Dylla, Brenda L; Hughes, John G; Lynch, David T; Greenwood-Quaintance, Kerryl E; Cheng, Allen C; Mandrekar, Jayawant N; Patel, Robin

    2016-01-05

    Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Copyright © 2016 Peel et al.

  15. Myo-inositol uptake by cultured calf retinal pigment epithelial cells: regulation by glucose

    International Nuclear Information System (INIS)

    Khatami, M.; Rockey, J.H.

    1986-01-01

    Confluent primary (P-1) or subcultured passage 2 or 3 (P-2, P-3) calf retinal pigment epithelial cells (RPE) were incubated with [ 3 H]-myo-inositol (MI, 100-200 μM) in balanced salt solution (BSS), for 5 to 60 min at 37 0 C. MI uptake into RPE (P-2, 5 days old) was saturable with K/sub m/ of 147 μM and V/sub max/ of 5.5 pmole/min/μg DNA. P-1 or P-2 incubated with 10 μM MI for 40 min accumulated MI against a concentration gradient ([MI]in/[MI]out > 20). Replacement of 150 mM NaCl in BSS by 150 mM choline-Cl reduced the uptake of MI by 87%. MI uptake was inhibited (39%) when cells were incubated in BSS in the absence of Ca Cl 2 . Transport of MI into RPE incubated in the presence of phloridzin, ouabain or 2,4-dinitrophenol (1 mM each) for 10 min was inhibited by 65, 37 and 21%, respectively. α-D-Glucose (20 mM) in the incubation media inhibited MI uptake into primary (or P-2) cultured RPE by 30 or 43% when cells were incubated for 10 or 60 min, respectively. The ability of RPE cells, grown in the presence of 50 mM glucose for 15-25 days, to concentrate MI (40 μM) was reduced up to 41%. Cultured RPE cells accumulated myo-inositol by an active transport system, sensitive to ouabain, DNP and phloridzin. High glucose in the incubation media or in the growth media inhibited the uptake of MI into calf RPE cells

  16. Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.

    Science.gov (United States)

    Gu, Liping; Ma, Yuhang; Gu, Mingyu; Zhang, Ying; Yan, Shuai; Li, Na; Wang, Yufan; Ding, Xiaoying; Yin, Jiajing; Fan, Nengguang; Peng, Yongde

    2015-09-01

    Spexin mRNA and protein are widely expressed in rat tissues and associate with weight loss in rodents of diet-induced obesity. Its location in endocrine and epithelial cells has also been suggested. Spexin is a novel peptide that involves weight loss in rodents of diet-induced obesity. Therefore, we aimed to examine its expression in human tissues and test whether spexin could have a role in glucose and lipid metabolism in type 2 diabetes mellitus (T2DM). The expression of the spexin gene and immunoreactivity in the adrenal gland, skin, stomach, small intestine, liver, thyroid, pancreatic islets, visceral fat, lung, colon, and kidney was higher than that in the muscle and connective tissue. Immunoreactive serum spexin levels were reduced in T2DM patients and correlated with fasting blood glucose (FBG, r=-0.686, Pepithelial tissues, indicating that spexin may be involved in physiological functions of endocrine and in several other tissues. Circulating spexin levels are low in T2DM patients and negatively related to blood glucose and lipids suggesting that the peptide may play a role in glucose and lipid metabolism in T2DM. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Tissue culture of three species of Laurencia complex

    Science.gov (United States)

    Shen, Songdong; Wu, Xunjian; Yan, Binlun; He, Lihong

    2010-05-01

    To establish a micropropagation system of three Laurencia complex species ( Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus) by tissue culture techniques, we studied the regeneration characteristics and optimal culture conditions of axenic algal fragments cultured on solid medium and in liquid medium. Regeneration structures were observed and counted regularly under a reverse microscope to investigate the regeneration process, polarity and optimal illumination, and temperature and salinity levels. The results show that in most cultures of the three species, we obtained bud regeneration on solidified medium with 0.5% agar and in liquid medium. Rhizoid-like regeneration was filamentous and developed from the lower cut surface of fragments in L. okamurai, but was discoid and developed from the apical back side of bud regeneration in L. tristicha and C. undulatus. Regeneration polarity was localized to the apical part of algal fronds in all three species, and on fragments cut from the basal part of algae buds could develop from both the upper and the lower cut surfaces. Buds could develop from both the medullary and the cortical portions in L. okamurai and C. undulatus, while in L. tristicha, buds only emerged from the cortex. The optimal culture conditions for L. okamurai were 4 500 lx, 20°C and 35 (salinity); for C. undulatus, 4 500 lx, 20°C and 30; and for L. tristicha, 4 500 lx, 25°C and 30.

  18. HYPOLIPIDEMIC EFFECT OF ARGLABIN IN HEPATOMA TISSUE CULTURE

    Directory of Open Access Journals (Sweden)

    A. V. Ratkin

    2015-01-01

    Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone Arglabin in hepatoma tissue culture (HTC.Materials and methods. In this study we’ve evaluated the effect of sesquiterpene γ-lactone Arglabin and gemfibrozil (reference drug on the lipid content in the hepatoma tissue culture (HTC which were incubated with a fat emulsion “Lipofundin” by fluorescent method with vital dye Nile Red. The cell viability was investigated using the MTT-test and staining by Trypan blue.Results. Cultivation of cell cultures of rat’s hepatoma cell line HTC with Arglabin and gemfibrozil in concentrations from 10 to 50 μmol and from 0.25 to 0.5 mmol, respectively, had no cytotoxic effect. HTC cell viability did not change compared with the corresponding rate in the control culture. Experimental hyperlipidemia in hepatoma culture was induced by the addition in the incubation medium of fat emulsion “Lipofundin” in a final concentration of 0.05 %. The fluorescence intensity of Nile Red in the cells was increased 4-fold (p < 0.05, which indicates a significant accumulation of lipids in the cytosol of cells. In these steady-state Arglabin and gemfibrozil at concentrations 75–100 μM and 0.25–1.0 mM, respectively, reduced the content of lipid in cells. Conclusion. In the model of hyperlipidemia induced by lipofundin, sesquiterpene γ-lactone Arglabin prevents the accumulation of lipids in the HTC cell line, as evidenced by a decrease in Nile Red fluorescence. However hypolipidemic effect of Arglabin is associated with cytotoxic effects, which is typical for anticancer drugs.

  19. Addressing the instability of DNA nanostructures in tissue culture.

    Science.gov (United States)

    Hahn, Jaeseung; Wickham, Shelley F J; Shih, William M; Perrault, Steven D

    2014-09-23

    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable

  20. A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

    Science.gov (United States)

    Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

    2011-05-01

    A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  1. Changes in responsiveness of rat tracheal epithelial cells to growth factors during preneoplastic transformation in cell culture

    International Nuclear Information System (INIS)

    Thomassen, D.G.

    1988-01-01

    Preneoplastic rat tracheal epithelial (RTE) cell lines require fewer growth factors for clonal proliferation in culture than normal cells. Serum-free media missing various combinations of growth factors (e.g., cholera toxin, serum albumin, epidermal growth factor, hydrocortisone) required for proliferation of normal, but not preneoplastic, RTE cells can be used to select for carcinogen-induced preneoplastic variants having an increased proliferative potential in culture. These results suggest that reductions in growth factor requirements are primary events in the carcinogenic process. (author)

  2. Immunohistochemical Localization of Fibrinogen C Domain Containing 1 on Epithelial and Mucosal Surfaces in Human Tissues

    DEFF Research Database (Denmark)

    von Huth, Sebastian; Moeller, Jesper B; Schlosser, Anders

    2018-01-01

    Fibrinogen C domain containing 1 (FIBCD1) is a transmembrane receptor that binds chitin and other acetylated compounds with high affinity. FIBCD1 has previously been shown to be present in the epithelium of the gastrointestinal tract. In the present study, we performed a detailed analysis...... high expression of FIBCD1 and also mesodermal-derived cells in the genitourinary system and ectodermal-derived epidermis and sebaceous glands cells expressed FIBCD1. In some columnar epithelial cells, for example, in the salivary gland and gall bladder, the FIBCD1 expression was clearly polarized...

  3. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  4. Anaerobic Cultures from Preserved Tissues of Baby Mammoth

    Science.gov (United States)

    Pikuta, Elena V.; Hoover, Richard B.; Fisher, Daniel

    2011-01-01

    Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 4 C. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that prevents other bacteria from over-dominating a system. Permafrost and lactic acid preserved the body of this one-month old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete such specimen ever recovered. The diversity of novel anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here we discuss the specifics of the isolation of new strains, differentiation from trivial contamination, and preliminary results for the characterization of cultures.

  5. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  6. Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing.

    Science.gov (United States)

    Lendeckel, Derik; Eymann, Christine; Emicke, Philipp; Daeschlein, Georg; Darm, Katrin; O'Neil, Serena; Beule, Achim G; von Woedtke, Thomas; Völker, Uwe; Weltmann, Klaus-Dieter; Jünger, Michael; Hosemann, Werner; Scharf, Christian

    2015-01-01

    The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine.

  7. Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

    Directory of Open Access Journals (Sweden)

    Urszula Adamczyk-Rogozińska

    2014-01-01

    Full Text Available The conditions for the regeneration of plants through organogenesis from callus tissues of Menyanthes trifoliata are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M containing indole-3-acetic acid (IAA 0,5 mg/l and 6-benzyladenine (BA 1 mg/l or zeatin (2 mg/l. Under these conditions ca 7 shoots (mostly 1 cm or more in length per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

  8. A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage.

    Science.gov (United States)

    Shaban, Lamyaa; Chen, Ying; Fasciano, Alyssa C; Lin, Yinan; Kaplan, David L; Kumamoto, Carol A; Mecsas, Joan

    2018-04-01

    Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O 2 conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  10. Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  11. Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

    DEFF Research Database (Denmark)

    Jantzen, Kim; Roursgaard, Martin; Madsen, Claus Desler

    2012-01-01

    Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung...... treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine...... relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells....

  12. Immunohistochemical correlation of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-2 in tobacco associated epithelial dysplasia.

    Science.gov (United States)

    Bajracharya, Dipshikha; Shrestha, Bijayatha; Kamath, Asha; Menon, Aparna; Radhakrishnan, Raghu

    2014-01-01

    To study the immunohistochemical expression of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase-2 in different histological grades of tobacco associated epithelial dysplasia and correlate the association between these proteases. Potentially malignant oral disorders (PMODs) progressing to oral cancer are related to the severity of epithelial dysplasia. A retrospective immunohistochemical study was carried out on 30 clinically and histologically proven cases of leukoplakia with dysplasia and 10 cases of normal buccal mucosa using anti-MMP-2 and anti-TIMP-2 monoclonal antibodies. Mann Whitney U test, for comparing the expression of both MMP-2 and TIMP-2 in normal mucosa with dysplasia, was highly significant (P correlation between MMP-2 and TIMP-2 through different grades of dysplasia and cells observed showed positive correlation. Concomitant increase in the expression of both MMP-2 and TIMP-2 suggested that the activation of MMP-2 is dependent on TIMP-2 acting as a cofactor. Changes in TIMP-2 levels are considered important because they directly affect the level of MMP-2 activity.

  13. Immunohistochemical Correlation of Matrix Metalloproteinase-2 and Tissue Inhibitors of Metalloproteinase-2 in Tobacco Associated Epithelial Dysplasia

    Directory of Open Access Journals (Sweden)

    Dipshikha Bajracharya

    2014-01-01

    Full Text Available Aim. To study the immunohistochemical expression of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase-2 in different histological grades of tobacco associated epithelial dysplasia and correlate the association between these proteases. Potentially malignant oral disorders (PMODs progressing to oral cancer are related to the severity of epithelial dysplasia. Methods. A retrospective immunohistochemical study was carried out on 30 clinically and histologically proven cases of leukoplakia with dysplasia and 10 cases of normal buccal mucosa using anti-MMP-2 and anti-TIMP-2 monoclonal antibodies. Results. Mann Whitney U test, for comparing the expression of both MMP-2 and TIMP-2 in normal mucosa with dysplasia, was highly significant (P<0.001. Kruskal-Wallis test to compare the median score of MMP-2 and TIMP-2 in different grades of dysplasia showed statistical significance (P<0.001, and a Spearman’s correlation between MMP-2 and TIMP-2 through different grades of dysplasia and cells observed showed positive correlation. Conclusion. Concomitant increase in the expression of both MMP-2 and TIMP-2 suggested that the activation of MMP-2 is dependent on TIMP-2 acting as a cofactor. Changes in TIMP-2 levels are considered important because they directly affect the level of MMP-2 activity.

  14. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

    Science.gov (United States)

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

    2014-02-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

  15. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...... with mass production. The last part of this thesis also includes perspectives on how to expand the latest designed device to facilitate culturing of tissue and co-culturing of cells....

  16. Tobacco clones derived from tissue culture with supersensitivity to ozone

    International Nuclear Information System (INIS)

    Sun, E.J.; Kang, H.W.

    2003-01-01

    New tobacco clones supersensitive to ozone were obtained from tissue culture. - At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference

  17. On factors modifying reparative regeneration of epithelial tissue of small intestine in the presence of intestinal syndrome

    International Nuclear Information System (INIS)

    Kudryavtsev, V.D.

    1980-01-01

    In experiments on Wistar rats irradiated in dosages of 1000 and 1200 rad, the possibility of reparative regeneration of cryptae was demonstrated in the case when ''intestinal death'' was prevented by therapeutic means (kanamycin mixed with Ringer-Lock's solution). Shielding of part of the abdomen and extensive bone marrow region, and transplantation of homologous bone marrow elicit a stimulatory effect on postradiation recovery of small intestine epithelial tissue. When radiation dose increases up to 1400 rad reepithelization of the exposed region occurs only with the protection of 50-60% of the abdomen. The regenerating cryptae do not appear after irradiation of the whole body or whole abdomen though life expectancy of rats increases up to 6-7 days due to the therapeutic cure

  18. Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

    International Nuclear Information System (INIS)

    Wan, X. Steven; Ware, Jeffrey H.; Zhou, Zhaozong; Donahue, Jeremiah J.; Guan, Jun; Kennedy, Ann R.

    2006-01-01

    Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, α-lipoic acid, L-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, γ-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects

  19. Immunohistochemical expression of epithelial cell adhesion molecule (EpCAM) in mucoepidermoid carcinoma compared to normal salivary gland tissues.

    Science.gov (United States)

    Kamal, Noura M; Salem, Hend M; Dahmoush, Heba M

    2017-07-01

    Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumor which displays biological, histological and clinical diversity thus representing a challenge for its diagnosis and management. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein identified as a tumor specific antigen due to its frequent overexpression in the majority of epithelial carcinomas and its correlation with prognosis. It is considered to be a promising biomarker used as a therapeutic target already in ongoing clinical trials. The purpose of this study was to investigate the pattern, cellular characterization and level of EpCAM expression in MEC and demonstrate its correlation with histologic grading which may benefit future clinical trials using EpCAM targeted therapy. 48 specimens (12 normal salivary gland tissue and 36 MEC) were collected and EpCAM membranous expression was evaluated by immunohistochemistry. Total immunoscore (TIS) was evaluated, the term 'EpCAM overexpression' was given for tissues showing a total immunoscore >4. A highly significant difference was observed between TIS percent values in control and different grades of MEC (p<0.001). High grade MEC (HG-MEC) was the highest EpCAM expressor. In addition, EpCAM expression pattern differed among the different grades. EpCAM expression was detected in MEC, and its overexpression correlated with increasing the histological grade. The diffuse membranous expression in HG-MEC could be of diagnostic value in relation to the patchy expression observed in both low grade and intermediate grade MEC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

    Science.gov (United States)

    Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

    2017-01-01

    Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Expression of preoperative KISS1 gene in tumor tissue with epithelial ovarian cancer and its prognostic value.

    Science.gov (United States)

    Cao, Fang; Chen, Liping; Liu, Manhua; Lin, Weiwei; Ji, Jinlong; You, Jun; Qiao, Fenghai; Liu, Hongbin

    2016-11-01

    Our study aimed to elucidate the role of Kisspeptin (KISS1) in tumor tissues of patients with epithelial ovarian cancer (EOC) and investigate the prognostic value of this biomarker.Forty EOC patients and 20 uterine fibroids female patients with healthy ovaries undergoing cytoreductive surgery between January 2010 and January 2014 in our hospital were enrolled in this study. KISS1 expression in tumor and normal tissues was detected. Correlations between clinic-pathologic variables and KISS1 expression in EOC tissues and the prognostic value of KISS1 for overall survival were evaluated.During the follow-up of 11.2 to 62.1 months, the overall survival rate and mean survival time were 28.9% (11/38) and 38.35 ± 2.84 months. Preoperative KISS1 mRNA was higher in tumor tissue than in normal tissue (P <0.001), and it was associated with histologic grade of tumor, surgical FIGO stage, metastasis, and residual tumor size (all P <0.05). Multivariate survival analysis indicated significant influence of residual tumor size (HR = 2.357, P = 0.039) and preoperative KISS1 mRNA (HR = 0.0001, P <0.001) on mean survival time. Patients with low KISS1 mRNA expression had shorter survival time than those with high expression (P = 0.001).Preoperative KISS1 mRNA was a potential prognostic biomarker for EOC, and high preoperative KISS1 expression indicated a favorable prognosis.

  2. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Directory of Open Access Journals (Sweden)

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  3. Expression and clinical implication of Beclin1, HMGB1, p62, survivin, BRCA1 and ERCC1 in epithelial ovarian tumor tissues.

    Science.gov (United States)

    Ju, L-L; Zhao, C Y; Ye, K-F; Yang, H; Zhang, J

    2016-05-01

    The aim of the present study is to investigate the differential expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 protein in epithelial ovarian cancer (EOC) and to evaluate the relationship between autophagy and platinum resistance of EOC patients during platinum-based chemotherapy with the protein expression. Expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 were detected with immunohistochemistry in 60 patients, including 39 with epithelial ovarian cancer (EOC), 13 benign epithelial ovarian tumor tissue (BET) and 8 borderline ovarian tumor tissue. Beclin, p62 and ERCC1 expression was significantly higher in the EOC than the BET (p0.05). BRCA1 expression was lower in EOC than BET (pepithelial ovarian cancer.

  4. A novel culture method reveals unique neural stem/progenitors in mature porcine iris tissues that differentiate into neuronal and rod photoreceptor-like cells.

    Science.gov (United States)

    Royall, Lars N; Lea, Daniel; Matsushita, Tamami; Takeda, Taka-Aki; Taketani, Shigeru; Araki, Masasuke

    2017-11-15

    Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

    1999-01-01

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  6. Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

    Science.gov (United States)

    Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

    2012-01-01

    Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and

  7. Palifermin for the protection and regeneration of epithelial tissues following injury: new findings in basic research and pre-clinical models.

    Science.gov (United States)

    Finch, Paul W; Mark Cross, Lawrence J; McAuley, Daniel F; Farrell, Catherine L

    2013-09-01

    Keratinocyte growth factor (KGF) is a paracrine-acting epithelial mitogen produced by cells of mesenchymal origin, that plays an important role in protecting and repairing epithelial tissues. Pre-clinical data initially demonstrated that a recombinant truncated KGF (palifermin) could reduce gastrointestinal injury and mortality resulting from a variety of toxic exposures. Furthermore, the use of palifermin in patients with hematological malignancies reduced the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. Based upon these findings, as well as the observation that KGF receptors are expressed in many, if not all, epithelial tissues, pre-clinical studies have been conducted to determine the efficacy of palifermin in protecting different epithelial tissues from toxic injury in an attempt to model various clinical situations in which it might prove to be of benefit in limiting tissue damage. In this article, we review these studies to provide the pre-clinical background for clinical trials that are described in the accompanying article and the rationale for additional clinical applications of palifermin. © 2013 The Authors. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  8. Inference of Cell Mechanics in Heterogeneous Epithelial Tissue Based on Multivariate Clone Shape Quantification

    Science.gov (United States)

    Tsuboi, Alice; Umetsu, Daiki; Kuranaga, Erina; Fujimoto, Koichi

    2017-01-01

    Cell populations in multicellular organisms show genetic and non-genetic heterogeneity, even in undifferentiated tissues of multipotent cells during development and tumorigenesis. The heterogeneity causes difference of mechanical properties, such as, cell bond tension or adhesion, at the cell–cell interface, which determine the shape of clonal population boundaries via cell sorting or mixing. The boundary shape could alter the degree of cell–cell contacts and thus influence the physiological consequences of sorting or mixing at the boundary (e.g., tumor suppression or progression), suggesting that the cell mechanics could help clarify the physiology of heterogeneous tissues. While precise inference of mechanical tension loaded at each cell–cell contacts has been extensively developed, there has been little progress on how to distinguish the population-boundary geometry and identify the cause of geometry in heterogeneous tissues. We developed a pipeline by combining multivariate analysis of clone shape with tissue mechanical simulations. We examined clones with four different genotypes within Drosophila wing imaginal discs: wild-type, tartan (trn) overexpression, hibris (hbs) overexpression, and Eph RNAi. Although the clones were previously known to exhibit smoothed or convoluted morphologies, their mechanical properties were unknown. By applying a multivariate analysis to multiple criteria used to quantify the clone shapes based on individual cell shapes, we found the optimal criteria to distinguish not only among the four genotypes, but also non-genetic heterogeneity from genetic one. The efficient segregation of clone shape enabled us to quantitatively compare experimental data with tissue mechanical simulations. As a result, we identified the mechanical basis contributed to clone shape of distinct genotypes. The present pipeline will promote the understanding of the functions of mechanical interactions in heterogeneous tissue in a non-invasive manner. PMID

  9. Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    International Nuclear Information System (INIS)

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

  10. Micropropagation and maintenance of phytoplasmas in tissue culture.

    Science.gov (United States)

    Bertaccini, Assunta; Paltrinieri, Samanta; Martini, Marta; Tedeschi, Mara; Contaldo, Nicoletta

    2013-01-01

    Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

  11. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Mytych, Jennifer, E-mail: jennifermytych@gmail.com [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland)

    2017-01-15

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  12. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    International Nuclear Information System (INIS)

    Mytych, Jennifer; Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek

    2017-01-01

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  13. Tissue culture and micropropagation for forest biomass production

    Energy Technology Data Exchange (ETDEWEB)

    Mason, E.; Maine, F.W.

    1984-09-01

    An increase in forest production will be necessary in the future when wood becomes a major renewable source of energy and chemicals along with its traditional role of fibre source. This increase could eventually by achieved be proper selection and breeding of trees. Clonal forestry by vegetative propagation of cuttings is becoming a viable alternative to a seedling-based forestry with many advantages, and cutting could be used to quickly propagate large numbers of clones of control-pollinated seedlings. Most forest trees are propagated sexually and seed orchards were started in the US and Canada in the last 40-50 years for breeding purposes. Forests could ultimately be established with improved seedlings instead of from seed with unknown genetic potential, or by natural regeneration. Micropropagation is the term used to refer to the propagation of plants raised by tissue culture methods rather than from seeds or cuttings. Many clonal plantlets could be regenerated asexually in the laboratory and eventually transplanted to permanent sites. In addition the technology could be developed to produce new variants from somatic cells. Tissue culture is a technique which may be useful for plant propagation where conventional methods are inadequate or unsuitable. However, traditional studies of field planting observed over long periods of time would still be necessary. This document has the object of informing those who may wish to know more about these techniques in relation to practical application, and require a general overview rather than experimental details, which are given in an annotated bilbiography. 274 refs., 2 figs., 1 tab.

  14. Connective tissue growth factor is a positive regulator of epithelial-mesenchymal transition and promotes the adhesion with gastric cancer cells in human peritoneal mesothelial cells.

    Science.gov (United States)

    Jiang, Cheng-Gang; Lv, Ling; Liu, Fu-Rong; Wang, Zhen-Ning; Na, Di; Li, Feng; Li, Jia-Bin; Sun, Zhe; Xu, Hui-Mian

    2013-01-01

    Connective tissue growth factor (CTGF) is involved in human cancer development and progression. Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. In this study, we wished to investigate the role of CTGF in EMT of peritoneal mesothelial cells and the effects of CTGF on adhesion of gastric cancer cells to mesothelial cells. Human peritoneal mesothelial cells (HPMCs) were cultured with TGF-β1 or various concentrations of CTGF for different time. The EMT process was monitored by morphology. Real-time RT-PCR and Western blot were used to evaluate the expression of vimentin, α-SMA , E-cadherin and β-catenin. RNA interference was used to achieve selective and specific knockdown of CTGF. We demonstrated that CTGF induced EMT of mesothelial cells in a dose- and time-dependent manner. HPMCs were exposed to TGF-β1 also underwent EMT which was associated with the induction of CTGF expression. Transfection with CTGF siRNA was able to reverse the EMT partially after treatment of TGF-β1. Moreover, the induced EMT of HPMCs was associated with an increased adhesion of gastric cancer cells to mesothelial cells. These findings suggest that CTGF is not only an important mediator but a potent activator of EMT in peritoneal mesothelial cells, which in turn promotes gastric cancer cell adhesion to peritoneum. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Organotypic culture of human bone marrow adipose tissue.

    Science.gov (United States)

    Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

    2010-04-01

    The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

  16. Tissue culture of black pepper (piper nigrum l.) in Pakistan

    International Nuclear Information System (INIS)

    Hussain, A.; Naz, S.; Nazir, H.; Shinwari, Z.K.

    2011-01-01

    Black pepper (Piper nigrum L.) the 'King of Spices' is a universal table condiment. It is extensively used in Pakistani cuisines and herbal medicines and imported in bulk from neighboring countries. The black pepper vine is generally cultivated by seed because other vegetative propagation methods are slow and time consuming. Therefore the tissue culture technique is considered more efficient and reliable method for rapid and mass propagation of this economically important plant. The present study was initiated to develop protocol for micro-propagation of black pepper vine. The stem, leaf and shoot tip explants from mature vine were cultured on MS medium supplemented with different concentrations of plant growth regulators (2,4-D, BA, IBA). Best callus was produced on MS medium with 1.5 mg/l BA by shoot tip explant. Shoot regeneration was excellent on MS medium with 0.5 mg/l BA. The plantlets formed were rooted best on 1.5 mg/l IBA. The rooted plants were transplanted in soil medium and acclimatized in growth room. The plants raised were test planted under the local conditions of Hattar. (author)

  17. Coordination of Septate Junctions Assembly and Completion of Cytokinesis in Proliferative Epithelial Tissues.

    Science.gov (United States)

    Daniel, Emeline; Daudé, Marion; Kolotuev, Irina; Charish, Kristi; Auld, Vanessa; Le Borgne, Roland

    2018-05-07

    How permeability barrier function is maintained when epithelial cells divide is largely unknown. Here, we have investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. We report that, following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. We propose that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells: the protective effects of lactoferrin

    International Nuclear Information System (INIS)

    Shimmura, Shigeto; Suematsu, Makoto; Shimoyama, Masaru; Oguchi, Yoshihisa; Ishimura, Yuzuru

    1996-01-01

    Acute exposure to suprathreshold ultraviolet B radiation (UV-B) is known to cause photokeratitis resulting from the necrosis and shedding of corneal epithelial cells. However, the corneal effects of low dose UV-B in the environmental range is less clear. In this study, subthreshold UV-B was demonstrated to cause non-necrotic peroxide formation in cultured corneal epithelial cells, which was attenuated by the major tear protein lactoferrin. Intracellular oxidative insults and cell viability of rabbit corneal epithelial cells (RCEC) were assessed by dual-color digital microfluorography using carboxydichlorofluorescin (CDCFH) diacetate bis (acetoxymethyl) ester, a hydroperoxide-sensitive fluoroprobe, and propidium iodode (PI) respectively. The magnitude of UV-induced oxidative insults was calibrated by concentrations of exogenously applied H 2 O 2 which evoke compatible levels of CDCFH oxidation. Exposure of RCEC to low-dose UV-B (2.0 mJ cm -2 at 313 nm, 10.0 mJ cm -2 total UV-B) caused intracellular oxidative changes which were equivalent to those elicited by 240 μM hydrogen peroxide under the conditions of the study. The changes were dose dependent, non-necrotic, and were partially inhibited by lactoferrin ( 1 mg ml -1 ) but not by iron-saturated lactoferrin. Pretreatment with deferoxamine (2 mΜ) or catalase (100 U ml -1 ) also attenuated the UV-induced oxidative stress. The results indicate that UV-B comparable to solar irradiation levels causes significant intracellular peroxide formation in corneal epithelial cells, and that lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation. (Author)

  19. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.

  20. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    International Nuclear Information System (INIS)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs

  1. Culture of primary epithelial adenoma cells from familial adenomatous polyposis patients

    Czech Academy of Sciences Publication Activity Database

    Fostira, F.; Apessos, A.; Oikonomou, E.; Kouklis, P.; Baratsis, S.; Manifikos, G.; Anděra, Ladislav; Yannoukakos, D.; Pintzas, A.; Nasioulas, G.

    2008-01-01

    Roč. 28, 2A (2008), s. 843-846 ISSN 0250-7005 Institutional research plan: CEZ:AV0Z50520514 Keywords : colorectal neoplasia * adenomatous polyposis coli * epithelial cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.390, year: 2008

  2. The use of tissue culture techniques to detect irradiated vegetables

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Sharabi, N.E.; Nabulsi, I

    2001-01-01

    the ability of two tissue culture methods, callus and vegetable growth induction, to detect irradiated vegetables was evaluated. Potato tubers, carrot roots, garlic cloves and onion bulbs were subjected to various gamma radiation doses (0, 25, 100, 150, 250, 500, 750, and 1000 Gy). Irradiated vegetables were cultured in vitro and in vivo (pots). Gamma irradiation significantly reduced callus-forming ability especially in carrot and potato where no callus was observed in doses higher than 50 Gy. Length of shoots and roots growing from irradiated garlic and onion explants was considerably reduced starting from the 25 Gy dose. No roots were formed on garlic explants at any irradiation dose. Garlic leaves growing from irradiated explants were spotted with purple to brown spots. The intensity of these spots increased as gamma ray dosage increased. In the pot experiment, potato plant appeared in the control only. On the contrary, a complete sprouting of garlic and onion was seen in all irradiation treatments. It was not possible to distinguish between the various irradiation treatments and the control 3 days after planting in pots. The two in vitro techniques, tested in our study, may effectively be used to detect irradiated vegetables and estimate the range of doses used. The callus formation method is more useful for potato and carrot, since regeneration of shoots in vitro from these two plants takes along time, making this method unpractical. The other technique is very useful in the case of onion and garlic since it is rapid. The two techniques can be used with most of the vegetables that can be cultured in vitro. (Author)

  3. Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

    Science.gov (United States)

    Ciesiółka, S; Bryja, A; Budna, J; Kranc, W; Chachuła, A; Bukowska, D; Piotrowska, H; Porowski, L; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation

  4. Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Mee-Hae Kim

    2017-10-01

    Full Text Available In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL, which can be set to 37 °C and 5% CO2, and uses a gas-permeable resin (poly- dimethylsiloxane as the vessel wall. RPE cells were seeded at 5.0 × 104 cells/cm2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

  5. Cells in human postmortem brain tissue slices remain alive for several weeks in culture

    NARCIS (Netherlands)

    Verwer, Ronald W. H.; Hermens, Wim T. J. M. C.; Dijkhuizen, PaulaA; ter Brake, Olivier; Baker, Robert E.; Salehi, Ahmad; Sluiter, Arja A.; Kok, Marloes J. M.; Muller, Linda J.; Verhaagen, Joost; Swaab, Dick F.

    2002-01-01

    Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue

  6. Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

    Science.gov (United States)

    Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

    2016-02-01

    To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

  7. Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles.

    Science.gov (United States)

    Merz, Lea; Höbel, Sabrina; Kallendrusch, Sonja; Ewe, Alexander; Bechmann, Ingo; Franke, Heike; Merz, Felicitas; Aigner, Achim

    2017-03-01

    The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350μm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of

  8. Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

    OpenAIRE

    Beales, Ian LP

    2002-01-01

    Abstract Background Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study. Methods ...

  9. Effects of Apollo 12 lunar material on lipid levels of tobacco tissue and slash pine cultures

    Science.gov (United States)

    Weete, J. D.

    1972-01-01

    Investigations of the lipid components of pine tissues (Pinus elloitii) are discussed, emphasizing fatty acids and steroids. The response by slash pine tissue cultures to growth in contact with Apollo lunar soil, earth basalt, and Iowa soil is studied. Tissue cultures of tobacco grown for 12 weeks in contact with lunar material from Apollo 12 flight contained 21 to 35 percent more total pigment than control tissues. No differences were noted in the fresh or dry weight of the experimental and control samples.

  10. Mass micropropagation of pineapple tissue culture using bioreactor technology

    International Nuclear Information System (INIS)

    Irwan Syafri; Amir Hamzah Harun; Rusli Ibrahim

    2005-01-01

    Pineapple (ananas comosus) is the most important fruit in terms of revenue earner in this country. The export of the canned pineapple is about 2 million standard cases annually valued at RM 60 million, while the export of fresh pineapple is about 40,000 tonnes worth about RM 10 million. The industry for canning is however, an ailing industry with production on the decline since the 70s. Scaling up the pineapple propagation using in vitro methods seems to be possible solutions for the lack of planting material. Temporary immersion system (TIS) has been described by Teisson and Alvard (1995) for plant tissue culture propagation. This system, also known as RITA, has been successfully used with embryogenic tissues of banana (Alvard et al 1993), coffee (Berthouly 1991), rubber (Etienne et al 1993) and sugarcane (Lorenzo et al 1998). In this study, the system has been set up with a potential capacity of 3 manifolds with 10 RITA each, to multiply meristem explants at different immersion periods. The system was compared with the conventional micropropagation system on solid medium. Both systems were treated with MS media containing 2.5 mg/l BAP and 0.1 NAA. In TIS the shoots were able to multiplied faster in comparison with solid media. The multiplication rates were increased up to 1:3 to 1:5 compared to normal propagation on solid media. The results show that TIS not only increase the propagation rates of pineapple but could also be adapted to reduce implementation costs to establish low-cost propagation systems. (Author)

  11. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    International Nuclear Information System (INIS)

    Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

    2013-01-01

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment

  12. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    Energy Technology Data Exchange (ETDEWEB)

    Katano, Takahito [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Ootani, Akifumi [Department of Gastroenterology and GI Endoscopy Center, Shin-Kokura Hospital, Federation of National Public Service Personnel Mutual Aid Associations, 1-3-1 Kanada, Kokurakita-ku, Kitakyushu 803-0816 (Japan); Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Mizoshita, Tsutomu, E-mail: tmizoshi@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Toda, Shuji [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    2013-03-22

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

  13. NMR-based metabolomics of mammalian cell and tissue cultures

    International Nuclear Information System (INIS)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan; Niemitz, Matthias; Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D.

    2011-01-01

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  14. Flowering of Woody Bamboo in Tissue Culture Systems

    Directory of Open Access Journals (Sweden)

    Jin-Ling Yuan

    2017-09-01

    Full Text Available Flowering and subsequent seed set are not only normal activities in the life of most plants, but constitute the very reason for their existence. Woody bamboos can take a long time to flower, even over 100 years. This makes it difficult to breed bamboo, since flowering time cannot be predicted and passing through each generation takes too long. Another unique characteristic of woody bamboo is that a bamboo stand will often flower synchronously, both disrupting the supply chain within the bamboo industry and affecting local ecology. Therefore, an understanding of the mechanism that initiates bamboo flowering is important not only for biology research, but also for the bamboo industry. Induction of flowering in vitro is an effective way to both shorten the flowering period and control the flowering time, and has been shown for several species of bamboo. The use of controlled tissue culture systems allows investigation into the mechanism of bamboo flowering and facilitates selective breeding. Here, after a brief introduction of flowering in bamboo, we review the research on in vitro flowering of bamboo, including our current understanding of the effects of plant growth regulators and medium components on flower induction and how in vitro bamboo flowers can be used in research.

  15. NMR-based metabolomics of mammalian cell and tissue cultures

    Energy Technology Data Exchange (ETDEWEB)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan [Bristol-Myers Squibb Company (United States); Niemitz, Matthias [PERCH Solutions Ltd. (Finland); Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D., E-mail: michael.reily@bms.com [Bristol-Myers Squibb Company (United States)

    2011-04-15

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  16. Epithelial control of gut-associated lymphoid tissue formation through p38α-dependent restraint of NF-κB signaling

    Science.gov (United States)

    Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

    2015-01-01

    The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. Here we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a non-cell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. PMID:26792803

  17. Epithelial Control of Gut-Associated Lymphoid Tissue Formation through p38α-Dependent Restraint of NF-κB Signaling.

    Science.gov (United States)

    Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

    2016-03-01

    The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. In this study, we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a noncell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are most likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. Copyright © 2016 by The American Association of Immunologists, Inc.

  18. Sub-epithelial connective tissue graft for root coverage in nonsmokers and smokers: A pilot comparative clinical study

    Directory of Open Access Journals (Sweden)

    Chini Doraswamy Dwarakanath

    2016-01-01

    Full Text Available Background: Gingival recession is a common condition and is more prevalent in smokers. It is widely believed that root coverage procedures in smokers result in less desirable outcome compared to nonsmokers', and there are few controlled studies in literature to support this finding. Therefore, the purpose of this study was to evaluate and compare the outcome of root coverage with sub-epithelial connective tissue graft (SCTG in nonsmokers and smokers. Materials and Methods: A sample of twenty subjects, 10 nonsmokers and 10 smokers were selected each with at least 1 Miller's Class I or II recession on a single rooted tooth. Clinical measurements of probing depth, clinical attachment level (CAL, gingival recession total surface area (GRTSA, depth of recession (RD, width of recession (RW, and width of keratinized tissue were determined at baseline, 3, and 6 months after surgery. Results: The treatment of gingival recession with SCTG and coronally advanced flap showed a decrease in the GRTSA, RD, RW, and an increase in CAL and width of keratinized gingiva in both the groups. However, the intergroup comparison of the clinical parameters showed no statistical significance. About 6 out of 10 nonsmokers (60% and 3 smokers (30% showed complete root coverage. The mean percentage of root coverage of 71.2% in nonsmokers and 38% in smokers was observed. Conclusion: The results of the present study suggest that smoking may negatively influence gingival recession reduction and CAL gain. In addition, smokers may exhibit fewer chances of complete root coverage. Overall, nonsmokers showed better improvements in all the parameters compared to smokers at the end of 6 months.

  19. Localization of trefoil factor family peptide 3 (TFF3) in epithelial tissues originating from the three germ layers of developing mouse embryo.

    Science.gov (United States)

    Bijelić, Nikola; Belovari, Tatjana; Tolušić Levak, Maja; Baus Lončar, Mirela

    2017-08-20

    Trefoil factor family (TFF) peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old) were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3,3'-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation.

  20. Localization of trefoil factor family peptide 3 (TFF3 in epithelial tissues originating from the three germ layers of developing mouse embryo

    Directory of Open Access Journals (Sweden)

    Nikola Bijelić

    2017-08-01

    Full Text Available Trefoil factor family (TFF peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3,3'-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation.

  1. Use of UV-irradiated bacteriophage T6 to kill extracellular bacteria in tissue culture infectivity assays

    International Nuclear Information System (INIS)

    Shaw, D.R.; Maurelli, A.T.; Goguen, J.D.; Straley, S.C.; Curtiss, R. III

    1983-01-01

    The authors have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique described has the following characteristics: (a) bacterial killing is complete within 15 min at 37 0 C, with a >10 3 -fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues. (Auth.)

  2. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from [U- 14 C]glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state

  3. Correlation of Hypoxia and Pro-senescence Protein Expression in Green Sea Turtle (Chelonia mydas Lung Epithelial and Dermal Fibroblast Cell Culture

    Directory of Open Access Journals (Sweden)

    Anggraini Barlian

    2018-03-01

    Full Text Available Recent studies have shown hypoxia-induced gene expression correlated with cellular senescence. HIF-1α (hypoxia-inducible factor 1-alpha, p53, and pRB were induced under hypoxia and correlated with cellular senescence. The localization and expression of HIF-1α, p53, and pRB in Chelonia mydas lung epithelial and dermal fibroblast cell cultures were analyzed under normoxic and hypoxic conditions (at 4 and 24 hours. Human dermal fibroblast was used for comparison purposes. Protein localization was analyzed with immunocytochemistry, while protein expression was analyzed with the Western blot and enhanced chemiluminescence (ECL method. HIF-1α, p53, and pRB were localized in the nuclei of the C. mydas cell cultures treated with hypoxia. The C. mydas lung epithelial cell cultures had a higher increase of HIF-1α expression than the human dermal fibroblast cell culture. The hypoxic conditions did not affect p53 expression significantly in C. mydas lung epithelial and dermal fibroblast cell cultures. Meanwhile, pRB expression changed significantly under hypoxia in the C. mydas dermal fibroblast cells. Expression of p53 and pRB in the human cell cultures was higher than in the C. mydas cell cultures. This research suggests that C. mydas and human cell cultures have different pro-senescence protein expression responses under hypoxic conditions.

  4. Dose Optimization of Calcusol™ and Calcium Oxalate Monohydrate (COM on Primary Renal Epithelial Cells Cultures of Mice ( Mus musculus

    Directory of Open Access Journals (Sweden)

    Ahmad Soni

    2014-05-01

    Full Text Available Kidney stones are one of the urologic diseases that have plagued mankind for centuries. The main constituents of stones in the kidney are calcium oxalate monohydrate (COM crystals. Nowadays, there are varieties of drugs and treatments that can be made to minimize the grievances due to kidney stone disease. The treatment can be done either by using chemicals or traditional medicine. Calcusol™ is one of the popular herbal products that have been used by Indonesian people in curing the kidney stone disease. The main constituent that was contained in Calcusol™ is an extract of the tempuyung leaves (Sonchus arvensis L., which is expected could cure the kidney stone disease. This study used primary cultured renal epithelial cells of mice to determine the optimal dose of Calcusol™ and the optimal dose of COM. The primary Kidney epithelial cell were treated with Calcusol™ and COM at various doses. The analysis of the cell death either by necrosis or apoptosis pathways was analyzed by flow cytometric analysis. The results that were obtained is the percentage of cell death that is then analyzed by using a complete randomized design (CRD One Way Anova. Based on the results that were obtained, it is known that the optimal dose of Calcusol™ in vitro were ranging from 75 ppm to 100 ppm, whereas the optimal dose of COM suggested for 500 ppm.

  5. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and equipment. 864.2240 Section 864.2240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products...

  6. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and components. 864.2220 Section 864.2220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture...

  7. Advances in tissue engineering through stem cell-based co-culture.

    Science.gov (United States)

    Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-05-01

    Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Clonal differences in generation times of GPK epithelial cells in monolayer culture.

    Science.gov (United States)

    Riley, P A; Hola, M

    1980-01-01

    Pedigrees of cells in eight clones of guinea pig keratocyte (GPK) cells in monolayer culture were analyzed from a time-lapse film. The generation times and the position in the field of observation were recorded up to the sixth generation when the cultures were still subconfluent. Statistical analysis of the results indicates that the position in the culture has less significance than the clonal origin of the cell in determining the interval between successive mitoses.

  9. Apical polarity in three-dimensional culture systems: where to now?

    Energy Technology Data Exchange (ETDEWEB)

    Inman, J.L.; Bissell, Mina

    2010-01-21

    Delineation of the mechanisms that establish and maintain the polarity of epithelial tissues is essential to understanding morphogenesis, tissue specificity and cancer. Three-dimensional culture assays provide a useful platform for dissecting these processes but, as discussed in a recent study in BMC Biology on the culture of mammary gland epithelial cells, multiple parameters that influence the model must be taken into account.

  10. Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and pharmaceutical research

    Energy Technology Data Exchange (ETDEWEB)

    Minuth, Will W [Department of Molecular and Cellular Anatomy, University of Regensburg, D-93053 Regensburg, University Street 31 (Germany); Strehl, Raimund [Cellartis AB, S-41346 Goeteborg, Arvid Wallgrens Backe 20 (Sweden)

    2007-06-01

    Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container. (topical review)

  11. Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and pharmaceutical research

    International Nuclear Information System (INIS)

    Minuth, Will W; Strehl, Raimund

    2007-01-01

    Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container. (topical review)

  12. A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

    Directory of Open Access Journals (Sweden)

    Tobias Bohnenpoll

    2017-08-01

    Full Text Available The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH family of secreted proteins, Sonic hedgehog (SHH as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT.

  13. A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

    Science.gov (United States)

    Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

    2017-08-01

    The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

  14. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

    Science.gov (United States)

    Eiraku, Mototsugu; Sasai, Yoshiki

    2011-12-15

    Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

  15. Research progress in plant mutation by combining ion beam irradiations and tissue culture

    International Nuclear Information System (INIS)

    Zhou Linbin; Li Wenjian; Qu Ying; Li Ping

    2007-01-01

    About a new mutation breeding method which combines plant tissue culture technique with heavy ion beam irradiations were discussed in this paper with the principles, operation steps, molecular mechanisms, etc. The mutation method developed a few advantages coming from plant tissue culture, which can produce offspring by asexual ways. Meanwhile, using this method, the study of biological effects of high energy particles with different linear energy transfer values on plant tissues or cells can be explored and optimized in theory or practice. (authors)

  16. Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

    Directory of Open Access Journals (Sweden)

    Saw Yu-Ting

    2012-08-01

    Full Text Available Abstract Background Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Methods Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Results Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. Conclusions The results of our study indicate that ALDH enzyme expression and activity may be associated

  17. Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

    International Nuclear Information System (INIS)

    Saw, Yu-Ting; Thompson, David; Vasiliou, Vasilis; Berkowitz, Ross S; Ng, Shu-Wing; Yang, Junzheng; Ng, Shu-Kay; Liu, Shubai; Singh, Surendra; Singh, Margit; Welch, William R; Tsuda, Hiroshi; Fong, Wing-Ping

    2012-01-01

    Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to

  18. Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells

    Science.gov (United States)

    Emam, Aufaugh; Carter, William G; Lingwood, Clifford

    2010-01-01

    Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed PMID:21270937

  19. Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

    International Nuclear Information System (INIS)

    Choudhary, S.; Xiao, T.; Srivastava, S.; Zhang, W.; Chan, L.L.; Vergara, L.A.; Van Kuijk, F.J.G.M.; Ansari, N.H.

    2005-01-01

    Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H 2 O 2 , 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H 2 O 2 -, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H 2 O 2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3 H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST) (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD

  20. Effects of Lutein on Hyperosmoticity-Induced Upregulation of IL-6 in Cultured Corneal Epithelial Cells and Its Relevant Signal Pathways

    Directory of Open Access Journals (Sweden)

    Shih-Chun Chao

    2016-01-01

    Full Text Available Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye.

  1. An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

    Directory of Open Access Journals (Sweden)

    Kajohnkiart Janebodin

    2013-01-01

    Full Text Available Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10, decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

  2. Revision washout decreases implant capsule tissue culture positivity: a multicenter study.

    Science.gov (United States)

    Henry, Gerard D; Carson, Culley C; Wilson, Steven K; Wiygul, Jeremy; Tornehl, Chris; Cleves, Mario A; Simmons, Caroline J; Donatucci, Craig F

    2008-01-01

    Positive cultures, visible biofilm and confocal micrography confirm bacterial presence on clinically uninfected inflatable penile prostheses at revision surgery. Salvage irrigation has been proved to rescue patients with clinically infected inflatable penile prostheses. Similar washout at revision for noninfectious reasons significantly lowers subsequent infection rates. We investigated a larger series of patients for positive culture rates and evaluated implant capsule tissue culture rates before and after revision washout. At 4 institutions a total of 148 patients with inflatable penile prostheses underwent revision surgery for noninfectious reasons between June 2001 and September 2005. Swab cultures of the fluid around the pump and visible biofilm were obtained. Also, in 65 patients a wedge of tissue from the capsule that forms around the pump was cultured. After implant removal revision washout of the implant spaces was performed and a second wedge of tissue was cultured. Of the 148 patients 97 (66%) had positive bacterial swab cultures of the fluid around the pump or biofilm. A total of 124 isolates were cultured. Of the 65 implant capsule tissue cultures obtained before washout 28 (43%) were positive for bacteria, while 16 (25%) obtained after revision washout were positive. Positive cultures and visible bacterial biofilm are present on clinically uninfected inflatable penile prostheses at revision surgery in most patients. Revision washout appears to decrease the bacterial load on implant capsule tissue at revision surgery of inflatable penile prostheses for noninfectious reasons.

  3. Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses.

    Science.gov (United States)

    Punyadarsaniya, Darsaniya; Winter, Christine; Mork, Ann-Kathrin; Amiri, Mahdi; Naim, Hassan Y; Rautenschlein, Silke; Herrler, Georg

    2015-02-01

    Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. [Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

    Science.gov (United States)

    Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

    2016-06-25

    To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Ptissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (Ptissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

  5. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica

    2008-01-01

    (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures....... In BME-UV1, spermine significantly inhibited cell proliferation (Pcultured in the presence of all amines...

  6. Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

    Science.gov (United States)

    Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Nakamura, Ryosuke; Otsuki, Koshi; Murono, Shigeyuki; Omori, Koichi

    2017-07-01

    Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

  7. Propagation of jarrah (Eucalyptus marginata) by organ and tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, M.J.; McComb, J.A.

    1982-01-01

    Micropropagation methods are described for the production of clonal lines from Eucalyptus marginata (jarrah) seedlings. Nodal explants from mature trees can also yield shoot cultures, but a high frequency of contamination occurs among such explants. Uncontaminated callus cultures can be produced from mature trees by culturing stamen filaments and shoots can subsequently be regenerated from this callus. The rooting percentage of shoot cultures from either nodes or stamen callus of mature trees is low compared with that from seedling explants. Considerable variation was observed between trees in the ability of stamen callus to regenerate shoots and in the frequency of rooting. (Refs. 27)

  8. Expression of P-cadherin identifies prostate-specific-antigen-negative cells in epithelial tissues of male sexual accessory organs and in prostatic carcinomas. Implications for prostate cancer biology.

    OpenAIRE

    Soler, A. P.; Harner, G. D.; Knudsen, K. A.; McBrearty, F. X.; Grujic, E.; Salazar, H.; Han, A. C.; Keshgegian, A. A.

    1997-01-01

    Cadherins constitute a family of calcium-dependent cell-cell adhesion molecules the individual members of which are essential for the sorting of cells into tissues during development. In this study, we examined the expression of E-cadherin, N-cadherin, and P-cadherin in tissues obtained from radical prostatectomies. Epithelial cells of prostatic glands, ejaculatory ducts, and seminal vesicles expressed E-cadherin but not N-cadherin. P-cadherin was expressed in epithelial cells of the seminal ...

  9. Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

    Directory of Open Access Journals (Sweden)

    Kamal R. Acharya

    2017-12-01

    Full Text Available Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38. Likewise, almost all tissue culture (61/64 or histopathology (52/58 positives were detected with good to moderate agreement (Cohen’s kappa statistic and no significant difference to the reference tests (McNemar’s Chi-square test. Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07. Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

  10. Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

    Science.gov (United States)

    Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

    2014-05-01

    Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

    Energy Technology Data Exchange (ETDEWEB)

    Sluis, C.

    1980-09-01

    The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media and rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.

  12. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  13. Application of tissue culture to cashew ( Anacardium occidentale L ...

    African Journals Online (AJOL)

    Summary of the previous works on the in vitro culture of cashew is highlighted with emphasis on the critical factors that influence the explants response and plantlet regeneration. The recalcitrant nature of cashew has been attributed to the limited success recorded so far in the in vitro culture of the crop and abnormal ...

  14. Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.

    Science.gov (United States)

    Yi, Jin-Kyu; Mehrazarin, Shebli; Oh, Ju-Eun; Bhalla, Anu; Oo, Jenessa; Chen, Wei; Lee, Min; Kim, Reuben H; Shin, Ki-Hyuk; Park, No-Hee; Kang, Mo K

    2014-11-01

    Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid.

    Directory of Open Access Journals (Sweden)

    Robert A Hirst

    Full Text Available The diagnosis of primary ciliary dyskinesia (PCD requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n  111 was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture.Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

  16. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

    Science.gov (United States)

    Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

    2017-08-07

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

  17. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  18. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Science.gov (United States)

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  19. Equine ovarian tissue viability after cryopreservation and in vitro culture

    Science.gov (United States)

    The efficiency of several cryoprotective agents were compared using both slow-freezing and vitrification methods. Results indicate that the viability of ovarian tissue cells increases when DMSO (slow-freezing) and ethylene glycol (vitrification) are used....

  20. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

    Science.gov (United States)

    Pilgrim, Matthew G; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C; Messinger, Jeffrey D; Read, Russell W; Guidry, Clyde; Curcio, Christine A

    2017-02-01

    Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

  1. Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions

    NARCIS (Netherlands)

    Hiemstra, P.S.; Grootaers, G.G.; Does, A.M. van der; Krul, C.A.M.; Kooter, I.M.

    2018-01-01

    The epithelium that covers the conducting airways and alveoli is a primary target for inhaled toxic substances, and therefore a focus in inhalation toxicology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the

  2. Specificity in calcium oxalate adherence to papillary epithelial cells in culture

    International Nuclear Information System (INIS)

    Riese, R.J.; Riese, J.W.; Kleinman, J.G.; Wiessner, J.H.; Mandel, G.S.; Mandel, N.S.

    1988-01-01

    Attachment of microcystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with [ 14 C]CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a β value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/α ox value of 287 μg/cm 2 . On incubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/α AP value of 93 μg/cm 2 . Microcystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding

  3. [18S-25S rDNA variation in tissue culture of some Gentiana L. species].

    Science.gov (United States)

    Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

    2007-01-01

    18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

  4. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  5. Mary Jane Hogue (1883-1962): A pioneer in human brain tissue culture.

    Science.gov (United States)

    Zottoli, Steven J; Seyfarth, Ernst-August

    2018-05-16

    The ability to maintain human brain explants in tissue culture was a critical step in the use of these cells for the study of central nervous system disorders. Ross G. Harrison (1870-1959) was the first to successfully maintain frog medullary tissue in culture in 1907, but it took another 38 years before successful culture of human brain tissue was accomplished. One of the pioneers in this achievement was Mary Jane Hogue (1883-1962). Hogue was born into a Quaker family in 1883 in West Chester, Pennsylvania, and received her undergraduate degree from Goucher College in Baltimore, Maryland. Research with the developmental biologist Theodor Boveri (1862-1915) in Würzburg, Germany, resulted in her Ph.D. (1909). Hogue transitioned from studying protozoa to the culture of human brain tissue in the 1940s and 1950s, when she was one of the first to culture cells from human fetal, infant, and adult brain explants. We review Hogue's pioneering contributions to the study of human brain cells in culture, her putative identification of progenitor neuroblast and/or glioblast cells, and her use of the cultures to study the cytopathogenic effects of poliovirus. We also put Hogue's work in perspective by discussing how other women pioneers in tissue culture influenced Hogue and her research.

  6. Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

    Science.gov (United States)

    Ammar, David A; Kahook, Malik Y

    2011-10-01

    We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6). BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p < 0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p < 0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p < 0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%). These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

  7. Retinal Pigment Epithelial Cell Culture and Cooperation of L-carnitine in Reducing Stress Induced Cellular Damage

    International Nuclear Information System (INIS)

    Shamsi, Farrukh A.; Al-Rajhi, Ali A.; Athmanathan, S.; Boulton, M.; Chaudhry, Imtiaz A.

    2006-01-01

    Purpose was to show that L-carnitine (LC) is capable of reducing non-oxidative stress in the retinal pigment epithelial cells (RPE) of the human eye. The RPE cells were cultured from donor eyes, obtained immediately after post-mortem. The interaction between bovine serum albumin (BSA) and non-oxidative (sodium hydroxide and methyl methane sulphonate) stress-inducers was observed by recording the change in the absorption profiles of the interacting molecules after incubation in light for 5 hours and after treatment with LC. The isolated and cultured RPE cells from the human eyes were treated with sodium hydroxide or methyl methane sulphonate and/or LC for 5 hours under light, and the qualitative effect on cell morphology after treatment was analyzed by staining cells with Giemsa and visualization by light microscopy. The cell morphology was also qualitatively analyzed by scanning electron microscopy (SEM). L-carnitine and stress-inducers interact with BSA and bring about changes in the spectral profile of the interacted molecules. Light microscopy as well as SEM show that the changes in the cellular morphology, induced by 100 uM concentrations of non-oxidative stress-inducers, are considerably reduced in the presence of 100 uM LC. However, L-carnitine alone does not cause any qualitative damage to the cell morphology during incubation under similar conditions. The results give a preliminary indication that LC has ability to reduce the changes brought about by the non-oxidative stress-inducers in the RPF cells in culture. (author)

  8. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    Science.gov (United States)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  9. The use of cultured epithelial autograft in the treatment of major burn injuries: a critical review of the literature.

    Science.gov (United States)

    Wood, F M; Kolybaba, M L; Allen, P

    2006-06-01

    The need to achieve rapid wound closure in patients with massive burns and limited skin donor sites led to the investigation of in vitro cellular expansion of keratinocytes. The use of cultured epithelial autografts (CEA) was first reported in the treatment of major burns in 1981. Since that time, support for the use of CEA has varied, ranging from 'a useful agent' to having 'no demonstrable effect on the outcome of extensively burned patients'. This critical review of the literature examines issues associated with the use of CEA and the introduction of the technology into clinical practice. The factors potentially limiting the use of cultured CEA are the time necessary to culture CEA sheets, the reliability of 'take', vulnerability of grafts on the newly healed surface, long-term durability and the cost implications of such treatment. The available literature was located and critically evaluated using the Australian National Health and Medical Research Council Guidelines. In the identified literature, the level of evidence to support the use of CEA in major burn injures is limited and often restricted to case studies and case series with no Level 1 evidence currently available. The main question arising 'Does CEA have a role in the treatment of major burns?' has proven difficult to answer due to the wide variation in both the quality of study design and the findings. At best, the literature review has highlighted areas of concern that have hindered the successful use of CEA. Our review critically evaluates the use of CEA and explores the advances in techniques towards attempting to improve reliable clinical implementation of CEA. The need for higher level research into the use of CEA is emphasised by this review.

  10. The effect of plant growth regulators on optimization of tissue culture ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... ISSN 1684–5315 © 2010 Academic Journals ... tissue culture system in Malaysian upland rice ... Scientists believe that using new cultivars which have potential ..... providing the financial support and to Firouzeh Ashjazadeh ...

  11. How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture.

    Science.gov (United States)

    Haldeman, Janice H.; Ellis, Jane P.

    1988-01-01

    Presents techniques used for disinfestation of plant material, preparation of equipment and media, and laboratory procedures for tissue culture using cauliflower. Details methods for preparing solutions and plant propagation by cloning. (CW)

  12. Silk Film Topography Directs Collective Epithelial Cell Migration

    Science.gov (United States)

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  13. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  14. Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors.

    Science.gov (United States)

    Saegeman, V; Verhaegen, J; Lismont, D; Verduyckt, B; De Rijdt, T; Ectors, N

    2009-02-01

    Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

  15. Canine osteosarcoma karyotypes from an original tumor, its metastasis, and tumor cells in tissue culture

    International Nuclear Information System (INIS)

    Taylor, N.; Shifrine, M.; Wolf, H.G.; Trommershausen-Smith, A.

    1975-01-01

    Radiation-induced osteosarcoma, its metastasis, and cells grown in tissue culture were karyotyped. Both hypodiploid and hyperdiploid stem lines were observed. The hypodiploid line contained 45-55 chromosomes with 10 to 15 abnormal metacentric and submetacentric chromosomes and one subtelocentric marker. The hyperdiploid line contained 90 to 105 chromosomes with 20 to 30 abnormal metacentric and submetacentric chromosomes with two subtelocentric markers. Karyotypic analysis can be used to monitor osteosarcomas maintained in tissue culture

  16. Substrate specific hydrolysis of aromatic and aromatic-aliphatic esters in orchid tissue cultures

    Directory of Open Access Journals (Sweden)

    Agnieszka Mironowicz

    2014-01-01

    Full Text Available We found that tissue cultures of higher plants were able, similarly as microorganisms, to transform low-molecular-weight chemical compounds. In tissue cultures of orchids (Cymbidium 'Saint Pierre' and Dendrobium phalaenopsis acetates of phenols and aromatic-aliphatic alcohols were hydrolyzed, whereas methyl esters of aromatic and aromatic-aliphatic acids did not undergo this reaction. Acetates of racemic aromatic-aliphatic alcohols were hydrolyzed with distinct enantiospecificity.

  17. Cloning higher plants from aseptically cultured tissues and cells

    Science.gov (United States)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  18. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

    Science.gov (United States)

    Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe

    2012-04-01

    IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

  19. In-vivo nonlinear optical microscopy (NLOM) of epithelial-connective tissue interface (ECTI) reveals quantitative measures of neoplasia in hamster oral mucosa.

    Science.gov (United States)

    Pal, Rahul; Yang, Jinping; Ortiz, Daniel; Qiu, Suimin; Resto, Vicente; McCammon, Susan; Vargas, Gracie

    2015-01-01

    The epithelial-connective tissue interface (ECTI) plays an integral role in epithelial neoplasia, including oral squamous cell carcinoma (OSCC). This interface undergoes significant alterations due to hyperproliferating epithelium that supports the transformation of normal epithelium to precancers and cancer. We present a method based on nonlinear optical microscopy to directly assess the ECTI and quantify dysplastic alterations using a hamster model for oral carcinogenesis. Neoplastic and non-neoplastic normal mucosa were imaged in-vivo by both multiphoton autofluorescence microscopy (MPAM) and second harmonic generation microscopy (SHGM) to obtain cross-sectional reconstructions of the oral epithelium and lamina propria. Imaged sites were biopsied and processed for histopathological grading and measurement of ECTI parameters. An ECTI shape parameter was calculated based on deviation from the linear geometry (ΔLinearity) seen in normal mucosa was measured using MPAM-SHGM and histology. The ECTI was readily visible in MPAM-SHGM and quantitative shape analysis showed ECTI deformation in dysplasia but not in normal mucosa. ΔLinearity was significantly (p tissue with 87.9% sensitivity and 97.6% specificity, while calculations from histology provided 96.4% sensitivity and 85.7% specificity. Among other quantifiable architectural changes, a progressive statistically significant increase in epithelial thickness was seen with increasing grade of dysplasia. MPAM-SHGM provides new noninvasive ways for direct characterization of ECTI which may be used in preclinical studies to investigate the role of this interface in early transformation. Further development of the method may also lead to new diagnostic approaches to differentiate non-neoplastic tissue from precancers and neoplasia, possibly with other cellular and layer based indicators of abnormality.

  20. Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

    Science.gov (United States)

    Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

    2008-10-01

    Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

  1. Apollo 12 lunar material - Effects on lipid levels of tobacco tissue cultures.

    Science.gov (United States)

    Weete, J. D.; Walkinshaw, C. H.; Laseter, J. L.

    1972-01-01

    Tobacco tissue cultures grown in contact with lunar material from Apollo 12, for a 12-week period, resulted in fluctuations of both the relative and absolute concentrations of endogenous sterols and fatty acids. The experimental tissues contained higher concentrations of sterols than the controls did. The ratio of campesterol to stigmasterol was greater than 1 in control tissues, but less than 1 in the experimental tissues after 3 weeks. High relative concentrations (17.1 to 22.2 per cent) of an unidentified compound or compounds were found only in control tissues that were 3 to 9 weeks of age.

  2. [Research progress of co-culture system for constructing vascularized tissue engineered bone].

    Science.gov (United States)

    Fu, Weili; Xiang, Zhou

    2014-02-01

    To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

  3. Effect of hGC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice.

    Science.gov (United States)

    Song, Lin; Zhou, Xin; Jia, Hong-Jun; Du, Mei; Zhang, Jin-Ling; Li, Liang

    2016-08-01

    To study the effect of hGC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice. BABL/c nude mice were selected as experimental animals and gastric cancer tumor-bearing mice model were established by subcutaneous injection of gastric cancer cells, randomly divided into different intervention groups. hGC-MSCs group were given different amounts of gastric cancer cells for subcutaneous injection, PBS group was given equal volume of PBS for subcutaneous injection. Then tumor tissue volume were determined, tumor-bearing mice were killed and tumor tissues were collected, mRNA expression of proliferation, invasion, EMT-related molecules were determined. 4, 8, 12, 16, 20 d after intervention, tumor tissue volume of hGC-MSCs group were significantly higher than those of PBS group and the more the number of hGC-MSCs, the higher the tumor tissue volume; mRNA contents of Ki-67, PCNA, Bcl-2, MMP-2, MMP-7, MMP-9, MMP-14, N-cadherin, vimentin, Snail and Twist in tumor tissue of hGC-MSCs group were higher than those of PBS group, and mRNA contents of Bax, TIMP1, TIMP2 and E-cadherin were lower than those of PBS group. hGC-MSCs from human gastric cancer tissue can promote the tumor growth in gastric cancer tumor-bearing mice, and the molecular mechanism includes promoting cell proliferation, invasion and epithelial-mesenchymal transition. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  4. Plant Regeneration Through Tissue Culture Of Pear Millet ...

    African Journals Online (AJOL)

    1. 1. 2,5), MS(5) and N6(1.100.25) culture media, calli embryogenic potential and fertile plants regeneration were conserved for more than 12 months. Characteristics of regenerated plants were similar to control. It appears that dissected shoot ...

  5. Tissue culture as a plant production technique for horticultural crops

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-18

    Aug 18, 2009 ... Recovery of regenerants from transformed cells. - Cell culture .... methods. Micropropagation techniques. Micropropagation is a simple concept. The basic pro- tocols were well established by the 1960s and a whole research field and ... the environment are naturally contaminated on their sur- faces (and ...

  6. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten

    1998-01-01

    in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase...

  7. Co-culture systems-based strategies for articular cartilage tissue engineering.

    Science.gov (United States)

    Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

    2018-03-01

    Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

  8. The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

    Science.gov (United States)

    Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

    2006-06-01

    Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

  9. Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

    Science.gov (United States)

    Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

    2009-04-01

    Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

  10. MicroRNA-145 Inhibits Cell Migration and Invasion and Regulates Epithelial-Mesenchymal Transition (EMT) by Targeting Connective Tissue Growth Factor (CTGF) in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Wang, Xiao-Jing; Zhang, Bing; Chen, Hua

    2016-10-23

    BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (Ptissues was than in normal adjacent esophageal tissues (Ptissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

  11. A core invasiveness gene signature reflects epithelial-to-mesenchymal transition but not metastatic potential in breast cancer cell lines and tissue samples.

    Directory of Open Access Journals (Sweden)

    Melike Marsan

    Full Text Available INTRODUCTION: Metastases remain the primary cause of cancer-related death. The acquisition of invasive tumour cell behaviour is thought to be a cornerstone of the metastatic cascade. Therefore, gene signatures related to invasiveness could aid in stratifying patients according to their prognostic profile. In the present study we aimed at identifying an invasiveness gene signature and investigated its biological relevance in breast cancer. METHODS & RESULTS: We collected a set of published gene signatures related to cell motility and invasion. Using this collection, we identified 16 genes that were represented at a higher frequency than observed by coincidence, hereafter named the core invasiveness gene signature. Principal component analysis showed that these overrepresented genes were able to segregate invasive and non-invasive breast cancer cell lines, outperforming sets of 16 randomly selected genes (all P<0.001. When applied onto additional data sets, the expression of the core invasiveness gene signature was significantly elevated in cell lines forced to undergo epithelial-mesenchymal transition. The link between core invasiveness gene expression and epithelial-mesenchymal transition was also confirmed in a dataset consisting of 2420 human breast cancer samples. Univariate and multivariate Cox regression analysis demonstrated that CIG expression is not associated with a shorter distant metastasis free survival interval (HR = 0.956, 95%C.I. = 0.896-1.019, P = 0.186. DISCUSSION: These data demonstrate that we have identified a set of core invasiveness genes, the expression of which is associated with epithelial-mesenchymal transition in breast cancer cell lines and in human tissue samples. Despite the connection between epithelial-mesenchymal transition and invasive tumour cell behaviour, we were unable to demonstrate a link between the core invasiveness gene signature and enhanced metastatic potential.

  12. [Effective productions of plant secondary metabolites having antitumor activity by plant cell and tissue cultures].

    Science.gov (United States)

    Taniguchi, Shoko

    2005-06-01

    Methods for the effective production of plant secondary metabolites with antitumor activity using plant cell and tissue cultures were developed. The factors in tannin productivity were investigated using culture strains producing different types of hydrolyzable tannins, i.e., gallotannins (mixture of galloylglucoses), ellagi-, and dehydroellagitannins. Production of ellagi- and dehydroellagitannins was affected by the concentrations and ratio of nitrogen sources in the medium. The formation of oligomeric ellagitannins in shoots of Oenothera tetraptera was correlated with the differentiation of tissues. Cultured cells of Eriobotrya japonica producing ursane- and oleanane-type triterpenes with antitumor activities were also established.

  13. Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

    Science.gov (United States)

    Varettas, Kerry

    2013-12-01

    Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

  14. The basic design and requirement for plant tissue culture laboratory in MINT

    International Nuclear Information System (INIS)

    Azraf Azman; Rosli Darmawan; Rusli Ibrahim; Mohd Nazir Basiran; Azhar Mohamad; Mohamed Najli Mohamed Yasin; Shuhaimi Shamsuddin

    2005-01-01

    The production of multiple species plantlets involves a relatively complex process and it is a highly specialized operation. Tissue culture technology is rapidly becoming a commercialized method for propagating new cultivars, rare species and difficult-to-propagate plant. Not only are skills and knowledge essential but the laboratory itself also plays an important role to ensure the successful growth of the plantlets. To produce quality plantlets, plant tissue culture laboratories should fulfill the basic requirements. The laboratory should have proper building and layout which comprise of media preparation and washing room, sterilization or autoclave room, transfer room and culture or growth room. The scope of this paper is to compare these fundamental requirements with the plant tissue culture laboratory in MINT. All the basic needs and differences will be discussed and the proposal for corrective actions will be presented. (Author)

  15. TCUP: A novel hAT transposon active in maize tissue culture

    Directory of Open Access Journals (Sweden)

    Alan eSmith

    2012-01-01

    Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  16. Histological and Thermometric Examination of Soft Tissue De-Epithelialization Using Digitally Controlled Er:YAG Laser Handpiece: An Ex Vivo Study.

    Science.gov (United States)

    Grzech-Leśniak, Kinga; Matys, Jacek; Jurczyszyn, Kamil; Ziółkowski, Piotr; Dominiak, Marzena; Brugnera Junior, Aldo; Romeo, Umberto

    2018-06-01

    The purpose of this study was histological and thermometric examination of soft tissue de-epithelialization using digitally controlled laser handpiece (DCLH) - X-Runner. Commonly used techniques for de-epithelialization include scalpel, abrasion with diamond bur, or a combination of the two. Despite being simple, inexpensive and effective, these techniques are invasive and may produce unwanted side effects. It is important to look for alternative techniques using novel tools, which are minimally invasive and effective. 114 porcine samples sized 6 × 6 mm were collected from the attached gingiva (AG) of the alveolar process of the mandible using 15C scalpel blade. The samples were irradiated by means of Er:YAG laser (LightWalker, Fotona, Slovenia), using X-Runner and HO 2 handpieces at different parameters; 80, 100, and 140 mJ/20 Hz in time of 6 or 16 sec, respectively. The temperature was measured with a K-type thermocouple. For the histopathological analysis of efficiency of epithelium removal and thermal injury, 3 random samples were de-epithelialized with an HO 2 handpiece, and 9 random samples with an X-Runner handpiece with different parameters. For the samples irradiated with DCLH, we have used three different settings, which resulted in removing 1 to 3 layers of the soft tissue. The efficiency of epithelium removal and the rise of temperature were analyzed. DCLH has induced significantly lower temperature increase compared with HO 2 at each energy to frequency ratio. The histological examination revealed total epithelium removal when HO 2 handpiece was used at 100 and 140 mJ/20 Hz and when DCLH was used for two- and threefold lasing at 80, 100, and 140 mJ/20 Hz. Er:YAG laser with DCLH handpiece may be an efficient tool in epithelium removal without excessive thermal damage.

  17. Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.

    Science.gov (United States)

    Hall, M O; Prieto, A L; Obin, M S; Abrams, T A; Burgess, B L; Heeb, M J; Agnew, B J

    2001-10-01

    The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye. Copyright 2001 Academic Press.

  18. Tissue culture regeneration and radiation induced mutagenesis in banana

    International Nuclear Information System (INIS)

    Kulkarni, V.M.; Ganapathi, T.R.

    2009-01-01

    Radiation induced mutagenesis is an important tool for banana genetic improvement. At BARC, protocols for shoo-tip multiplication of commercial banana varieties have been developed and transferred to user agencies for commercial production. Excellent embryogenic cell suspensions were established in banana cvs. Rasthali and Rajeli, and were maintained at low temperatures for long-term storage. Normal plantlets were successfully regenerated from these cell suspensions. The cell suspensions and shoot-tip cultures were gamma-irradiated for mutagenesis. The mutagenized populations were field screened and a few interesting mutants have been isolated. The existence of genetic variation was confirmed using DNA markers. Further evaluation of these mutants is in progress. (author)

  19. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.

    Science.gov (United States)

    Cifola, Ingrid; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A

    2011-06-13

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  20. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

    International Nuclear Information System (INIS)

    Cifola, Ingrid; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A

    2011-01-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  1. Uptake and metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by cultured renal tubular epithelial cells (RTEC)

    International Nuclear Information System (INIS)

    Gordon, J.A.; Spector, A.A.

    1986-01-01

    To determine if 12-HETE, a lipoxygenase product that mediates inflammation and tissue injury, can interact with RTEC, confluent Madin Darby Canine Kidney (MDCK) cells were incubated for 2-16 hr with 1.0 μM [ 3 H]-12-HETE. Initial uptake of 12-HETE was rapid; at 16 hrs. 70% of the 12-HETE uptake was incorporated into phospholipids (PL). The distribution among the choline, ethanolamine, inositol, and serine PL was 36, 36, 20 and 8%, respectively. Incubation of MDCK cells with 0.5 to 5.0 μM [ 3 H]-12-HETE for 1 hr indicated linear uptake without evidence of saturation. Incubation with 1.0 μM 12-HETE and 0.25-10.0 μM arachidonic acid for 1 hr revealed no competition for uptake at the lower concentrations but a 40% reduction in 12-HETE uptake at 10.0 μM. Polarity of 12-HETE uptake was indicated by a preference of the basolateral surface over the apical surface by 1.4. After 2 hr, analysis of the medium by reverse phase HPLC revealed that 12-HETE was converted to three polar metabolites which eluted at 25.9, 29.4 and 31.3 min respectively; 12-HETE eluted at 37.5 min. The appearance of these polar metabolites was not prevented by ibuprofen (50 μM) nordihydroguaiaretic acid (30 μM), allopurinol (15 mM), or butylated hydroxytoluene (20 μM). These findings suggest that the lipoxygenase product 12-HETE may affect RTEC through incorporation into membrane PL and/or conversion to polar metabolites

  2. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Antibiotic effects against periodontal bacteria in organ cultured tissue.

    Science.gov (United States)

    Takeshita, Masaaki; Haraguchi, Akira; Miura, Mayumi; Hamachi, Takafumi; Fukuda, Takao; Sanui, Terukazu; Takano, Aiko; Nishimura, Fusanori

    2017-02-01

    Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony-forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

  4. Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

    Science.gov (United States)

    Cassells, Alan C

    2012-01-01

    The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

  5. Correlation of clinical data with fallopian tube specimen immune cells and tissue culture capacity.

    Science.gov (United States)

    Ramraj, Satish Kumar; Smith, Katie M; Janakiram, Naveena B; Toal, Coralee; Raman, Ankita; Benbrook, Doris Mangiaracina

    2018-06-01

    Human fallopian tube fimbria secretory epithelial cells (hFTSECs) are considered an origin of ovarian cancer and methods for their culture from fallopian tube specimens have been reported. Our objective was to determine whether characteristics of the donors or surgeries were associated with the capacities of fimbria specimens to generate hFTSEC cultures or their immune profiles. There were no surgical complications attributable to fallopian tube removal. Attempts to establish primary hFTSEC cultures were successful in 37 of 55 specimens (67%). Success rates did not differ significantly between specimens grouped by patient or surgery characteristics. Established cultures could be revived after cryopreservation and none became contaminated with microorganisms. Two cultures evaluated for long term growth senesced between passages 10 and 15. M1 macrophages were the predominant cell type, while all other immune cells were present at much lower percentages. IL-10 and TGF-β exhibited opposing trends with M1 and M2 macrophages. Plasma IL-10 levels exhibited significant positive correlation with patient age. In conclusion, fallopian tube fimbria specimens exhibit a pro-inflammatory phenotype and can be used to provide a source of hFTSECs that can be cultured for a limited time regardless of the donor patient age or race, or the type of surgery performed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

    Science.gov (United States)

    Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit

    2014-04-01

    Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

  7. Micropropagation of six Paulownia genotypes through tissue culture

    Directory of Open Access Journals (Sweden)

    Lydia Shtereva

    2014-12-01

    Full Text Available We investigated the effect of genotype and culture medium on the in vitro germination and development of plantlets from seeds of 6 different Paulownia genotypes (P. tomentosa, hybrid lines P. tomentosa P. fortunei (Mega, Ganter and Caroline, P. elongata and hybrid line P. elongata P. fortunei. Nodal and shoot tip explants were used for micropropagation of Paulownia genotypes by manipulating plant growth regulators. The highest germination percentage for all genotypes was obtained for seeds inoculated on medium supplemented with 50 mg*L GA3 (MSG2. On Thidiazuron containing media, the explants of hybrid line P. elongata P. fortunei exhibited the highest frequency of axillary shoot proliferation following by P. tomentosa P. fortunei. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several economically valuable Paulownia genotypes.

  8. Effect of induced mutagenesis in rice tissue culture

    International Nuclear Information System (INIS)

    Maddumage, R.

    1994-01-01

    The influence of chemical mutagens and ionising radiation on growth, regenerative capacity of rice callus culture and the effect o9f mutagens on frequency and spectrum of mutant regenerants, derived from calli and determination of approximate semi-lethal dose of each mutagen on rice calli was studied. Intact mature de-husked grains and pieces of primordial particles of four varieties were used as explants in the experiment. Organogenesis was induced using MS media supplemented with agar. After thirty days calluses were subjected to varying concentrations/dosage of mutagens. The effect of mutagens on growth of callus was stimulative in low concentration/doses at short exposure, but in higher concentration/doses at longer exposure it was oppressive. In x-radiation treatment all the studied doses showed only stimulative effect on growth. The effect of mutagenic treatment on regenerative capacity was negative. No specificity was found even between two chemical mutagens of their action on studied characters

  9. Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine

    Directory of Open Access Journals (Sweden)

    Eva Maier

    2017-12-01

    Full Text Available Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2 activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2. The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER, of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

  10. Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine.

    Science.gov (United States)

    Maier, Eva; Anderson, Rachel C; Roy, Nicole C

    2017-12-12

    Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

  11. Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

    Science.gov (United States)

    Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

    2014-01-01

    Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

  12. Studies of Bystander Effect and Intercellular Communication in Human Epithelial Cell Cultures Irradiated with X-rays

    International Nuclear Information System (INIS)

    Romppanen, E.; Trott, K. R.; Musatonen, R.; Leszcznski, D.; Belyakov, O.

    2004-01-01

    The bystander effect is a phenomenon whereby biological consequences of irradiation are expressed in nonexposed cells in the vicinity of exposed cells. Two main pathways have been proposed to mediate the bystander effect: Gap Junction Intercellular Communication (GJIC) and medium borne soluble factors dependent mechanisms. The present study was designed to evaluate the relative contributions of gap junction intercellular communication and of soluble extracellular factors on the bystander effects of low dose X-ray irradiation. HaCaT human epithelial cell monolayers were exposed to X-ray using specially constructed shield, which cover 95% or 56% or 0% of the cells from the radiation. To evaluate whether the GJIC is involved in transmission of the bystander signal from irradiated to nonirradiated cells, irradiations were performed in presence or absence of GJIC inhibitor lindane. The cytochalasin B block technique was used to quantify fractions of micronucleated cells 48 hours after the irradiation. Our results suggest that more micronucleated cells are induced in partially shielded monolayers than expected according to back extrapolation of the data from open field irradiation. Treatment with lindane considerably reduced amount of the bystander damage. We demonstrated that fraction of micronucleated cells after X-rays irradiation of 5% of cells with 1 Gy was 0.07±0.08 (without lindane) and 0.05±0.004 (in presence of lindane). Irradiation of 100% of cells with the same dose resulted in 0.023±0.04 /without lindane) and 0.013±0.02 (in presence of lindane) fractions of micronucleated cells. Comparison with open field data showed that the fraction of micronucleated cells after irradiation of 5% of the cell culture was 5-10 times greater than the estimated fraction assuming no bystander effect. Irradiation of 44% of cells ded not demonstrate a pronounced bystander effect. (Author) 20 refs

  13. Project on production of mutants by irradiation of in vitro cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

    International Nuclear Information System (INIS)

    Guzman, E.V. de

    1975-01-01

    Fruit pulp tissue, ovary segments with or without ovules and sections from shoot tips of banana were used for studies on growth stimulating or morphogenetic effects of irradiation. Irradiation at 0.1-1.0 kR tended to induce faster callus growth in the otherwise slow-growing cultures. The physical condition and composition of the culture media especially with respect to growth regulators were studied, as were techniques to overcome discoloration of explants, the best choice of plant tissue for explant, and radiation effects on growth and morphogenesis. Due to the difficulty of callus induction with coconut, only the effects of irradiation on embryos cultured in vitro were studied. They were irradiated at various stages of development, i.e. during the early and final stage of liquid culture, and several days after transfer to a solid medium. Adverse effects of irradiation became evident only during the subsequent growth in solid, during the latter stage of which morphological changes were observed. Whereas irradiation of the liquid as well as solid media up to 50 kR had no adverse effect; survival and development became adversely affected at a dose of 1 kR

  14. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    Science.gov (United States)

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  15. Comparison of radiosensitivities of human autologous normal and neoplastic thyroid epithelial cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Kopecky, K.J.; Hiraoka, T.; Ezaki, H.; Clifton, K.H.

    1986-01-01

    Studies were conducted to examine differences between the radiosensitivities of normal and neoplastic epithelial cells of the human thyroid. Freshly excised thyroid tissues from the tumours of eight patients with papillary carcinoma (PC) and five with follicular adenoma (FA) were cultured in vitro separately from normal thyroid tissue obtained from the surgical margins of the same patients. Plating efficiency of unirradiated control tissue was lower, on average for tumour tissue compared with normal tissue. Radiosensitivity, measured by the 37% inactivation dose D 0 , was greater for carcinoma tissue than for normal tissue in seven out of eight PC cases. Adenomatous tissue was less radiosensitive than normal tissue in four out of five FA cases. This is the first report comparing the radiosensitivity of autologous normal and abnormal epithelial tissue from the human thyroid. (author)

  16. Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation

    Directory of Open Access Journals (Sweden)

    Yoshinobu Takahashi

    2018-05-01

    Full Text Available Summary: Clinical transplantation of tissue fragments, including islets, faces a critical challenge because of a lack of effective strategies that ensure efficient engraftment through the timely integration of vascular networks. We recently developed a complex organoid engineering method by “self-condensation” culture based on mesenchymal cell-dependent contraction, thereby enabling dissociated heterotypic lineages including endothelial cells to self-organize in a spatiotemporal manner. Here, we report the successful adaptation of this method for generating complex tissues from diverse tissue fragments derived from various organs, including pancreatic islets. The self-condensation of human and mouse islets with endothelial cells not only promoted functionalization in culture but also massively improved post-transplant engraftment. Therapeutically, fulminant diabetic mice were more efficiently treated by a vascularized islet transplant compared with the conventional approach. Given the general limitations of post-transplant vascularization associated with 3D tissue-based therapy, our approach offers a promising means of enhancing efficacy in the context of therapeutic tissue transplantation. : Takahashi et al. report on generating vascularized islet tissue from humans and mice. After transplantation, vascularized islets significantly improve survival of diabetic mice, demonstrating the quick normalization of blood glucose compared with conventional islet transplantation. Keywords: tissue engineering, tissue-based therapy, vascularization, islet transplantation, organoid

  17. The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

    Science.gov (United States)

    Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

    2013-09-24

    The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial

  18. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

    Science.gov (United States)

    Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

    2015-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

  19. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    Science.gov (United States)

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  20. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    International Nuclear Information System (INIS)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

    1990-01-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

  1. Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand.

    Science.gov (United States)

    Harkin, Damien G; Apel, Andrew J; Di Girolamo, Nick; Watson, Stephanie; Brown, Karl; Daniell, Mark D; McGhee, J Jane; McGhee, Charles N J

    2013-04-01

    Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases. © 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.

  2. Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

    International Nuclear Information System (INIS)

    Minagawa, T.; Nakaya, C.; Iida, H.

    1974-01-01

    Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

  3. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    International Nuclear Information System (INIS)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B.

    1990-01-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients

  4. Structure and component alteration of rabbit Achilles tendon in tissue culture.

    Science.gov (United States)

    Hosaka, Yoshinao; Ueda, Hiromi; Yamasaki, Tadatsugu; Suzuki, Daisuke; Matsuda, Naoya; Takehana, Kazushige

    2005-12-01

    The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO(2) for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons.

  5. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients<