Journal articles on the topic 'Epithelium Cultures and culture media'
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1
Johnson, L. G., K. G. Dickman, K. L. Moore, L. J. Mandel, and R. C. Boucher. "Enhanced Na+ transport in an air-liquid interface culture system." American Journal of Physiology-Lung Cellular and Molecular Physiology 264, no. 6 (June 1, 1993): L560—L565. http://dx.doi.org/10.1152/ajplung.1993.264.6.l560.
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Use of the air-liquid interface culture technique has produced improved morphological differentiation of rodent, canine, and human tracheal epithelia. We have investigated the effect of this culture technique on ion transport activities of cultured canine bronchial epithelia. These cells were isolated from excised airways by enzymatic digestion and plated on permeable collagen membrane substrates. All cultures were maintained utilizing standard culture techniques, by bathing both apical and basolateral sides with hormone supplemented, serum-free media until confluent (days 4–6). Half of the cultures were converted to air-liquid interface cultures (ALIC) by gentle aspiration of the apical medium and half were continued under standard technique culture (STC) conditions. After three additional days, preparations cultured under both conditions were mounted in modified Ussing chambers where bioelectric properties were measured under short-circuit conditions. Mean short-circuit current (Isc) was significantly greater in ALIC (-91.3 +/- 7.84 microA/cm2) than in STC (-54.8 +/- 5.03 microA/cm2). The sodium channel blocker, amiloride, reduced Isc by 68.4 +/- 5.0% in STC and by 84.8 +/- 3.0% in ALIC. 22Na and 36Cl fluxes confirmed the presence of enhanced sodium absorption in ALIC when compared with STC. The depth of the apical fluid, measured by microelectrodes during ALIC, was approximately 15 microns. Studies of cellular metabolism demonstrated a shift in metabolism from an anaerobic to an oxidative pattern in ALIC. This change in the pattern of metabolism suggests that the ALIC technique enhanced sodium transport in canine bronchial epithelia by increasing oxygen delivery to the epithelium.
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Senanayake, P. deS, A. Calabro, K. Nishiyama, J. G. Hu, D. Bok, and J. G. Hollyfield. "Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface." Journal of Cell Science 114, no. 1 (January 1, 2001): 199–205. http://dx.doi.org/10.1242/jcs.114.1.199.
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Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan-specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.
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Morimoto, Keisuke, Hiroshi Mishima, Teruo Nishida, and Toshifumi Otori. "Role of Urokinase Type Plasminogen Activator (u-PA) in Corneal Epithelial Migration." Thrombosis and Haemostasis 69, no. 04 (1993): 387–91. http://dx.doi.org/10.1055/s-0038-1651617.
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SummaryThe role of plasminogen activator (PA) in the migration of comeal reepithelialization was studied. Rabbit corneal blocks were cultured, and both the extent of epithelial migration over the exposed corneal stroma and the activity of PA released into the culture media were measured. A significant, direct correlation between epithelial migration and PA activity in the medium was observed, even when the migration was stimulated by fibronectin or EGF, or was inhibited by cytochalasin B or cycloheximide. Zymography confirmed that the PA released into the culture medium was of the urokinase type (u-PA). Immunohistochemical studies showed that u-PA and plasmin(ogen) were present at the leading edge of the migrating epithelium. Studies of corneal cell cultures indicated that epithelial cells rather than endothelial cells or fibroblasts were the source of the u-PA. The addition of anti-human u-PA IgG or protease inhibitors retarded the migration of the comeal epithelium in a dose-dependent manner, indicating that u-PA activity is essential for the migration of the corneal epithelium. These findings suggest that the migration of corneal epithelial cells requires not only cell attachment to the extracellular matrix through the fibronectin but also degradation of the fibronectin by the release of cellular u-PA.
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Seeber, Judith W., Michaela Zorn-Kruppa, Simone Lombardi-Borgia, Heike Scholz, Anna K. Manzer, Brigitte Rusche, Monika Schäfer-Korting, and Maria Engelke. "Characterisation of Human Corneal Epithelial Cell Cultures Maintained under Serum-free Conditions." Alternatives to Laboratory Animals 36, no. 5 (November 2008): 569–83. http://dx.doi.org/10.1177/026119290803600512.
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Three-dimensional tissue constructs have been proposed as in vitro screening models for ocular irritancy. Based on our previous studies, in which a full-thickness corneal model based exclusively on SV40-immortalised cell lines was generated, we have currently evaluated the effects of a range of commercially-available cell culture media on several cellular parameters in cultures of a human corneal epithelial (HCE) cell line. This cell line was used in an attempt to establish a rational basis for the development of serum-free culture media for the assembly and long-term tissue culture of full-thickness corneal models. Briefly, we investigated the impact of serum-free culture on the proliferation, morphology, barrier function and cytokine expression of HCE cells. The number of cell layers and the epithelial differentiation were evaluated by histology. Barrier properties were characterised via the determination of transepithelial electrical resistance (TEER), fluorescein permeation, and the expression of the tight junction-related protein, zona occludin 1 (ZO-1). The cytokine expression pattern in response to serum-free culture was measured by using an antibody array system. Our results revealed that both the morphology and the barrier function of the epithelial constructs were comparable to those of human donor corneas, when serum-free media were supplemented with ascorbic acid, calcium, hydrocortisone and retinoic acid. Under these conditions, the artificial epithelium based on serum-free HCE cultures represented a valid model for the natural ocular surface.
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Borzenok, Sergey A., Boris E. Malyugin, Maxim Y. Gerasimov, Dmitriy S. Ostrovskiy, and Anna V. Shatskikh. "Feeder-Free Cell Culture of Labial Oral Mucosal Epithelium for Tissue-Engineering and Regenerative Medicine." Annals of the Russian academy of medical sciences 75, no. 5 (December 27, 2020): 561–70. http://dx.doi.org/10.15690/vramn1357.
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Background.The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions.Aims to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering.Methods.Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patients lip mucosa were used to set the explant (n=18) and enzymatic (n=17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06mM Calcium contained 5% fetal bovine serum, 5 g/ml human insulin, 5 g/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression.Results.The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p=0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.13.6 m thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p=0.001) in comparison with the enzymatic method. Stable cultures had a cobble-stone morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mMCa++medium (p=0.0006).Conclusions.Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.
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Kelly, Scott P., and Chris M. Wood. "Effect of cortisol on the physiology of cultured pavement cell epithelia from freshwater trout gills." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 281, no. 3 (September 1, 2001): R811—R820. http://dx.doi.org/10.1152/ajpregu.2001.281.3.r811.
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Cortisol had dose-dependent effects on the electrophysiological, permeability, and ion-transporting properties of cultured pavement cell epithelia derived from freshwater rainbow trout gills and grown on cell culture filter supports. Under both symmetrical (L15 media apical/L15 media basolateral) and asymmetrical (freshwater apical/L15 media basolateral) culture conditions, cortisol treatment elevated transepithelial resistance, whereas permeability of epithelia to a paracellular permeability marker (polyethylene glycol-4000) decreased. Cortisol did not alter the Na+-K+-ATPase activity or the total protein content of the cultured preparations. During 24-h exposure to asymmetrical conditions, the net loss rates of both Na+ and Cl− to the water decreased with increasing cortisol dose, an important adaptation to dilute media. Unidirectional Na+ and Cl− flux measurements and the application of the Ussing flux-ratio criterion revealed cortisol-induced active uptake of both Na+ and Cl− under symmetrical culture conditions together with an increase in transepithelial potential (positive on the basolateral side). Under asymmetrical conditions, cortisol did not promote active ion transport across the epithelium. These experiments provide evidence for the direct action of cortisol on cultured pavement cell epithelia and, in particular, emphasize the importance of cortisol for limiting epithelial permeability.
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Romberger, D. J., P. Pladsen, L. Claassen, M. Yoshida, J. D. Beckmann, and S. I. Rennard. "Insulin modulation of bronchial epithelial cell fibronectin in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 2 (February 1, 1995): L230—L238. http://dx.doi.org/10.1152/ajplung.1995.268.2.l230.
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Fibronectin (Fn) is involved in the migration of epithelial cells in re-epithelialization of wounds. Epithelial cell-derived Fn is particularly potent as a chemotactic factor for bronchial epithelial cells (BECs) in vitro. Thus modulation of airway epithelial cell Fn may be a key aspect of airway repair. Insulin is both an important growth factor and known chemotactic factor for cultured BECs. We postulated that insulin may modulate Fn production of cultured BECs. We examined this hypothesis utilizing bovine BECs in culture with serum-free media with and without insulin. BECs grown in media without insulin released more Fn into culture supernatants and contained more Fn in cell layers than cells grown with insulin. Labeling of cells with [35S]methionine demonstrated an increase in new protein production and Fn mRNA expression was increased. Increased Fn in BEC cultures without insulin was associated with an increase in active transforming growth factor-beta (TGF-beta) release as measured by a standard bioassay. Increased BEC Fn in cultures without insulin was partially inhibited by exposure of cultures to TGF-beta antibody. Thus insulin appears to modulate BEC Fn production in vitro in part through a TGF-beta-dependent mechanism. Insulin may be involved in airway repair mechanisms through modulation of epithelial cell Fn production.
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Chato-Astrain, Jesús, David Sánchez-Porras, Óscar Darío García-García, Claudia Vairo, María Villar-Vidal, Silvia Villullas, Indalecio Sánchez-Montesinos, Fernando Campos, Ingrid Garzón, and Miguel Alaminos. "Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers." Biomedicines 9, no. 11 (November 6, 2021): 1634. http://dx.doi.org/10.3390/biomedicines9111634.
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Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.
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Ferrara, N., D. K. Fujii, P. C. Goldsmith, J. H. Widdicombe, and R. I. Weiner. "Transport epithelial characteristics of cultured bovine pituitary follicular cells." American Journal of Physiology-Endocrinology and Metabolism 252, no. 3 (March 1, 1987): E304—E312. http://dx.doi.org/10.1152/ajpendo.1987.252.3.e304.
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Confluent monolayers of polygonal epithelioid cells were obtained from enzymatically and mechanically dispersed bovine anterior pituitaries (AP) and pars tuberali (PT). The ultrastructure of the cells composing the monolayer was consistent with the follicular or folliculostellate cells (FC) of the pituitary, i.e., lack of secretory granules; formation of follicles in culture; interdigitations with neighboring cells with numerous tight junctions; presence of extensive microfilaments; and sparse rough endoplasmic reticulum and Golgi apparatus. Culture media from monolayers of first passage cultures contained little if any of the AP hormones' luteinizing hormone, prolactin, and ACTH. Shortly after reaching confluency, regions of the monolayer bulge away from the surface of the culture dish to form domes. Dome formation has been described only with cultures of cells that function as transport epithelia in vivo. FC cultured on polycarbonate filters were placed in Ussing chambers. A transepithelial potential difference of approximately 1.1 mV and a resistance greater than 300 omega cm2 were detectable 4–5 days after plating. The short-circuit current (Isc) was decreased 70% by amiloride applied to the mucosal surface and further decreased by the addition of ouabain at the serosal surface. The beta-adrenergic agonist isoproterenol increased the Isc and this action was prevented by a beta-antagonist. These observations indicate that pituitary FC in culture behave as a transport epithelium. Considering the organization of FC in the AP and PT, they suggest a regulatory role for FC in the maintenance of the ionic composition of the interstitial fluid of the pituitary gland.
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Kouthouridis, Sonya, Julie Goepp, Carolina Martini, Elizabeth Matthes, John W. Hanrahan, and Christopher Moraes. "Oxygenation as a driving factor in epithelial differentiation at the air–liquid interface." Integrative Biology 13, no. 3 (March 2021): 61–72. http://dx.doi.org/10.1093/intbio/zyab002.
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Abstract Culture at the air–liquid interface is broadly accepted as necessary for differentiation of cultured epithelial cells towards an in vivo-like phenotype. However, air–liquid interface cultures are expensive, laborious and challenging to scale for increased throughput applications. Deconstructing the microenvironmental parameters that drive these differentiation processes could circumvent these limitations, and here we hypothesize that reduced oxygenation due to diffusion limitations in liquid media limits differentiation in submerged cultures; and that this phenotype can be rescued by recreating normoxic conditions at the epithelial monolayer, even under submerged conditions. Guided by computational models, hyperoxygenation of atmospheric conditions was applied to manipulate oxygenation at the monolayer surface. The impact of this rescue condition was confirmed by assessing protein expression of hypoxia-sensitive markers. Differentiation of primary human bronchial epithelial cells isolated from healthy patients was then assessed in air–liquid interface, submerged and hyperoxygenated submerged culture conditions. Markers of differentiation, including epithelial layer thickness, tight junction formation, ciliated surface area and functional capacity for mucociliary clearance, were assessed and found to improve significantly in hyperoxygenated submerged cultures, beyond standard air–liquid interface or submerged culture conditions. These results demonstrate that an air–liquid interface is not necessary to produce highly differentiated epithelial structures, and that increased availability of oxygen and nutrient media can be leveraged as important strategies to improve epithelial differentiation for applications in respiratory toxicology and therapeutic development.
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Zachos, Nicholas C. "Abstract IA008: Modeling immune-epithelial interactions in health and disease using human intestinal enteroid co-cultures." Cancer Research 82, no. 23_Supplement_1 (December 1, 2022): IA008. http://dx.doi.org/10.1158/1538-7445.crc22-ia008.
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Abstract Human intestinal enteroids derived from Lgr5+ actively dividing adult stem cells offer a relevant ex vivo system to study biological processes of the human small intestine and colon. While these primary cultures recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids), they are limited by the lack of associated cell types, including immunological cells, nerves, stromal cells that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium by conducting lumial surveillance, support barrier function, and deploy effector functions in response to injury/damage. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Human intestinal enteroids can generated from biopsies or resected tissue from any segment of the human intestine/colon and maintained in long-term cultures in a basement membrane-rich matrix supplemented with growth factor-conditioned media necessary to maintain intestinal stem cell homeostasis. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC) and co-cultured with human intestinal enteroids/colonoids grown as confluent monolayers on permeable supports (e.g., Transwells). This configuration allows for controlled access to the apical side of the epithelium to mimic the gut luminal environment as well as the basolateral side occupied by immune cells to mimic the mucosal environment. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. Citation Format: Nicholas C. Zachos. Modeling immune-epithelial interactions in health and disease using human intestinal enteroid co-cultures [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA008.
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Alpert, S. E., R. W. Walenga, C. M. Kramer, C. L. Silski, and P. B. Davis. "Tracheal epithelial cell fatty acid composition modulates prostaglandin E2 and cAMP production." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 2 (February 1, 1992): L192—L197. http://dx.doi.org/10.1152/ajplung.1992.262.2.l192.
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Tracheal epithelial (TE) cells from both rabbits and humans, when cultured in defined serum-free media without lipid supplements, develop fatty acid profiles significantly different from freshly isolated epithelium, including a markedly decreased cellular content of arachidonic acid (AA). In rabbit TE cells, supplementation of media with a phospholipid-rich lipoprotein extract (Excyte III) plus 1 microM bovine serum albumin-complexed AA (Excyte/AA) restored the fatty acid composition of the cultured cells more similar to that of native airway epithelium than did supplementation of media with 5% fetal bovine serum (FBS). In human TE cells, Excyte/AA or 5% FBS increased AA content, but neither lipid supplement completely “normalized” the fatty acid profiles. Compared with lipid-unsupplemented cultures, basal production of prostaglandin E2 (PGE2) was increased by approximately four- to eightfold in rabbit and human TE cells supplemented with 5% FBS or Excyte/AA. In Excyte/AA-supplemented human TE cells, PGE2 production induced by 5 microM calcium ionophore A23187 was more than threefold greater than that of companion ionophore-stimulated unsupplemented monolayers, but PGE2 production was similar in both culture conditions in response to 10 microM exogenous AA. Thus increased cellular content and availability of AA, rather than changes in cyclooxygenase activity, appear to be responsible for the elevated PGE2 production in Excyte/AA-supplemented human TE cells. Secondary effects of lipid supplementation were also observed; Excyte/AA-supplemented human TE cells produced significantly less adenosine 3',5'-cyclic monophosphate (cAMP) in response to exogenous PGE2 and isoproterenol than did lipid-unsupplemented cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Armstrong, J., T. Matainaho, E. J. Cragoe, G. J. Huxham, J. R. Bourke, and S. W. Manley. "Bidirectional ion transport in thyroid: secretion of anions by monolayer cultures that absorb sodium." American Journal of Physiology-Endocrinology and Metabolism 262, no. 1 (January 1, 1992): E40—E45. http://dx.doi.org/10.1152/ajpendo.1992.262.1.e40.
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Cultured porcine thyroid cell monolayers transport Na+ in an apical-to-basal direction, resulting in the development of a basal-positive transepithelial potential difference (TEP) and the formation of domes (fluid-filled elevations of the cell layer above the culture dish substrate). Stimulation by prostaglandin E2 (PGE2) increases the magnitude of the TEP, the short-circuit current (Isc) measured in Transwell Ussing chambers, and the height of domes in cultures grown on impermeable substrates. A phenamil-resistant, PGE2-stimulated component of the Isc in Transwells and of the TEP in monolayers in conventional culture dishes was inhibitable by bumetanide, a diuretic drug that blocks NaKCl2 symporters, mediating active transport of Cl-. The rate of decrease in height of domes in cultures after addition of phenamil, presumably indicative of transport of fluid in a basal-to-apical direction, was also reduced by bumetanide. Studies with Transwells in Cl(-)-free, HCO(3-)-free or Cl(-)- and HCO(3-)-free media indicated that thyroid cells transported HCO3- as well as Cl- in a basal-to-apical direction. It was concluded that the thyroid epithelium is both sodium absorbing and anion secreting.
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Dale, Tina P., Emily Borg D’anastasi, Mohammed Haris, and Nicholas R. Forsyth. "Rock Inhibitor Y-27632 Enables Feeder-Free, Unlimited Expansion of Sus scrofa domesticus Swine Airway Stem Cells to Facilitate Respiratory Research." Stem Cells International 2019 (November 23, 2019): 1–15. http://dx.doi.org/10.1155/2019/3010656.
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Current limitations in the efficacy of treatments for chronic respiratory disorders position them as prospective regenerative medicine therapeutic targets. A substantial barrier to these ambitions is that research requires large numbers of cells whose acquisition is hindered by the limited availability of human tissue samples leading to an overreliance on physiologically dissimilar rodents. The development of cell culture strategies for airway cells from large mammals will more effectively support the transition from basic research to clinical therapy. Using readily available porcine lungs, we isolated conducting airway tissue and subsequently a large number of porcine airway epithelial cells (pAECs) using a digestion and mechanical scraping technique. Cells were cultured in a variety of culture media formulations, both foetal bovine serum-containing and serum-free media, in air (21%) and physiological (2%) oxygen tension and in the presence and absence of Rho kinase inhibitor Y-27362 (RI). Cell number at isolation and subsequent population doublings were determined; cells were characterised during culture and following differentiation by immunofluorescence, histology, and IL-8 ELISA. Cells were positive for epithelial markers (pan-cytokeratin and E-cadherin) and negative for fibroblastic markers (vimentin and smooth muscle actin). Supplementation of cultures with Y-27632 allowed for unlimited expansion whilst sustaining an epithelial phenotype. Early passage pAECs readily produced differentiated air-liquid interface (ALI) cultures with a capacity for mucociliary differentiation retained after substantial expansion, strongly modulated by the culture condition applied. Primary pAECs will be a useful tool to further respiratory-oriented research whilst RI-expanded pAECs are a promising tool, particularly with further optimisation of culture conditions.
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Zagon, I. S., J. W. Sassani, and P. J. McLaughlin. "Opioid growth factor modulates corneal epithelial outgrowth in tissue culture." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 268, no. 4 (April 1, 1995): R942—R950. http://dx.doi.org/10.1152/ajpregu.1995.268.4.r942.
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In addition to neuromodulation, endogenous opioid peptides serve as growth factors. To determine involvement of opioids in the homeostatic renewal and repair of the corneal epithelium, epithelial outgrowths from 3-mm explants of rabbit cornea were investigated. Blockade of opioid-receptor interaction by the potent opioid antagonist naltrexone (NTX) for 7 days significantly increased the extent of outgrowths and the number and labeling index (DNA synthesis) of epithelial cells, relative to control levels. Outgrowths exposed to the opioid growth factor (OGF) [Met5]enkephalin for 7 days were subnormal in extent and labeling index and displayed alterations in architectural pattern. The effects of OGF on epithelial outgrowth were blocked by concomitant exposure to the opioid antagonist naloxone; naloxone alone had no effect on growth at the concentration utilized. NTX and OGF were active in both serum-containing and serum-free cultures. Immunocytochemical investigations showed that both OGF and its opioid receptor zeta (zeta) were present in epithelial cells growing in control media. The results indicate that an endogenous opioid peptide and its receptor are present in mammalian corneal epithelium and serve to modulate cell proliferation, migration, and organization.
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Das, S. R., N. Bhardwaj, and P. Gouras. "Synthesis of retinoids by human retinal epithelium and transfer to rod outer segments." Biochemical Journal 268, no. 1 (May 15, 1990): 201–6. http://dx.doi.org/10.1042/bj2680201.
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The synthesis and release of 11-cis-retinoids by primary cultures of human retinal pigment epithelium (RPE) and the transfer of these retinoids to co-incubated human rod outer segments (ROS) were studied. Monolayers of 2-3-week-old cultured RPE incorporate tritiated all-trans-retinol, esterify it to the corresponding retinyl palmitate, form 11-cis-retinol and 11-cis-retinaldehyde and release retinaldehyde into the culture medium. The ratio of 11-cis to all-trans isomers of retinol, retinyl palmitate and retinaldehyde formed in the cells along with retinaldehyde released and incorporated into the ROS progressively increases, indicating a progressive increase in the concentration of 11-cis isomer from the time it is formed in RPE cells until its transfer to ROS. Incorporation of 11-cis-retinaldehyde into the ROS is directly related to the amount of albumin present in the media, suggesting the transfer of retinoids from RPE to photoreceptor to be a protein-mediated process. Events leading to isomerization, esterification, oxidation and release of retinoids by human RPE and incorporation of retinoids into ROS can therefore be examined in vitro.
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Bagher, Mariam, Oskar Rosmark, Linda Elowsson Rendin, Annika Nybom, Sebastian Wasserstrom, Catharina Müller, Xiao-Hong Zhou, et al. "Crosstalk between Mast Cells and Lung Fibroblasts Is Modified by Alveolar Extracellular Matrix and Influences Epithelial Migration." International Journal of Molecular Sciences 22, no. 2 (January 6, 2021): 506. http://dx.doi.org/10.3390/ijms22020506.
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Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.
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Gstraunthaler, Gerhard, Thomas Seppi, and Walter Pfaller. "Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures." Cellular Physiology and Biochemistry 9, no. 3 (1999): 150–72. http://dx.doi.org/10.1159/000016312.
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Jovov, B., N. K. Wills, and S. A. Lewis. "A spectroscopic method for assessing confluence of epithelial cell cultures." American Journal of Physiology-Cell Physiology 261, no. 6 (December 1, 1991): C1196—C1203. http://dx.doi.org/10.1152/ajpcell.1991.261.6.c1196.
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We describe a convenient nonelectrophysiological technique for assessing cell proliferation and subsequent tight junction formation for epithelial monolayers grown on permeable supports. The method involves the use of phenol red (PR), a standard pH indicator in most cell culture media. In addition, we report a systematic error in a commercially available system for measuring transepithelial electrical properties. Briefly, the flux of PR across the epithelium was measured from the serosal solution into the mucosal solution. The mucosal solution was first replaced with a PR-free solution and then collected at timed intervals. The PR concentration was measured using a spectrophotometer set at the isosbestic point for PR (479 nm). PR flux was then calculated and used as an index of the permeability of the epithelium to PR. This method was tested using the renal epithelial cell line A6. After cell seeding, PR flux decreased in two phases: an initial large decrease, associated with cell growth and monolayer confluence, and a second decrease associated with tight junction formation [assessed by measuring transepithelial conductance (Gt)]. In addition to monitoring tight junction formation, PR flux measurements were also used to estimate the net movement of solution by the epithelial cells between the mucosal and serosal compartments. For convenience, Gt was initially measured in culture dishes using a commercially available “chopstick” electrode system. However, the chopstick system yielded Gt values that were on average 51% lower than values for the same preparations when measured in standard Ussing-type chambers. The discrepancy was due to a nonuniform current field produced by the chopstick electrodes.
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Pezzulo, Alejandro A., Timothy D. Starner, Todd E. Scheetz, Geri L. Traver, Ann E. Tilley, Ben-Gary Harvey, Ronald G. Crystal, Paul B. McCray, and Joseph Zabner. "The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia." American Journal of Physiology-Lung Cellular and Molecular Physiology 300, no. 1 (January 2011): L25—L31. http://dx.doi.org/10.1152/ajplung.00256.2010.
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Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.
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Roh, Terrence, Ying Chen, Harry Paul, Chengchen Guo, and David Kaplan. "P140 3D BIOENGINEERED TISSUE MODEL OF THE LARGE INTESTINE TO STUDY INFLAMMATORY BOWEL DISEASE." Inflammatory Bowel Diseases 26, Supplement_1 (January 2020): S34—S35. http://dx.doi.org/10.1093/ibd/zaa010.087.
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Abstract An in vitro model of intestine epithelium with an immune compartment was bioengineered to mimic immunologic responses seen in inflammatory bowel disease [1]. While aspects of intestinal immunity can be modeled in transwells and 2D culture systems, 3D tissue models improve physiological relevance by providing a 3D substrate which enable migration of macrophages towards the epithelium. An intestinal epithelium comprised of non-transformed human colon organoid cells and a subepithelial layer laden with monocyte-derived macrophages was bioengineered to mimic native intestinal mucosa cell organization using spongy silk scaffolds. Confluent epithelial monolayers with microvilli, a mucus layer, and infiltration of macrophages to the basal side of the epithelium were observed. Inflammation, induced by E. coli O111:B4 lipopolysaccharide and interferon γ resulted in morphology changes to the epithelium, resulting in ball-like structures, decreased epithelial coverage, and migration of macrophages to the epithelium. Analysis of cytokines present in the inflamed tissue model demonstrated significantly upregulated secretion of pro-inflammatory cytokines associated with active inflammatory bowel disease, including CXCL10, IL-1β, IL-6, MCP-2, and MIP-1β. The macrophage layer enhanced epithelial and biochemical responses to inflammatory stimuli, and this new tissue system may be useful to study and develop potential therapies for inflammatory bowel disease. References: 6 Roh, T.T., et al., 3D bioengineered tissue model of the large intestine to study inflammatory bowel disease. Biomaterials, 2019: p. 119517. 7 In, J., et al., Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cellular and molecular gastroenterology and hepatology, 2015. 2(1): p. 48–62.e3. 8 Chen, Y., et al., In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses. PLoS ONE, 2017. 12(11): p. e0187880. Colonoid and macrophage cultivation scheme in the 3D bilayer system. (A) Human monocytes were isolated from whole blood and human colonoids from large intestine biopsies were cultured according to established protocols [2]. (B) Cell suspensions of colonoids were seeded on the film surface on the inner silk scaffold and monocyte-derived macrophages were seeded throughout the porous outer silk scaffold using established protocols [3]. (C) The model is cultured for 3 weeks total with 2 weeks in High WNT media and 1 week in differentiation media based on established protocol. Colonoids are present in the model throughout the 3 week culture time. 2 sets of macrophages are added with the first set added after the first week of culture and the second set replacing the first set after the second week.
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Lee, Jonathan K., Jessica Bloom, Arantzazu Zubeldia-Plazaola, James C. Garbe, Martha R. Stampfer, and Mark A. LaBarge. "Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures." PLOS ONE 13, no. 10 (October 1, 2018): e0204645. http://dx.doi.org/10.1371/journal.pone.0204645.
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Wilkinson, Peter J., and Richard H. Clothier. "Comparison of an Animal Product Free Medium and Normal Growth Supplement on the Growth and Barrier Integrity of a Human Corneal Epithelial Cell Line." Alternatives to Laboratory Animals 33, no. 5 (October 2005): 509–18. http://dx.doi.org/10.1177/026119290503300514.
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With the development of defined media for general and specific use with cell cultures, and concern over the use of human cells and over potential prion infections associated with growth factor extracts such as bovine pituitary extract, an animal product-free medium has become available. The basic keratinocyte defined medium can be used with a choice of animal product-containing or animal product-free supplements. Human corneal epithelia cell lines were cultured in the media with these two types of supplement, and compared in terms of their growth rates, their capacity to form tight barriers, and calcium regulation of the location of a junction-associated protein, zonula occludins-1 (ZO-1). The growth rates were not different in the two media, as long as the recommended coating was applied to the culture flask for the animal product-free medium. The barrier function was equally effective for confluent cultures seeded at the same densities. A calcium concentration of 100μM or above resulted in ZO-1 localisation at the cell membrane in either medium. Hence, cultures in the media are comparable, when the coating is employed. Further experiments are being conducted to establish the comparability of responses to chronic treatment with surfactants.
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Grote, Jan J., Gerben J. M. Tjebbes, Saco C. Hesseling, and Clemens A. Van Blitterswijk. "Effect of HA-1A Monoclonal IGM Antibody on Endotoxin-Induced Proliferation of Cultured Rat Middle Ear Epithelium." Annals of Otology, Rhinology & Laryngology 104, no. 3 (March 1995): 226–30. http://dx.doi.org/10.1177/000348949510400308.
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The effect of human monoclonal antibody HA-1A (Centoxin) on the effect of endotoxin on cultured rat middle ear epithelium was investigated. The addition of endotoxin to the standard culture medium revealed a concentration-related proliferative effect on cultured rat middle ear epithelium, leading to cobblestone cells, cell tracks, and stratification of epithelium, whereas rat middle ear epithelium cultured in standard medium grew as a monolayer composed of flat polygonal cells. Addition of HA-1A to standard medium supplemented with endotoxin gave rise to a statistically significant suppression of the proliferative effects of endotoxin on these cells. The morphology of rat middle ear epithelium cultured in the presence of HA-1A and endotoxin showed that these cells still had a tendency to form cobblestone-like cells and cell tracks, but to a substantially lower degree. The present results support the hypothesis that HA-1A suppresses the proliferative and morphological effects of endotoxin on rat middle ear epithelium and may play an important role in the pathogenesis of otitis media.
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Packard, Mary J. "The effect of strain of donor eggs, culture conditions, and experimental protocol on release of eggshell calcium by the chick chorioallantoic membrane in vitro." Canadian Journal of Zoology 75, no. 9 (September 1, 1997): 1474–78. http://dx.doi.org/10.1139/z97-770.
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Factors affecting release of calcium by the chick chorioallantoic membrane in vitro were assessed by culturing explants of shell with and without the chorioallantoic epithelium. Explants were removed from eggs on day 16 of incubation and cultured using techniques similar to those used to examine resorption of bone in vitro. The region of an egg from which an explant is removed, the strain of donor eggs used, and addition of bovine serum albumin to the culture medium did not affect release of calcium. In contrast, exposure of explants to a variety of different culture media (DMEM, Medium 199, BGJb, and RPMI) introduced considerable variation in calcium release. Calcium release in vitro was also examined by removing the blunt end of a fertile egg as well as the embryo and yolk. The remaining preparation was filled with culture medium, covered, and cultured for two 24-h periods, with a change of medium after the first 24 h. Preparations from which the chorioallantoic membrane had been removed released very little calcium during culture, whereas those with the epithelium in place released considerable quantities. However, release of calcium by preparations with the membrane declined during the second interval. When surface area is taken into account, such preparations mobilize less calcium than explants cultured under similar conditions.
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Rosca, Aliona S., Joana Castro, and Nuno Cerca. "Evaluation of different culture media to support in vitro growth and biofilm formation of bacterial vaginosis-associated anaerobes." PeerJ 8 (September 10, 2020): e9917. http://dx.doi.org/10.7717/peerj.9917.
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Background Bacterial vaginosis (BV) is one of the most common vaginal infections worldwide. It is associated with the presence of a dense polymicrobial biofilm on the vaginal epithelium, formed mainly by Gardnerella species. The biofilm also contains other anaerobic species, but little is known about their role in BV development. Aim To evaluate the influence of different culture media on the planktonic and biofilm growth of six cultivable anaerobes frequently associated with BV, namely Gardnerella sp., Atopobium vaginae, Lactobacillus iners, Mobiluncus curtisii, Peptostreptococcus anaerobius and Prevotella bivia. Methods A total of nine different culture media compositions, including commercially available and chemically defined media simulating genital tract secretions, were tested in this study. Planktonic cultures and biofilms were grown under anaerobic conditions (10% carbon dioxide, 10% helium and 80% nitrogen). Planktonic growth was assessed by optical density measurements, and biofilm formation was quantified by crystal violet staining. Results Significant planktonic growth was observed for Gardnerella sp., A. vaginae and L. iners in New York City III broth, with or without ascorbic acid supplementation. Biofilm quantification showed high in vitro biofilm growth for Gardnerella sp., P. anaerobius and P. bivia in almost all culture media excluding Brucella broth. Contrary, only New York City III broth was able to promote biofilm formation for A. vaginae, L. iners and M. curtisii. Conclusions Our data demonstrate that New York City III broth relative to the other tested media is the most conducive for future studies addressing polymicrobial biofilms development as this culture medium allowed the formation of significant levels of single-species biofilms.
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Wilson, P. D., M. A. Dillingham, R. Breckon, and R. J. Anderson. "Defined human renal tubular epithelia in culture: growth, characterization, and hormonal response." American Journal of Physiology-Renal Physiology 248, no. 3 (March 1, 1985): F436—F443. http://dx.doi.org/10.1152/ajprenal.1985.248.3.f436.
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Individually microdissected nephron segments of defined epithelial origin from human kidneys were cultured in vitro in the present studies. Nephron segments of proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb of Henle, and cortical collecting tubule were grown in defined media. Each cell type retained differentiated characteristics as assessed by ultrastructural morphology, marker enzyme profiles, and adenylate cyclase response to selected hormones. These studies demonstrate the feasibility of using primary cultures of well-defined segments of the human nephron to study human renal tubular epithelia in vitro.
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Leung, Clarus, Samuel J. Wadsworth, S. Jasemine Yang, and Delbert R. Dorscheid. "Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media." American Journal of Physiology-Lung Cellular and Molecular Physiology 318, no. 5 (May 1, 2020): L1063—L1073. http://dx.doi.org/10.1152/ajplung.00190.2019.
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The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.
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Ito, Ichiaki, Sam Jacob, Sumihiro Suzuki, Makiko Kobayashi, and Fujio Suzuki. "Intestinal epithelial cells differentiated from iPS cells (iPS-IECs) suppressed bacterial translocation in an organ model of alcohol-induced intestinal epithelial dysfunction." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 192.15. http://dx.doi.org/10.4049/jimmunol.202.supp.192.15.
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Abstract Microbiota translocated from the intestine causes infectious complications in alcoholics, frequently. In the present study, the effect of iPS-IEC transplantation on gut-associated bacterial translocation was investigated in an organoid model of intestinal epithelial burrier (IEB) dysfunction in patients with alcohol use disorder. iPS-IECs (a mixture of Paneth cells, goblet cells, enteroendocrine cells, and enterocytes) were differentiated from iPS cells, originated from human peripheral CD38+ hematopoietic stem cells. Human gut organoids (HGO) were generated from iPS cells in 3D cultures for 2 weeks. HGO were alcoholized with 0.2% ethanol for 2 weeks (A/HGO). iPSIECs (5 × 103 cells) were co-cultured with 100 A/HGO for 3 days. IEB functions of A/HGO were tested by severities of Enterococcus faecalis (E. faecalis) invasion into the organoids. The gene expressions of tight junction proteins and intestinal cell markers of A/HGO were determined by real-time PCR and Western blotting. In the results, E. faecalis added to culture media invaded the A/HGO, while the pathogen did not invade the HGO in the same cultures. However, the pathogen did not invade the A/HGO co-cultured with iPS-IECs. Reduced expression of tight junction protein mRNAs was shown in A/HGO, while these mRNAs were expressed in A/HGO co-cultured with iPS-IECs. LGR5 and NOTCH1 gene expressions in A/HGO were shown to be a minimum, while these genes were expressed sufficiently in A/HGO co-cultured with iPS-ICs. These results indicate that the alcohol-induced IEB dysfunction of A/HGO is significantly improved in cultures added with iPSIECs.
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Whigham, Amy, Brown Brandee, Avtandyl Kochiashvili, James L. Netterville, Brian B. Burkey, Robert J. Sinard, and Wendell G. Yarbrough. "Short-Term Culture and In-Vivo Modeling of Primary HNSCC." Otolaryngology–Head and Neck Surgery 139, no. 2_suppl (August 2008): P93—P94. http://dx.doi.org/10.1016/j.otohns.2008.05.502.
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Problem Head and neck squamous cell carcinoma (HNSCC) accounts for 4% of annual U.S. cancer deaths. In-vivo models exist using established HNSCC lines, but currently there is no such model that allows consistent growth of HNSCC from primary tumors. Methods Primary HNSCC tissue was obtained from 103 patients at biopsy/resection, disaggregated and seeded onto collagen-coated plates in keratinocyte growth media with 10% FBS, additives and antibiotics. After short-term growth in culture, cells were transferred to denuded rat tracheas and implanted subcutaneously in nude mice. Indirect immunofluorescent staining using antibodies specific to cytokeratin, vimentin and nuclear Ku was performed to determine cell lineage and origin. Results Cultured cells exhibited morphology consistent with epithelial or stromal derivation. 80% of cultures had viable cells present at 10 days and 24% were maintained 30 days or longer. 5 cultures (5%) proliferated after multiple passages and thrived on uncoated plates in the absence of mesenchymal cells. The xenograft model was able to successfully establish tumors in vivo from 59% of primary tumors. Immunostaining for nuclear Ku and cytokeratin confirmed human origin and epithelial cell lineage, respectively. Conclusion The high success rate indicates that selective growth and survival pressures for short-term culture of primary HNSCC may be considerably less than for establishment of cell lines. Additionally, the techniques permit tumor-derived epithelial and mesenchymal cells to be cultured simultaneously. Preliminary data for the in-vivo trachea xenograft model is promising. A luciferase lentiviral system has been created to allow monitoring of tumor growth in vivo with serial live animal imaging. Significance These short-term culture techniques may more accurately characterize both the biological diversity of HNSCC and tumor-stromal cell interactions. Once optimized, the trachea xenograft model can be used to determine the in-vivo response of a heterogeneous group of HNSCC to standard and novel therapies. Support Funds provided by an endowment for the Barry Baker Laboratory for Head and Neck Oncology, the Vanderbilt Ingram Cancer Center Endowed Professorship Fund, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.
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Baloglu, Emel, Kalpana Velineni, Ezgi Ermis-Kaya, and Heimo Mairbäurl. "Hypoxia Aggravates Inhibition of Alveolar Epithelial Na-Transport by Lipopolysaccharide-Stimulation of Alveolar Macrophages." International Journal of Molecular Sciences 23, no. 15 (July 27, 2022): 8315. http://dx.doi.org/10.3390/ijms23158315.
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Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS;1 µg/mL) and exposed to normoxia and hypoxia (1.5% O2). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISCΔamil); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.
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Koyama, S., S. I. Rennard, G. D. Leikauf, S. Shoji, S. Von Essen, L. Claassen, and R. A. Robbins. "Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation." Journal of Immunology 147, no. 12 (December 15, 1991): 4293–301. http://dx.doi.org/10.4049/jimmunol.147.12.4293.
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Abstract To test the effect of endotoxin on bronchial epithelial cells (BEC), BEC were isolated from bovine lungs and cultured in the presence of bacterial endotoxin. The BEC culture supernatant fluids were harvested, and neutrophil chemotactic activity (NCA) was determined with a blindwell chamber technique; cytotoxicity determined by lactate dehydrogenase release and BEC proliferation determined by Coulter counting. Endotoxin caused a dose- and time-dependent release of NCA from BEC cultures compared with media alone (82.3 +/- 8.1 vs 12.0 +/- 3.1 cells/high power field, p less than 0.001). To further characterize this activity, reverse phase HPLC analysis of release eicosanoid metabolites after [3H]arachidonic acid incorporation was performed. Endotoxin stimulated the release of the neutrophil chemoattractants, leukotriene B4 and 12-hydroxyeicosatetraenoic acids. Endotoxin also resulted in a dose and time dependent release of lactate dehydrogenase (42.9 +/- 4.2 vs 20.2 +/- 2.2 U/liter, p less than 0.001) although higher doses were required to cause cytotoxicity than to stimulate chemotaxis. Finally, endotoxin resulted in a dose dependent inhibition of BEC proliferation (176 x 10(3) +/- 16 x 10(3) vs 1,080 x 10(3) +/- 38 x 10(3) cells/ml measured at day 14, p less than 0.001). These data suggest that bacterial release of endotoxin may contribute to the pathophysiologic changes observed in bronchial inflammation by stimulating BEC to release NCA, denuding airway epithelium by causing cytotoxicity of BEC, and inhibiting epithelial repair by inhibiting BEC proliferation.
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Atherton, Hazel C., Gareth Jones, and Henry Danahay. "IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation." American Journal of Physiology-Lung Cellular and Molecular Physiology 285, no. 3 (September 2003): L730—L739. http://dx.doi.org/10.1152/ajplung.00089.2003.
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In addition to a direct proinflammatory role, IL-13 has been demonstrated to induce a goblet cell metaplastic phenotype in the airway epithelium in vivo. We have studied the direct effects of IL-13 (and IL-4) on well-differentiated, air-liquid interface cultures of human bronchial epithelial cells (HBEs) and provide a quantitative assessment of the development of a mucus hypersecretory phenotype induced by these cytokines. Using Alcian blue staining of goblet cells and immunohistochemical detection of MUC5AC, we found that IL-13 (and IL-4) induced increases in the goblet cell density (GCD) of the HBE cultures. The effects of these cytokines were critically dependent on concentration: 1 ng/ml routinely induced a 5- to 10-fold increase in GCD that was associated with a hypersecretory ion transport phenotype. Paradoxically, 10 ng/ml of either cytokine induced a profound reduction in GCD. Removal of EGF from the culture media or treatment of the cells with AG-1478 [a potent inhibitor of EGF receptor tyrosine kinase (EGFR-TK)] demonstrated that the EGFR-TK pathway was key to the regulation of the basal GCD but that it was not involved in the IL-13-driven increase. The IL-13-driven increase in GCD was, however, sensitive to inhibition of MEK (PD-98059, U-0126), p38 MAPK (SB-202190), and phosphatidylinositol (PtdIns) 3-kinase (LY-294002). These data support the concept that IL-13 is in part able to induce a mucus hypersecretory phenotype through a direct interaction with the airway epithelium and that MAP kinase and PtdIns 3-kinase signaling pathways are involved.
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Pastuła, Agnieszka, Moritz Middelhoff, Anna Brandtner, Moritz Tobiasch, Bettina Höhl, Andreas H. Nuber, Ihsan Ekin Demir, et al. "Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche." Stem Cells International 2016 (2016): 1–16. http://dx.doi.org/10.1155/2016/3710836.
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The gastrointestinal epithelium is characterized by a high turnover of cells and intestinal stem cells predominantly reside at the bottom of crypts and their progeny serve to maintain normal intestinal homeostasis. Accumulating evidence demonstrates the pivotal role of a niche surrounding intestinal stem cells in crypts, which consists of cellular and soluble components and creates an environment constantly influencing the fate of stem cells. Here we describe different 3D culture systems to culture gastrointestinal epithelium that should enable us to study the stem cell nichein vitroin the future: organoid culture and multilayered systems such as organotypic cell culture and culture of intestinal tissue fragmentsex vivo. These methods mimic thein vivosituationin vitroby creating 3D culture conditions that reflect the physiological situation of intestinal crypts. Modifications of the composition of the culture media as well as coculturing epithelial organoids with previously described cellular components such as myofibroblasts, collagen, and neurons show the impact of the methods applied to investigate niche interactionsin vitro. We further present a novel method to isolate labeled nerves from the enteric nervous system using Dclk1-CreGFP mice.
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O'Brodovich, H., L. Berry, M. D'Costa, R. Burrows, and M. Andrew. "Influence of fetal pulmonary epithelium on thrombin activity." American Journal of Physiology-Lung Cellular and Molecular Physiology 261, no. 4 (October 1, 1991): L262—L270. http://dx.doi.org/10.1152/ajplung.1991.261.4.l262.
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Epithelial injury and intra-alveolar fibrin are present in lung injury. To determine whether healthy fetal and neonatal lung epithelium could regulate thrombin activity (hence fibrin formation) we collected amniotic and postnatal endotracheal tube fluids from humans and directly sampled lung and amniotic fluids from fetal guinea piglets, rabbit pups, and lambs. The coagulant properties of the cell surface and media conditioned by rat fetal type II alveolar epithelium were assessed. All fluids contained glycosaminoglycans (GAGs), but mass and biological assays demonstrated only some (heparan sulfate and to a lesser extent dermatan sulfate) had antithrombin activity. The presence of proteoglycans (greater than 1,000 kDa) yielding active GAGs with less than 100 kDa after base elimination were demonstrated by Sepharose CL4B chromatography. Epithelial-derived fluids contained a factor VII-dependent procoagulant activity, but concentrated conditioned media overlying primary cultures of type II epithelium demonstrated a net antithrombin effect. These studies demonstrate that the lungs of human and nonprimate mammalian fetuses and fetal type II epithelium secrete GAGs, some of which possess antithrombin activity, which would oppose intra-alveolar fibrin formation.
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Nilsson, Mikael, Johanna Husmark, Bengt Nilsson, Lars-Erik Tisell, and Lars E. Ericson. "Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function." European Journal of Endocrinology 135, no. 4 (October 1996): 469–80. http://dx.doi.org/10.1530/eje.0.1350469.
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Nilsson M, Husmark J, Nilsson B, Tisell L-E, Ericson LE. Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function. Eur J Endocrinol 1996;135:469–80. ISSN 0804–4643 Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell™) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4–6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200–500Ω·cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 Ω·cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM- or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma stimulation for 48 h. We conclude that human thyrocytes form a tight and polarized epithelium when cultured on permeable filters. The polarized structure and function of the cells are positively regulated by TSH. The culture system may be useful in studies addressing the role of the epithelial phenotype (cell polarity and tight barrier) in normal thyroid function as well as in pathological processes in the thyroid, such as autoimmunity, cell transformation and tumor progression. Mikael Nilsson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden
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Wasano, K., K. C. Kim, R. M. Niles, and J. S. Brody. "Membrane differentiation markers of airway epithelial secretory cells." Journal of Histochemistry & Cytochemistry 36, no. 2 (February 1988): 167–78. http://dx.doi.org/10.1177/36.2.3335774.
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We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.
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Andersen, Knut-Jan, Erik Ilsø Christensen, and Hogne Vik. "Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing." Alternatives to Laboratory Animals 21, no. 2 (April 1993): 191–95. http://dx.doi.org/10.1177/026119299302100212.
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The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.
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Boucher, R. C., and E. H. Larsen. "Comparison of ion transport by cultured secretory and absorptive canine airway epithelia." American Journal of Physiology-Cell Physiology 254, no. 4 (April 1, 1988): C535—C547. http://dx.doi.org/10.1152/ajpcell.1988.254.4.c535.
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The use of primary cell culture techniques to predict the function of native respiratory epithelia was tested in studies of dog airway epithelia. Epithelial cells from Cl- secretory (tracheal) and Na+ absorptive (bronchial) airway regions were isolated by enzymatic digestion, plated on collagen matrices, and maintained in serum-free, hormone-supplemented media. Transepithelial and intracellular studies showed that both the tracheal and bronchial culture preparations exhibited bioelectric parameters quantitatively similar to those of intact tissues. Similar to the native tissue, the tracheal preparation exhibited an equivalent short-circuit circuit (Ieq) that was sensitive to inhibitors of Cl- transport (bumetanide, diphenylamine carboxylic acid) but was insensitive to an inhibitor of Na+ transport, amiloride. In contrast, the bronchial preparation, like the native tissue, exhibited an Ieq sensitive to amiloride but insensitive to bumetanide. As compared with the trachea, the bronchial (absorptive) epithelium is characterized by 1) a large amiloride-sensitive cellular conductance and 2) a relatively depolarized basolateral membrane. We conclude that this primary cell culture technique provides epithelial preparations comparable to the native tissue and suitable for quantitative studies of regional differences in ion transport function.
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Ourahmane, Amine, Xiaohong Cui, Li He, Meaghan Catron, Dirk Dittmer, Ahmed Al Qaffasaa, Mark Schleiss, Laura Hertel, and Michael McVoy. "Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus." Viruses 11, no. 3 (March 5, 2019): 221. http://dx.doi.org/10.3390/v11030221.
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Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.
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Wille, Diana, Joana Heinzelmann, Astrid Kehlen, Marc Lütgehetmann, Dominik S. Nörz, Udo Siebolts, Anke Mueller, et al. "New Safety Aspects in Corneal Donation—Studies on SARS-CoV-2-Positive Corneal Donors." Journal of Clinical Medicine 11, no. 12 (June 9, 2022): 3312. http://dx.doi.org/10.3390/jcm11123312.
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In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT–PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT–PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT–PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT–PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.
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Gruenert, D. C., W. E. Finkbeiner, and J. H. Widdicombe. "Culture and transformation of human airway epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 3 (March 1, 1995): L347—L360. http://dx.doi.org/10.1152/ajplung.1995.268.3.l347.
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The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease.
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Rees, W. D., N. Telkar, D. Lin, M. Wong, C. Poloni, A. Fathi, M. Kobor, N. Zachos, and S. Ted. "A8 REPEATED SUBMERGENCE OF AIR-LIQUID INTERFACE COLONOID CULTURES IMPAIRS INFLAMMATORY AND REGENERATIVE RESPONSES." Journal of the Canadian Association of Gastroenterology 5, Supplement_1 (February 21, 2022): 9–10. http://dx.doi.org/10.1093/jcag/gwab049.007.
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Abstract Background Damage in the intestinal epithelium is repaired via de-differentiation of mature intestinal epithelial cells to a stem-like state. Indeed, literature has primarily focused on acute forms of intestinal damage, but there is a lack of models to study how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). A previous report found that growth of mouse intestinal organoids in air-liquid interface (ALI) follows by submergence caused differentiation and reversible injury, but this has not been demonstrated in human cells or with repeated cycles of injury. Understanding how chronic damage alters human intestinal stem cell fate and function is imperative to developing novel therapies that repair the epithelium in people with IBD Aims To develop a robust in vitro model to differentiate and damage human intestinal epithelial cells, with or without the addition of bacterial flagellin to mimic pathogen exposure. Methods Human colonoid monolayers were seeded on Transwell inserts for 10 days until fully confluent and then differentiated by removing the apical media to create ALI growth conditions for 7 days. To induce damage, media was added to the apical side of the Transwell, with or without the addition of flagellin in the basolateral compartment. Following submergence induced damage, the apical media was removed and collected for chemokine analysis, and the cells were grown back in ALI for 3 days to recover them from injury. This cycle was repeated 5 times to induce chronic damage. Cells were collected for qPCR analysis, immunofluorescence imaging, RNA sequencing and DNA methylation analysis Results Repeated rounds of damage impaired the ability of intestinal epithelial cells (IECs) to respond to TLR stimulation (a decrease in basolateral IL-8 with each round), likely due to a decrease in TLR signaling pathways, as demonstrated by GSEA and qPCR. Chronic submergence damage led to an increase in differentiation of cells expressing MUC2, SLC26a3 and CHGA, and a decrease in stemness as shown by qPCR for BMI1, HOPX, and LGR5. After several rounds of damage, colonoid monolayers were unable to regrow as monolayers after passaging, likely due to a decrease in YAP signaling. We also identified mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. Conclusions We have developed a novel chronic damage model of recurrent IEC injury, which possibly mimics pathologies seen in people with inflammatory bowel disease. This model can be used to understand how chronic damage alters the ability of IECs to respond to pathogens and regenerate to repair and protect the epithelium from further damage. Funding Agencies CCC
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Gorr, Matthew W., Dane J. Youtz, Clayton M. Eichenseer, Korbin E. Smith, Timothy D. Nelin, Estelle Cormet-Boyaka, and Loren E. Wold. "In vitro particulate matter exposure causes direct and lung-mediated indirect effects on cardiomyocyte function." American Journal of Physiology-Heart and Circulatory Physiology 309, no. 1 (July 1, 2015): H53—H62. http://dx.doi.org/10.1152/ajpheart.00162.2015.
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Particulate matter (PM) exposure induces a pathological response from both the lungs and the cardiovascular system. PM is capable of both manifestation into the lung epithelium and entrance into the bloodstream. Therefore, PM has the capacity for both direct and lung-mediated indirect effects on the heart. In the present studies, we exposed isolated rat cardiomyocytes to ultrafine particulate matter (diesel exhaust particles, DEP) and examined their contractile function and calcium handling ability. In another set of experiments, lung epithelial cells (16HBE14o- or Calu-3) were cultured on permeable supports that allowed access to both the basal (serosal) and apical (mucosal) media; the basal media was used to culture cardiomyocytes to model the indirect, lung-mediated effects of PM on the heart. Both the direct and indirect treatments caused a reduction in contractility as evidenced by reduced percent sarcomere shortening and reduced calcium handling ability measured in field-stimulated cardiomyocytes. Treatment of cardiomyocytes with various anti-oxidants before culture with DEP was able to partially prevent the contractile dysfunction. The basal media from lung epithelial cells treated with PM contained several inflammatory cytokines, and we found that monocyte chemotactic protein-1 was a key trigger for cardiomyocyte dysfunction. These results indicate the presence of both direct and indirect effects of PM on cardiomyocyte function in vitro. Future work will focus on elucidating the mechanisms involved in these separate pathways using in vivo models of air pollution exposure.
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Alamri, Ahmad M., Keunsoo Kang, Svenja Groeneveld, Weisheng Wang, Xiaogang Zhong, Bhaskar Kallakury, Lothar Hennighausen, Xuefeng Liu, and Priscilla A. Furth. "Primary cancer cell culture: mammary-optimized vs conditional reprogramming." Endocrine-Related Cancer 23, no. 7 (July 2016): 535–54. http://dx.doi.org/10.1530/erc-16-0071.
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The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53. Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial–mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments.
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Timerbulatova, G. A., P. D. Dunaev, A. M. Dimiev, G. F. Gabidinova, N. N. Khaertdinov, R. F. Fakhrullin, S. V. Boichuk, and L. M. Fatkhutdinova. "Comparative characteristics of various fibrous materials in in vitro experiments." Kazan medical journal 102, no. 4 (August 8, 2021): 501–9. http://dx.doi.org/10.17816/kmj2021-501.
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Aim. Comparative assessment of the effect of fibrous materials on cell cultures RAW264.7 and BEAS-2B.
Methods. The effects of various fibrous materials single-walled carbon nanotubes of two types (SWCNT-1 and SWCNT-2), differing in morphological characteristics, and chrysotile asbestos as a positive control was assessed on two cell lines macrophages RAW 264.7 and human bronchial epithelium BEAS-2B cells. The studied materials concentration range for experiments on cells was selected taking into account the SWCNT content in the air of the working area and the subsequent modeling of SWCNT deposition in the human respiratory tract. Suspensions of the studied materials were prepared based on cell culture media by ultrasonication. Cytotoxicity assessment after 48 hours of incubation was performed by using the MTS colorimetric assay. The expression level of apoptosis markers was assessed by immunoblotting using the corresponding monoclonal antibodies. Visualization of SWCNT-1, SWCNT-2 and chrysotile asbestos in BEAS-2B cell cultures was carried out by improved dark-field microscopy.
Results. According to dark-field microscopy, all the studied fibrous materials were found on the surface or cytoplasm of the cells. SWCNT and chrysotile asbestos did not have a direct cytotoxic effect in the MTS assay and did not induce apoptosis according to the results of Western blotting in cell cultures of RAW264.7 macrophages and BEAS-2B bronchial epithelium. In the cells of the bronchial epithelium (BEAS-2B) that showed greater sensitivity, a slight increase in the expression of pro-apoptotic protein PARP, which was more pronounced for shorter SWCNT-2, was revealed.
Conclusion. Both types of SWCNTs, despite the differences in morphological characteristics, demonstrated similar effects in in vitro experiments; this result, with its further verification, can have an important practical application in justifying approaches to determining the safety criteria for single-walled carbon nanotubes as a class of nanomaterials of the same type.
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Hilmi, Abu, Ahmad Halim, Norhayati Noor, Chin Lim, Zamzuri Idris, Abdullah Pohchi, Hassan Asma, Shaik Wahab, Stephan Tiede, and Ralf Paus. "A simple culture method for epithelial stem cells derived from human hair follicle." Open Life Sciences 8, no. 5 (March 1, 2013): 432–39. http://dx.doi.org/10.2478/s11535-013-0149-6.
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AbstractThe challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.
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Simintiras, Constantine A., Thomas Fröhlich, Thozhukat Sathyapalan, Georg J. Arnold, Susanne E. Ulbrich, Henry J. Leese, and Roger G. Sturmey. "Modelling aspects of oviduct fluid formation in vitro." Reproduction 153, no. 1 (January 2017): 23–33. http://dx.doi.org/10.1530/rep-15-0508.
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Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage and genome activation. However, the composition and regulation of this critical environment remain rather poorly defined. This study uses anin vitropreparation of the bovine oviduct epithelium to investigate the formation and composition ofin vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 inivDOF and show that the amino acid and carbohydrate content resembles that of previously reportedin vivodata. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids withinivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully thein vivoenvironment and impacts onivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on thein vitrooviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviductin vitroand suggest a number of future research avenues, such as investigating epithelial–fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media.
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Zitta, K., S. Ulbrich, E. Wolf, and M. Boelhauve. "119 ENDOMETRIAL CO-CULTURE MODELS FOR THE IN VITRO INVESTIGATION OF EARLY EMBRYO - MATERNAL CROSSTALK IN CATTLE." Reproduction, Fertility and Development 20, no. 1 (2008): 139. http://dx.doi.org/10.1071/rdv20n1ab119.
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Cellular and molecular changes in the endometrium during the preimplantation period are orchestrated by a complex network of molecules. The endometrium consists mainly of epithelial (EC) and stromal cells (SC) which produce a variety of active factors, such as prostaglandins (PG), in response to hormones and embryonic signals. However, it is unknown whether and how EC and SC influence each other in their reaction to these endo- and paracrine stimuli. To address this question, we measured the PG secretion of separately cultured EC and SC, and of EC+SC co-cultures in an insert system (0.2 µm membrane pore size) allowing exchange of secreted factors, but no direct contact between EC and SC. Bovine endometrial cells were isolated from cow uterus by a combination of mechanical and enzymatic procedures on Day 8 of the estrous cycle (ovarian morphology). Isolated EC and SC (90% purity) were cultured in DMEM/F12 containing 10% fetal bovine serum at 37�C in an atmosphere of 5% CO2 in air. PGE2 and PGF2α concentrations in media from separately cultured EC and SC or co-cultured EC+SC were measured by specific ELISAs after 24 h of stimulation with (i) 100 nm oxytocin (OXT); (ii) 100 ng mL–1 interferon tau (IFNT); or (iii) the respective solvent as non-stimulated control. EC responded to OXT stimulation with a decreased PGE2/PGF2α ratio, although the difference was not significant (PGE2/PGF2α ratio non-stimulated: 2.4 � 0.9; PGE2/PGF2α ratio stimulated: 1.5 � 0.8; P > 0.05 v. non-stimulated control, evaluated by t-test). No differences in the ratios of PGE2/PGF2α release into the medium were observed when comparing OXT-treated SC with non-treated SC. Incubating co-cultures of EC and SC with OXT resulted in a statistically significant, 2.5-fold decrease in the PGE2/PGF2α ratio as compared to non-stimulated cells (0.8 � 0.2 v. 2.0 � 0.7; P < 0.05; t-test). In contrast, IFNT stimulation of EC and SC co-cultures shifted PG secretion toward the pregnancy protective PGE2 (PGE2/PGF2α ratio non-stimulated: 0.99 � 0.44; PGE2/PGF2α ratio stimulated: 2.36 � 0.96; P < 0.05 v. non-stimulated control, by t-test). Conclusions: Our findings indicate that interactions between EC and SC regulate the responsiveness of the co-culture system to OXT and the pregnancy recognition molecule IFNT. We propose that co-culture of both cell types should be used for studying mechanisms of early embryo–maternal communication.
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Morais, Christudas, Justin Westhuyzen, Betty Pat, Glenda Gobe, and Helen Healy. "High ambient glucose is effect neutral on cell death and proliferation in human proximal tubular epithelial cells." American Journal of Physiology-Renal Physiology 289, no. 2 (August 2005): F401—F409. http://dx.doi.org/10.1152/ajprenal.00408.2004.
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In vitro models of diabetic nephropathy that assess the role of hyperglycemia on proximal tubular cell turnover commonly compare cells in a high-glucose medium (25 or 30 mM) with a low-glucose medium (5 to 6.1 mM). Any cellular growth changes observed are usually attributed to the effect of high glucose. We hypothesize that in such experiments, glucose concentrations in the low-glucose medium may decline during the course of the experiments to levels that inhibit cell growth leading to the comparative conclusion that high glucose induces hyperplasia and/or hypertrophy. In this study, primary cultures of human proximal tubular epithelial cells (PTEC) and immortalized HK-2 cells were exposed to low (5 mM) or high (17, 30, or 47 mM) glucose for up to 6 days (PTEC) and 48 h (HK-2). When culture media were not replenished, low glucose induced a significant increase in necrosis and release of lactate dehydrogenase and a decrease in proliferation, metabolic activity, and protein content without any changes in apoptosis. High-glucose media failed to induce any of these changes. Glucose was undetectable in the low-glucose culture medium after 72 h. No significant differences were observed between any of the treatment groups when culture media were replenished daily. We conclude that regular replenishment of culture media is necessary to prevent the emergence of artifactual and misleading differences between high- and low-glucose groups. The current knowledge of the pathophysiology of high glucose based on cell culture systems may need to be reevaluated.