Primary Cell Culture of Turkey Intestinal Epithelial Cells

1996 ◽  
Vol 40 (1) ◽  
pp. 103 ◽  
Author(s):  
Akbar Ali ◽  
Donald L. Reynolds
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Primary Cell Culture ◽  
Intestinal Epithelial Cells ◽  
Primary Cell ◽  
Intestinal Epithelial

scholarly journals A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Coyne G. Drummond ◽  
Cheryl A. Nickerson ◽  
Carolyn B. Coyne
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Intestinal Epithelium ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Intestinal Epithelial ◽  
Cell Culture Model ◽  
Culture Model ◽  
Gastrointestinal Epithelium

ABSTRACT Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the epithelium lining the gastrointestinal tract early in infection. The lack of suitable in vivo and in vitro models to study CVB infection of the gastrointestinal epithelium has limited our understanding of the events that surround infection of these specialized cells. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of human intestinal epithelial cells that better models the gastrointestinal epithelium in vivo. By applying this 3-D model, which recapitulates many aspects of the gastrointestinal epithelium in vivo, to the study of CVB infection, our work provides a new cell system to model the mechanisms by which CVB infects the intestinal epithelium, which may have a profound impact on CVB pathogenesis. Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the epithelium lining the gastrointestinal tract early in infection. The lack of suitable in vivo and in vitro models to study CVB infection of the gastrointestinal epithelium has limited our understanding of the events that surround infection of these specialized cells. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of human intestinal epithelial cells that better models the gastrointestinal epithelium in vivo. By applying this 3-D model, which recapitulates many aspects of the gastrointestinal epithelium in vivo, to the study of CVB infection, our work provides a new cell system to model the mechanisms by which CVB infects the intestinal epithelium, which may have a profound impact on CVB pathogenesis. Podcast: A podcast concerning this article is available.

scholarly journals A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

1996 ◽  
Vol 18 (4) ◽  
pp. 269-281 ◽  
Author(s):  
Michael J. Rutten ◽  
Donald R. Campbell ◽  
Cheryl A. Luttropp ◽  
Wendy M. Fowler ◽  
Mitchell A. Hawkey ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Primary Cell Culture ◽  
Primary Cell ◽  
Gastric Mucous

Abstract 378: Cholecystokinin Upregulates Cholesterol Absorption in Human Intestinal Epithelial Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Zhongmao Guo ◽  
Ningya Zhang ◽  
Lemuel Dent ◽  
Emmanuel U Okoro ◽  
Hong Yang
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Plasma Cholesterol ◽  
Cholesterol Absorption ◽  
Adherent Cell ◽  
Cholesterol Transport ◽  
Suspension Cell Culture ◽  
Intestinal Epithelial ◽  
Intestinal Cholesterol Absorption

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. We previously reported that cholecystokinin (CCK) increases intestinal cholesterol absorption and plasma cholesterol level in mouse models. The goal of this study was to investigate the effect of CCK on cholesterol absorption and Niemann-Pick C1 Like 1 (NPC1L1) expression in human primary intestinal epithelial cells (HPIECs). Normal HPIECs were isolated from small bowl resection specimens, and purchased from the Lonza Group (Walkersville, MD) . Cholesterol absorption was determined by measuring transcellular cholesterol transport in adherent cell culture and cholesterol association and release in suspension cell culture. Surface NPC1L1 was isolated using a biotinylation kit and detected by western blotting. Our data demonstrate that HPIECs express both CCK receptor-1 and -2 (CCK1R; CCK2R). Treatment of HPIECs with 3 nM [Thr28, Nle31]-CCK for 60 min increased transcellular cholesterol transport, cholesterol association and release by ~38, 32 and 44%. Selective inhibition of CCK1R and CCK2R with antagonists (1 μM lorglumide or L365260) or selective knockdown of CCK1R and CCK2R with siRNAs attenuated CCK-induced cholesterol absorption. In the cells cultured on transwell membranes, CCK increased the level of NPC1L1 in the apical membrane by ~35% but did not alter the total NPC1L1 protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. These data imply that activation of CCK1R/2R enhances cholesterol absorption by induction of NPC1L membrane translocation. [This study was supported by NIH grants U54MD0007593, UL1TR000445, and SC1HL101431]

scholarly journals Expression of Selected Connexin and Aquaporin Genes and Real-Time Proliferation of Porcine Endometrial Luminal Epithelial Cells in Primary Culture Model

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Katarzyna Wojtanowicz-Markiewicz ◽  
Magdalena Kulus ◽  
Sandra Knap ◽  
Ievgenia Kocherova ◽  
Maurycy Jankowski ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Primary Cell Culture ◽  
Water Movement ◽  
Inorganic Ions ◽  
Primary Cell ◽  
Lag Phase ◽  
Luminal Epithelial Cells

Luminal epithelial cells are the first embryonic-maternal contact site undergoing very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume, with the help of aquaporins that provide a transcellular path of rapid water movement during the secretion and absorption of fluids, as well as connexins enabling the flow of inorganic ions and small molecules. In this work, we have examined how AQPs and Cx’s behave in luminal epithelium primary cell culture. Cells obtained from porcine specimen during slaughter were primarily in vitro cultured for 7 days. Their proliferation patterns were then analyzed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes of gene of interest expression were analyzed on each of the seven days of the porcine luminal primary cell culture. Our study showed that the significant changes were noted in the case of Cx43, whose level of protein expression and distribution increases after 120 hours of culture, when the cells enter the lag phase, and maintains an upward trend until the end of the culture. We noted an increase in AQP4, AQP7, AQP8, and AQP11 levels throughout the entire culture period, while the largest differences in expression were found in AQP3, AQP4, and AQP10. The obtained results could become a point of reference for further in vivo and clinical research. Experiments conducted with these proteins showed that they influence the endometrial fluid content during the oestrous cycle and participate in the process of angiogenesis, which intensifies during endometrial development.

scholarly journals Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

1995 ◽  
Vol 131 (6) ◽  
pp. 1587-1598 ◽  
Author(s):  
J Rak ◽  
Y Mitsuhashi ◽  
V Erdos ◽  
S N Huang ◽  
J Filmus ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Death ◽  
Epithelial Cells ◽  
Programmed Cell Death ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Intestinal Epithelial ◽  
Ras Oncogene ◽  
Multicellular Spheroids ◽  
Monolayer Cell Culture

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

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