ORIGINAL ARTICLE: Quantification and Comparison of Toll‐Like Receptor Expression and Responsiveness in Primary and Immortalized Human Female Lower Genital Tract Epithelia

Herbst-Kralovetz, Melissa M.; Quayle, Alison J.; Ficarra, Mercedes; Greene, Sheila; Rose, William A.; Chesson, Ralph R.; Spagnuolo, Rae Ann; Pyles, Richard B.

doi:10.1111/j.1600-0897.2007.00566.x

Cited by 125 publications

(175 citation statements)

References 43 publications

(75 reference statements)

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“…59 The expression of TLR4 in the epithelial cells of the human female genital tract is controversial. Some observed the presence of TLR4, [60][61][62] while others reported the absence. 63,64 In normal human intestinal epithelial cells, TLR2 and TLR4 are expressed at low level.…”

Section: Prrs Of Epithelial Cellsmentioning

confidence: 99%

The role of pattern recognition receptors in the innate recognition ofCandida albicans

Zheng

Wang

et al. 2015

Virulence

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Section: Prrs Of Epithelial Cellsmentioning

confidence: 99%

The role of pattern recognition receptors in the innate recognition ofCandida albicans

Zheng

Wang

et al. 2015

Virulence

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“…Upon stimulation with molecules derived from microbes that are recognized by the Toll-like Receptors (TLR), the aggregates respond and secrete pro-inflammatory cytokines, including IL-6 1 . Expression of the progesterone receptor (PR), a receptor that responds to hormone stimulation, can also be observed in the vaginal cells both at the RNA and protein levels (Figure 7 and Figure 8, respectively).…”

Section: Representative Resultsmentioning

confidence: 99%

“…The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type 1,[7][8][9][10][11][12][13] . Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability ( Figure 1D).…”

Section: Introductionmentioning

confidence: 99%

“…We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues [1][2][3][4][5][6] . The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions.…”

Section: Introductionmentioning

confidence: 99%

“…The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions ( Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial celltype specific properties.The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type 1,[7][8][9][10][11][12][13] . Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability ( Figure 1D).…”

mentioning

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Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Radtke

Herbst-Kralovetz

2012

JoVE

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Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues [1][2][3][4][5][6] . The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a highaspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads ( Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor ( Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions ( Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial celltype specific properties.The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type 1,[7][8][9][10][11][12][13] . Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability ( Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/d...

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3D Oral and Cervical Tissue Models for Studying Papillomavirus Host‐Pathogen Interactions

et al. 2020

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Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions.

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ORIGINAL ARTICLE: Quantification and Comparison of Toll‐Like Receptor Expression and Responsiveness in Primary and Immortalized Human Female Lower Genital Tract Epithelia

Abstract: TLR expression patterns were remarkably conserved across the study population and evaluated tissues indicating a predictable responsiveness to STI. The results support cautious use of immortalized cells for genital tract modeling.

Cited by 125 publications

References 43 publications

The role of pattern recognition receptors in the innate recognition ofCandida albicans

The role of pattern recognition receptors in the innate recognition ofCandida albicans

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

3D Oral and Cervical Tissue Models for Studying Papillomavirus Host‐Pathogen Interactions

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