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Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media.

ABSTRACT: Objectives:Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological model for the study of OM. Methods:We adapted a cell reprogramming protocol using irradiated fibroblast feeder medium in addition to Rho kinase inhibitor to proliferate pMEEC collected during cochlear implant surgery. Cells were plated on transwell membranes, proliferated with conditionally reprogrammed culture medium, and transferred to air-liquid interface (ALI). Cultures were maintained for 4?weeks at ALI, photos were taken and cell lysates and secretions were collected over time for characterization analysis using quantitative polymerase chain reaction, Western bolt, and proteomics. Keratins, MUC5B and MUC5AC mucins, and beta tubulin (TUBB) were analyzed at the mRNA and protein level. Results:Cultures took a mean of 2?weeks to proliferate before transwell plating and forming a tight epithelium at ALI from 2 to 4?weeks. Although mRNA expression of MUC5B, MUC5AC, TUBB, and keratin 5 (KRT5) were variable depending on the differentiation stage and the patient, both TUBB and KRT5 proteins were detected until week 2. Conclusion:We demonstrate a novel method to proliferate and differentiate pMEECs that express epithelial markers and that are able to secrete mucins for the study of OM. Level of Evidence:NA.

SUBMITTER: Chen Y

PROVIDER: S-EPMC6929573 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media.

Chen Yajun Y Val Stéphanie S Krueger Anna A Zhong Lydia L Panigrahi Aswini A Nino Gustavo G Wolf Seth S Preciado Diego D

Laryngoscope investigative otolaryngology 20191127 6

<h4>Objectives</h4>Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological ...[more]

PMID: 31890886

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Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 8 control samples and 9 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is from a single animal.

Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 6 control samples and 8 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is a pool of 4 animals

Project description:BackgroundProteins found in middle ear effusion play crucial roles in the physiological and pathological processes of otitis media with effusion (OME), influencing the etiology and clinical characteristics of this disease. The qualitative and quantitative composition of these proteins depending on the underlying pathogenesis of middle ear effusion. Understanding their physiological and pathological functions is of great importance.MethodsWe collected samples from 19 volunteers diagnosed with OME. After offline separation using high-pH reversed-phase liquid chromatography (RPLC), the pooled sample was subjected to LC-MS/MS analysis to obtain a comprehensive profile of the OME proteome. Functional analysis was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis (IPA) annotations. Data-independent acquisition (DIA) technology was utilized to analyze samples and fix whether the OME proteome could replicate the pathophysiological features associated with this disease. We conducted a differential proteomic analysis between patients with simple OME and patients who had received radiotherapy. The radiotherapy-reduced group was further divided into two subgroups: nasopharyngeal carcinoma (NPC) and other types of carcinoma. Parallel reaction monitoring (PRM) technology was used for validation of 36 differentially expressed proteins (DEPs).ResultsA number of 732 proteins were identified in the OME proteome database. Among them, 527 proteins were quantified using peak intensity-based semi-quantification (iBAQ), covering a wide dynamic range of approximately 8 orders of magnitude. Based on the functional analysis, we proposed a hypothetical mechanism of OME.ConclusionThis study managed to put up an inclusive analysis of the OME proteome, establishing the first human OME proteome database. We focused on differential proteomic analysis among different groups to gain a more comprehensive concept of the OME proteome and search for meaningful biomarkers.

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Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media.

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Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media.

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