Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media
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...
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doaj-55a1385d2fd64e789ffc97942da3b5d82020-11-25T01:22:54ZengWileyLaryngoscope Investigative Otolaryngology2378-80382019-12-014666367210.1002/lio2.319Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis mediaYajun Chen0Stéphanie Val1Anna Krueger2Lydia Zhong3Aswini Panigrahi4Gustavo Nino5Seth Wolf6Diego Preciado7Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.Center for Cancer and Immunology Research Children's National Health System Washington District of Columbia U.S.A.Division of Pulmonary Medicine Children's National Health System Washington District of Columbia U.S.A.Division of Pulmonary Medicine Children's National Health System Washington District of Columbia U.S.A.Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.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 NAhttps://doi.org/10.1002/lio2.319Differentiationotitis mediaprimary middle ear cell culturereprogramming |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yajun Chen Stéphanie Val Anna Krueger Lydia Zhong Aswini Panigrahi Gustavo Nino Seth Wolf Diego Preciado |
spellingShingle |
Yajun Chen Stéphanie Val Anna Krueger Lydia Zhong Aswini Panigrahi Gustavo Nino Seth Wolf Diego Preciado Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media Laryngoscope Investigative Otolaryngology Differentiation otitis media primary middle ear cell culture reprogramming |
author_facet |
Yajun Chen Stéphanie Val Anna Krueger Lydia Zhong Aswini Panigrahi Gustavo Nino Seth Wolf Diego Preciado |
author_sort |
Yajun Chen |
title |
Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media |
title_short |
Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media |
title_full |
Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media |
title_fullStr |
Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media |
title_full_unstemmed |
Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media |
title_sort |
human primary middle ear epithelial cell culture: a novel in vitro model to study otitis media |
publisher |
Wiley |
series |
Laryngoscope Investigative Otolaryngology |
issn |
2378-8038 |
publishDate |
2019-12-01 |
description |
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 |
topic |
Differentiation otitis media primary middle ear cell culture reprogramming |
url |
https://doi.org/10.1002/lio2.319 |
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