Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells
Abstract Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even sub...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-37828-0 |
| _version_ | 1797778956787646464 |
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| author | Nikhil T. Awatade Andrew T. Reid Kristy S. Nichol Kurtis F. Budden Punnam Chander Veerati Prabuddha S. Pathinayake Christopher L. Grainge Philip M. Hansbro Peter A. B. Wark |
| author_facet | Nikhil T. Awatade Andrew T. Reid Kristy S. Nichol Kurtis F. Budden Punnam Chander Veerati Prabuddha S. Pathinayake Christopher L. Grainge Philip M. Hansbro Peter A. B. Wark |
| author_sort | Nikhil T. Awatade |
| collection | DOAJ |
| description | Abstract Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)—(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions. |
| first_indexed | 2024-03-12T23:24:03Z |
| format | Article |
| id | doaj.art-34e398b88c08484c9891f298e967ba74 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-12T23:24:03Z |
| publishDate | 2023-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-34e398b88c08484c9891f298e967ba742023-07-16T11:13:41ZengNature PortfolioScientific Reports2045-23222023-07-0113111110.1038/s41598-023-37828-0Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cellsNikhil T. Awatade0Andrew T. Reid1Kristy S. Nichol2Kurtis F. Budden3Punnam Chander Veerati4Prabuddha S. Pathinayake5Christopher L. Grainge6Philip M. Hansbro7Peter A. B. Wark8School of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleSchool of Medicine and Public Health, University of NewcastleImmune Health Program, Hunter Medical Research Institute, University of NewcastleSchool of Medicine and Public Health, University of NewcastleAbstract Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)—(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.https://doi.org/10.1038/s41598-023-37828-0 |
| spellingShingle | Nikhil T. Awatade Andrew T. Reid Kristy S. Nichol Kurtis F. Budden Punnam Chander Veerati Prabuddha S. Pathinayake Christopher L. Grainge Philip M. Hansbro Peter A. B. Wark Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells Scientific Reports |
| title | Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells |
| title_full | Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells |
| title_fullStr | Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells |
| title_full_unstemmed | Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells |
| title_short | Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells |
| title_sort | comparison of commercially available differentiation media on cell morphology function and anti viral responses in conditionally reprogrammed human bronchial epithelial cells |
| url | https://doi.org/10.1038/s41598-023-37828-0 |
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